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Structure-guided insights into heterocyclic ring-cleavage catalysis of the non-heme Fe (II) dioxygenase NicX

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  • Institute of Plant Physiology and Ecology

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Biodegradation of aromatic and heterocyclic compounds requires an oxidative ring cleavage enzymatic step. Extensive biochemical research has yielded mechanistic insights about catabolism of aromatic substrates; yet much less is known about the reaction mechanisms underlying the cleavage of heterocyclic compounds such as pyridine-ring-containing ones like 2,5-hydroxy-pyridine (DHP). 2,5-Dihydroxypyridine dioxygenase (NicX) from Pseudomonas putida KT2440 uses a mononuclear nonheme Fe(II) to catalyze the oxidative pyridine ring cleavage reaction by transforming DHP into N-formylmaleamic acid (NFM). Herein, we report a crystal structure for the resting form of NicX, as well as a complex structure wherein DHP and NFM are trapped in different subunits. The resting state structure displays an octahedral coordination for Fe(II) with two histidine residues (His²⁶⁵ and His³¹⁸), a serine residue (Ser³⁰²), a carboxylate ligand (Asp³²⁰), and two water molecules. DHP does not bind as a ligand to Fe(II), yet its interactions with Leu¹⁰⁴ and His¹⁰⁵ function to guide and stabilize the substrate to the appropriate position to initiate the reaction. Additionally, combined structural and computational analyses lend support to an apical dioxygen catalytic mechanism. Our study thus deepens understanding of non-heme Fe(II) dioxygenases.
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ARTICLE
Structure-guided insights into heterocyclic
ring-cleavage catalysis of the non-heme Fe (II)
dioxygenase NicX
Gongquan Liu1,3, Yi-Lei Zhao 1,3, Fangyuan He2, Peng Zhang 2, Xingyu Ouyang1, Hongzhi Tang 1&
Ping Xu 1
Biodegradation of aromatic and heterocyclic compounds requires an oxidative ring cleavage
enzymatic step. Extensive biochemical research has yielded mechanistic insights about cat-
abolism of aromatic substrates; yet much less is known about the reaction mechanisms
underlying the cleavage of heterocyclic compounds such as pyridine-ring-containing ones like
2,5-hydroxy-pyridine (DHP). 2,5-Dihydroxypyridine dioxygenase (NicX) from Pseudomonas
putida KT2440 uses a mononuclear nonheme Fe(II) to catalyze the oxidative pyridine ring
cleavage reaction by transforming DHP into N-formylmaleamic acid (NFM). Herein, we
report a crystal structure for the resting form of NicX, as well as a complex structure wherein
DHP and NFM are trapped in different subunits. The resting state structure displays an
octahedral coordination for Fe(II) with two histidine residues (His265 and His318), a serine
residue (Ser302), a carboxylate ligand (Asp320), and two water molecules. DHP does not bind
as a ligand to Fe(II), yet its interactions with Leu104 and His105 function to guide and stabilize
the substrate to the appropriate position to initiate the reaction. Additionally, combined
structural and computational analyses lend support to an apical dioxygen catalytic
mechanism. Our study thus deepens understanding of non-heme Fe(II) dioxygenases.
https://doi.org/10.1038/s41467-021-21567-9 OPEN
1State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, and School of Life
Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, Peoples Republic of China. 2National Key Laboratory of Plant Molecular Genetics, CAS
Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai, Peoples Republic of China.
3
These authors contributed equally: Gongquan Liu, Yi-Lei Zhao. email: tanghongzhi@sjtu.edu.cn
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Pyridine rings are primary components of pyridoxyl deri-
vatives, natural plant alkaloids, and coenzymes. These
compounds are more soluble in water, meaning they can
spread into groundwater, which are hazardous to the health of
humans and other organisms1,2. A pyridine ring opening reaction
step is a common feature of most chemical and enzyme-based
degradation processes for such pollutants, yet relatively little is
known about such reactions, highlighting that gaining a clearer
understanding of pyridine ring opening should enable develop-
ment of management technologies.
2,5-Hydroxy-pyridine (DHP), a potential carcinogen, is a
metabolic intermediate in the catabolism of many pyridine
derivatives37, which showed to cause DNA strand scission8.
DHP is transformed to N-formylmaleamic acid (NFM) by a 2,5-
DHP dioxygenase, an enzyme known as NicX from Pseudomonas
putida KT2440 or Hpo from P. putida S165,9. A previous bio-
chemical study showed that this enzyme is a mononuclear non-
heme iron oxygenase9.
The superfamily of non-heme iron(II) enzymes catalyzes a
wide range of oxidative transformations, ranging from the cis-
dihydroxylation of arenes to the biosynthesis of antibiotics such
as isopenicillin and fosfomycin1013. These enzymes can be
classied into several different groups based on their structural
characteristics, reactivity, and specic requirements for catalysis,
among them: (I) Extradiol cleaving catechol dioxygenases,
(II) Rieske oxygenases, (III) Alpha-ketoglutarate dependent
enzymes, (IV) Cysteine dioxygenases, and (V) Pterin-dependent
hydroxylases13,14.
A phylogenetic analysis of non-heme Fe(II) dioxygenases
indicted that NicX is a member of a subclass of the non-heme
iron dependent oxygenases (Fig. 1)7. Unlike other known
non-heme Fe(II) enzymes, NicX catalyzes a pyridine ring-
cleavage7,15. Notably, many ring-cleavage dioxygenases have
been reported, including 2,3-HPCD from Brevibacterium
fuscum16, BphC from Pseudomonas sp. KKS10217, PcpA from
Sphingobium chlorophenolicum18,19, and PnpCD from Pseudo-
monas sp. WBC-320. The substrates for all of these enzymes
contain aromatic rings. NicX has strong specicity for its DHP
substrate; it cannot catalyze ring opening for pyridine-ring con-
taining phenols, hydroquinones, or catechols21. The typical
structural motif consists of a mononuclear iron(II) metal center
that is coordinated by two histidine residues and one carboxylate
ligand from either a glutamate or an aspartate residue. This
structural motif has been coined the 2-His-1-carboxylate
facial triad13. In addition, a His/His/His triad coordinated Fe(II)
has been found in cysteine dioxygenases and gentisate dioxy-
genase2224. Interestingly, NicX Ser302 coordinates the iron(II)
ion; a similar metal ion-interacting serine residue has been
reported for a dialkylglycine decarboxylase, Cu+-ATPases and for
transcriptional activators like CueR and GolS2528. Although
NicXs DHP dioxygenase activity was enzymologically char-
acterized in the 1970s, its structure has remained unsolved.
In this study, we present kinetics, mutational, and structural
studies of NicX and clarify how NicX selectively recognizes DHP.
We solved a resting homo-hexameric NicX structure as well as a
NicXDHPNFM complex structure. We found that four resi-
dues of NicX (His265, Ser302, His318, Asp320) coordinate the iron
(II) ion. We also found that Leu104 and His105 adapt different
conformations in DHP-bound monomers vs. NFM-bound
monomers. In addition, molecular modeling, molecular dynam-
ics simulations and quantum mechanics calculations were com-
bined with the crystallographic 3D structural data to propose the
Fig. 1 Phylogenetic analysis of NicX. Phylogenetic tree of NicX with selected dioxygenases constructed by using neighbor-joining method. GenBank
accession numbers or pdb numbers are shown at the end of each name. Bar represents 1.0 amino acid substitutions per site.
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plausible catalytic mechanisms of NicX. Our study of NicX dee-
pens understanding of non-heme Fe(II) dioxygenases.
Results
Structural determination and overall structure. Seeking to
better understand how NicX selectively recognizes its substrate
DHP, we adopted a selenomethionine (SeMet) phasing strategy in
which we initially determined the crystal structure of a SeMet-
substituted resting NicX to a resolution of 2.28 Å using the single-
wavelength anomalous dispersion (SAD) method (Table 1).
Subsequently, the crystal structures of NicX and complex
NicXDHPNFM were solved using coordinates of SeMet-NicX
(Table 1). There are six molecules in an asymmetric unit in all the
structures, consistent with gel ltration chromatography results
revealing that NicX occurs as a homohexamer in solution
(Fig. 2a)29.
The N-terminus of each NicX subunit has a 150-residue
domain comprising 12 αhelices and 19 β-strands (residues
2151): this domain mainly consists of continuous αβ motifs.
Each subunit also has a 199-residue C-terminal domain (residues
152350), which contains two antiparallel β-sheets (β5-β6-β10-
β13-β14-β18-β19/β7-β9-β11-β12-β15-β16-β17) (Fig. 2b).
Structure of the NicXDHPNFM complex. We examined the
aforementioned NicXDHPNFM complex structure and found
that only four subunits (C, D, E, and F) of the six constituent
molecules in this structure bind with substrate DHP; the other
subunits (A and B) contain the product NFM. This phenomenon
of heterogeneous monomer binding patterns for a multi-
homomeric enzyme is not surprising, with similar reports for
other metal iron-dependent dioxygenases3033. In the
NicXDHPNFM complex, each subunit contains a fully occu-
pied Fe(II), each of which coordinates with six ligands: four
residues and two waters (Fig. 2c). Notably, site where water 2 is
positioned exhibits elongated density in our models for one of the
DHP-bound subunits (subunit D). This density was modeled as a
water molecule, because the density for the possible oxygen is not
clear while the previously reported trend that non-heme Fe(II)
enzymes typically requires the presence of a substrate for oxygen
binding to occur34,35.
Strikingly, the arrangement of the 14 Å-deep pockets in the
NicX surface (Fig. 2d) changes based on the binding activity of a
given subunit. That is, NFM-bound subunits appear the same as
the resting state: there are apparently two channels at the enzyme
surface near the active site, which are separated by residues His105
and Glu308, in close proximity to Leu104 (openstate; Fig. 2e).
However, only one of two channels occurs in the DHP-bound
subunits (henceforth channel I,closestate; Fig. 2f).
Characteristics of the Fe (II) coordination in NicX. There is a
conspicuous, deep pocket (~14 Å in depth) on the surface of NicX
that harbors its catalytic active site (Fig. 2d). Here, a ferrous ion is
held in place via coordination involving four residues (His265,
Ser302, His318,and Asp320) and two waters. Interestingly, the
coordinating ligand Ser302 has not been previously reported in
studies of other ferrous ion-dependent dioxygenases (Fig. 2c). To
verify that these four residues directly participate in the iron
coordination (rather than crystal packing), we conducted alanine
screening mutation analysis. We observed a complete loss of
activity for the H265A, S302A, H318A, and D320A mutants in
enzymatic assays (Supplementary Table 1). Moreover, ICP-MS
analysis revealed iron signals for the wild type enzyme but no
such signals for any of these four mutant variants, and the cir-
cular dichroism analysis showed that the secondary structures of
the mutation variants were not changed (Supplementary Table 2,
Supplementary Table 3, and Supplementary Fig. 1). Collectively,
these results verify the direct participation of these four residues
in iron coordination with the C-terminal domain of NicX. It
should be noted that although crystals were soaked in the buffer
contain Fe2+, we were not able to experimentally determine the
oxidation state of the iron in the crystal structures.
Table 1 Data collection and renement statistics.
SeMet-NicX NicX NicX in complex with DHP and NFM
Data collection
Space group P2
1
2
1
2P2
1
2
1
2P2
1
2
1
2
Wavelength (Å) 0.97918 0.97892 0.97918
Cell dimensions
a, b, c (Å) 125.92, 144.13, 118.89 125.961, 143.75, 118.69 126.67, 145.51, 118.98
α,β,γ(°) 90.00, 90.00, 90.00 90.00, 90.00, 90.00 90.00, 90.00, 90.00
Number of molecules/asymmetric unit 6 6 6
Resolution range (Å) (outer shell) 502.28 (2.402.28) 50.002.00 (2.032.00) 502.20 (2.242.20)
Completeness (%) (outer shell) 99.2 (99.6) 100.0 (97.3) 99.8 (99.2)
Redundancy (outer shell) 6.0 (6.2) 12.3 (12.3) 12.8 (13.1)
Total observations 585,113 1,763,979 1,453,302
Unique reections 98,134 142,933 113,279
Wilson B factor (Å2) 50.13 22.91 27.51
R
merge
(%) (outer shell) 9.1 (65.1) 10.1 (95.3) 10.6 (95.9)
I/σ
I
(outer shell) 8.7 (3.0) 25 (2.67) 24.4 (3.7)
Renement
Resolution range (Å) 302.28 47.412.00 27.592.20
R
work
/R
free
(%) 21.1/26.7 17.12/21.33 18.3/23.8
Average B-factors(Å2)
Protein, metal ion, water, substrate/product 67.3, 97.2, 55.1, - 27.75, 38.04, 40.28, - 29.52, 45.6, 38.8, 42.9/55.1
Root mean square deviations
Bond angles (°), Bond lengths (Å) 0.014, 1.644 0.010, 1.625 0.008, 0.908
Number of atoms
Protein/substrate/water 16,371/0/241 16,334/0/1514 16,576/52/940
Ramachandran plot
Most favored, allowed, disallowed (%) 95.1, 4.7, 0.1 95.81, 3.66, 0.53 95.38, 4.23, 0.38
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Substrate and product binding in the complex structure.In
DHP-bound subunits of the complex, two hydrogen bonds are
formed between Glu177 and the N1 and the O1 of DHP (2.5 and
3.0 Å), one hydrogen bond is formed between His189 and O1 of
DHP (2.6 Å) (Fig. 3a). Thus, these hydrogen bond interactions
may have an indispensable effect on the proper pre-catalytic
positioning of the substrate. We explored the functional relevance
of Glu177 and His189 with alanine mutant variants of these resi-
dues (as well as a NicXE177A&H189A mutant) which we tested with
in vitro enzymatic assays with puried enzymes. Wild type NicX
was puried to more than 95% homogeneity after recombinant
expression in E. coli cells, and exhibited apparent K
m
and V
max
values for conversion of DHP to NFM of 94.9 ± 3.84 μM and
58.62 ± 0.95 U mg1(Supplementary Table 1), respectively. The
puried NicXE177A&H189A variant completely lost its catalytic
activity for DHP. The NicXE177A and NicXH189A variants enor-
mously reduced activities (reduced to only 2.7 and 6.9% of the
wild type), and exhibited 2.7-fold and 2.2-fold higher K
m
values
compared with the wild type, respectively.
It is intriguing that a NicXR293A variant completely lost activity
for DHP (Supplementary Table 1). Observation in structure that
the side chain of Arg293 is positioned about 4.3 Å distant from the
center of DHP pyridine ring, and this residue does not apparently
change its position or orientation in the resting or NFM-bound
subunits (Fig. 3a). Moreover, Arg293 forms two salt bridge
interactions with the side chain carboxyl group of the conrmed
Fe-binding residue Asp320 (2.7 and 3.5 Å) (Fig. 3a). Pursuing this,
we conducted ICP-MS assays and found that the NicXR293A
variant lost the ability to bind a ferrous ion (Supplementary
Table 2). This result indicates that the role of Arg293 may be
primarily steric, apparently functioning to position the Asp320
correctly for ligation.
Analysis of the ring-open product NFM in the active sites of
the A and B subunits of the complex structure revealed that the
pyridine ring of DHP has been cleaved between the C5DHP and
C6DHP carbons to form the product NFM (Fig. 3b). The
carboxide derived from the C2DHP carbon of the product
interacts particularly strongly with residues Glu177 (2.8 Å) and
His189 (2.9 Å) (Fig. 3b), so that the product stably binds to the
enzyme, additionally supported by NFMscis-carbon double
bond7. As mentioned above, Fig. 3b provides direct evidence of
ring ssure.
A conformational change of Leu104 and His105. A careful
examination of structure shows that there is a conformational
change for the Leu104 and His105 in the substrate-bound vs. both
the resting structures and the product-bound subunits (Fig. 3c,
Supplementary Movie 1). It is a surprise to nd that a hydro-
phobic path that goes straight to the active center of ferrous ion
when channel II is closed in the E subunit (Supplementary Fig. 2).
However, this hydrophobic path is blocked by Leu104 in resting
state or product NFM binding state, suggesting that a hydro-
phobic path appears induced by the conformational change of
Leu104His105 (Supplementary Fig. 2). Leu104 seems to interact
with DHP through the side chain pyridine ring (3.9 Å) via both
CH-π36 and van der Waals interactions (Fig. 3a). His105 forms a
hydrogen bond with the substrate DHP (3.1 Å) (Fig. 3a). We
performed in vitro assays with mutant variants to conrm
functional contributions from these residues in NicXs enzymatic
activity. The NicXH105A variant completely lost enzyme activity,
and the NicXL104A variant showed a dramatically decreased
activity (down to only 3% of the wild type) and exhibited a 1.2-
fold higher K
m
value compared with the wild type. Notably, the
NicXE308A variant lost about half of its activity, and had a K
m
value very similar to wild type NicX, results suggesting that
perhaps Glu308 does not participate directly in the recognition
of DHP, but rather affects enzymatic activity via an interaction
with His105.
Fig. 2 Structure of NicX. a Overall structure of NicX. Ribbon plot representation of the NicX hexamer. bOverall structure of a protomer of NicX. The α-
helices, β-sheets, and loops are in red, yellow, and green, respectively. Secondary structure elements of NicX are labeled. cCoordination sites of NicX-Fe
(II). The 2F
O
-F
C
electron density map is contoured at 1σ, colored in blue; water 1 molecule is opposite Asp320, while water 2 molecule is opposite Ser302;
green, red, blue, and purple represent carbon, oxygen, nitrogen, and Fe atoms, respectively. dThe 14 Å-deep substrate-binding pocket on the NicX surface,
two channels are observed. eTwo channels are separated by residues His105 and Glu308, observed in resting or NFM complex structure. fIn the DHP-
bound subunits, channel II was blocked by the Leu104 and His105 residues.
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Fig. 3 Active site of NicX. a Active site of NicX with bound DHP. The carbon atoms of DHP are in yellow. Green, red, blue, and purple represent carbon,
oxygen, nitrogen, and Fe atoms, respectively. bActive site of NicX with bound NFM. The carbon atoms of NFM are in yellow. Cyan, red, blue, and purple
represent carbon, oxygen, nitrogen, and Fe atoms, respectively. cA stereoview of the DHP bound subunit (green) and NFM bound subunit (cyan), the
superimposition was done on the whole subunit. Iron and solvent molecules are shown as purple and red spheres, respectively. DHP/NFM interacts with
residues were indicated as black dotted line, the coordination bonds of ferrous ion are shown as solid lines in purple, distances are given in angstroms.
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Based on structural analysis, it could be seen from the NFM
bound subunits that Cys76 is close to Leu104 at a distance of 4.9 Å,
away from His105 at a distance of 7.7 Å (Fig. 4a, b). In contrast, in
DHP bound subunits, Cys76 is close to His105 at a distance of
4.1 Å, away from Leu104 at a distance of 7.7 Å. This means that
the conformational changes of Leu104His105 may be inuenced
by Cys76 (Fig. 4a, b). It seems that the enzyme activity could also
be inuenced by Val175 in channel I but far away from active
sites. To verify the above idea, Val175 in channel I and Cys76 in
channel II were selected for mutation (Fig. 4a, b). The NicXV175F
variant activity reduced to only 21.8% of the wild type, but K
m
values did not change much compared with the wild type,
respectively (Supplementary Table 1). In contrast, the point
mutations of C76Q or C76E completely abolished the enzymatic
activity of NicX; however, it was notable that the NicXC76A
variant retained the capability of transforming DHP into NFM
(Supplementary Table 1). From the result of the mutation
experiments, it appears that channel II has a more signicant
effect on the enzyme activity. Thus, we speculate that the bulky
residues (glutamine or glutamic acid) take up more space than
cysteine, and affect the range of movement of Leu104 and His105,
causing it to be unable to guide and stabilize the substrate to the
appropriate position to initiate the reaction.
Structural and computational studies for the NicX mechanism.
Although NicX is the subtype dening member for non-heme
iron(II) ring-cleavage dioxygenases, its overall catalytic mechan-
ism bears some similarities with other non-heme iron(II) dioxy-
genases. Based on the studies of other Fe(II)-dependent
dioxygenases12,13,3032,37, and the computational analysis on our
structures, we proposed two possible equatorial and apical
dioxygen catalytic mechanisms, and calculated energetics of these
pathways (Fig. 5, Supplementary Fig. 3).
If the dioxygen takes the opposite position of Asp320
(equatorial position), there are two possibilities for substrates
to coordinate with metal ions: the hydroxyl group is at position 5
to chelate with Fe (II) (Pathway IA), or the N atom on the
pyridine ring can coordinate with Fe (II) (Pathway IB).
Subsequently, two one-electron transfers are needed to form the
peroxide intermediate, which can be followed by a Criegee
rearrangement to yield a 7-membered-ring lactone and a ring-
opened product; either of these scenarios would result in a
substrate-bound iron arrangement similar to the classic extradiol
catechol dioxygenases (Pathway IA & IB). Such a position would
suggest a trans-effect for Asp320 in promoting the subsequent O-O
cleavage reaction. However, this situation would require the DHP
substrate to drop from the hydrogen-bonding network comprising
His105Glu177His189, which would cause considerable destabili-
zation of the reacting substrateenzyme complex (Pathway IA &
IB) (Supplementary Fig. 4a, b). Alternatively, the dioxygen
molecule could be positioned opposite to Ser302 (apical
position), in which case the C5 and C6 atoms of DHP would
be adjacent to the dioxygen molecule. The apical arrangement
would be similar to a P450-like arrangement, and Ser302 could
exert a cystine-like catalytic role (Pathway II) (Supplementary
Fig. 4c). More interestingly, the pyridinyl N-H could also plausibly
involve in the proton transfer to activate the O-O cleavage in
Pathway II, equivalent to imidazolyl N-H of the histidine residue
in extradiol dioxygenases. Further investigation on the roles of
Arg293Asp320DHPN-H is needed to illustrate the details of
Fig. 4 A conformational change of L104 and H105. a In NFM bound subunits, Cys76 is close to Leu104 at a distance of 4.9 Å, away from His105 at a
distance of 7.7 Å. bIn DHP bound subunits, Cys76 is close to His105 at a distance of 4.1 Å, away from Leu104 at a distance of 7.7 Å. Yellow, red, blue, and
orange represent carbon, oxygen, nitrogen, and sulfur atoms, respectively.
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proton transfer for the O-O cleavage in the ring-open process. In a
preliminary study of the conversion of the quintet peroxide
intermediate to a 7-membered-ring lactone, both concerted, and
stepwise mechanisms were examined (Pathway II, Supplementary
Fig. 5). These calculations indicated a preference for a stepwise
O-O cleavage; and low-lying septet state might promote spin-ip
during the breaking and formation of multiple bonds (Supple-
mentary Fig. 5).
Discussion
Multiple crystal structures of dioxygenases that can catalyze the
ring-opening cleavage of aromatic compounds have been repor-
ted3032, but we are unaware of any reported crystal structures for
an enzyme capable of catalyzing cleavage of a pyridine ring. It is
notable that a phylogenetic analysis of non-heme Fe(II) dioxy-
genases indicated that NicX and Hpo comprise a subclass of non-
heme iron dependent oxygenases (Fig. 1). Despite extensive
efforts, we were unable to obtain a structure for Hpo, and
structural comparison using the DALI server failed to identify any
obvious homologs of full-length NicX.
Another notable observation from our study that a con-
formational change in the Leu104 and His105 residues induce a
major change in channel II; this change creates a hydrophobic
path that goes straight to the active ferrous ion. Based on in vitro
assays, we conrmed that His105 has a dramatic effect on enzyme
activity. Previous studies have proposed that His residues could
facilitate alkylperoxo intermediate breakdown, potentially via
protonation to form a gem-diol intermediate38. We therefore
examined whether His105 exerts this type of effect in NicXs
mechanism of action. However, we found that when His105 was
mutated into aprotic amino acids (e.g., our NicXH105M and
NicXH105F), these variants retained their catalytic activity for
producing NFM from DHP (Supplementary Table 1). We inter-
pret these results to rule out the participation of His105 in proton
transfer during NicX catalysis. Our analyzes support speculation
about two possible functions of the conformational change for
Leu104His105. The rst is the aforementioned creation of a
hydrophobic path that could greatly facility the direct delivery of
a dioxygen molecule to the iron center (Supplementary Fig. 2).
The second potential function of the conformational change
could be to guide and assist to bind the DHP substrate molecule
to a pre-reaction position, besides Glu177His189; this may facil-
itate activation of the dioxygen and promote reaction initiation.
Our site-directed mutagenesis results show that NicXC76Q and
NicXC76E variants are inactivated provides further (albeit indir-
ect) evidence of the contribution of Leu104 and His105 in orienting
the substrate DHP.
It is also fascinating to nd that the NicX ferrous center
coordinates with only four residues (His265, Ser302, His318, and
Asp320). Ser has been reported as a ligand for proteins including
dialkylglycine decarboxylase, Cu+-ATPases, and transcriptional
activators2528; however, it has not been reported in non-heme
dioxygenases, suggesting that NicX has a previously unknown
fold architecture and active site environment for non-heme iron
(II) dioxygenases. We examined site-directed mutant variants of
NicX and conrmed the indispensability of these four residues for
Fe(II) coordinating by using enzymatic assays, ICP-MS assays,
and circular dichroism analyzes.
It is intriguing that NicX has not been reported to have
activities for substrates other than DHP. Our study indicates
that this substrate specicity is strongly impacted by NicXs
substrate-binding sites, including His105,His
189,andGlu
177,
each of which is involved in the unique hydrogen bonding
network. The O1 atom of DHP interacts with the residues of
Glu177 and His189, and the N atom of DHP affects the electron
distribution over the pyridine ring. Given that N atoms are
more electronegative than carbon atoms, these factors appar-
ently contribute to controlling the ionic conguration of the
substrate DHP; thus the N atom of DHP is placed adjacent to
Fig. 5 Plausible pathways for NicX-catalyzed DHP degradation, in which dioxygen attacks either from the equatorial position (Pathway IA, colored in
pink; Pathway IB, colored in cyan), or the apical position (Pathway II, colored in light green). Pathway IA and IB denote that the nitrogen and oxygen
atoms of DHP bind the metal ferrous center, respectively. In Pathway II, the substrate DHP does not directly chelate with the ferrous ion. The possibly one-
electron transfer radical species are drawn in square brackets. The C6 is the most vulnerable position in DHP, with an f-value of 0.148 based on Fukui
function analysis in the upper left corner (C2, 0.067; C3, 0.062; C4, 0.052; C5, 0.090; C6, 0.148).
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the Glu177 side chain carboxyl group, rather than some other
orientation. We consider the different catalytic pathways of
NicX, wherein the dioxygen occupies the equatorial position
(Pathway I) or apical position (Pathway II); these pathways are
ultimately downhill energetically and cannot be distinguished
based on energetics alone. However, the specicsubstrate
orientation in the co-crystal structure motivated us to recon-
sider the possibility of Pathway II. First of all, the arrangement
in Pathway II would allow maintenance of the specic hydrogen
bonding network for holding the DHP substrate in place; in
contrast, the DHP molecule would have to undergo a big ip
and shift downwards by ~4 Å to bind the metal and contact the
equatorial dioxygen in Pathway I. Importantly, this would
require disturbing the specic hydrogen bond network
observed in the crystal structure. Alternatively, in Pathway II
the bridging peroxo can undergo a ~0.5 Å shift of the substrate
and a ~20° rotation of the substrate, a scenario that would
retain the intermediate in the hydrogen bond network. Second,
the rst one-electron transfer of the DHP substrate seems to
occur at the moiety of O-C =NbasedonthepKa(pK
a1
=8.56)
and Fukui function (f-=0.148) (Fig. 5), and the dioxygen
molecule could occupy the vacancy between C6---Fe and
mediate the ignition of the DHP-O
2
-Fetriad.Third,thecon-
sequent O-O cleavage could be promoted by neighboring pro-
ton donor candidates such as imidazolium of His105,neutral
form of Asp320, aromatic N-H of DHP, and even guanidinium
of Arg293 39,40. Moreover, a crystal structure-based CAVER
analysis indicated that the dioxygen tunnels terminate at a
position opposite to Ser302 rather than Asp320 (Supplementary
Fig. 2). Thus, both structural analysis and preliminary com-
putation lend support the apical hypothesis, but additional
theoretical calculations will be required for further validation
on these possible mechanisms.
In past studies, the mechanisms of homogentisate 1,2-dioxy-
genase (HGDO, PDB code 4AQ6 [https://doi.org/10.2210/
pdb4AQ6/pdb]) and homoprotocatechuate 2,3-dioxygenase
(HPCD, PDB code 2IGA [https://doi.org/10.2210/pdb5XRN/
pdb]) have been investigated by trapping different reaction
cycle intermediates in different subunits of a single crystal30,32.
HPCD is a prototypical type I extradiol dioxygenase acting on a
catechol-type substrate, while HGDO reacts with a benzene-type
substrate containing hydroxyl group in the para position. Both
enzymes share things in common: (i) both catalyze the oxidative
aromatic ring cleavage; (ii) both have a mononuclear iron(II)
metal center that is coordinated by two histidine residues and one
carboxylate ligand; (iii) their substrates act as dentate ligands
when bound to the active site Fe (II) ion; (iv) their mechanisms
both feature an attack of a superoxo ligand on their substrates at a
hydroxylated carbon. It should be emphasized that both our
crystal structure data and computational studies highlight dif-
ferences in the apparent reaction mechanism of NicX (Fig. 5)
compared to HPCD and HGDO. Specically, (i) NicX is able to
catalytically crack a pyridine ring substrate; (ii) NicX has a
mononuclear iron(II) metal center that is coordinated by two
histidine residues, one carboxylate and a serine residue; (iii) DHP
does not directly chelate ferrous ion; (iv) the reaction between
superoxide and DHP proceeds by reaction at the C6 atom of
DHP, not the OH-group carrying C5 atom. In light of these
differences, it is not surprising that our crystal structures and
computational studies indicate clear distinctions for the proposed
reaction mechanism of NicX vs. the reaction mechanisms of
HPCD and HGDO.
In summary, we here determined the crystal structure of a
pyridine ring-cleavage enzyme. Our structural and computational
studies of NicX from P. putida shed lights on the ring-cleavage
mechanisms used by dioxygenases; our study deepens
understanding about how non-home Fe(II) ring-cleavage dioxy-
genase family enzymes interact with their aromatic and hetero-
cyclic substrates.
Methods
Chemical reagents. 2,5-Dihydroxypyridine was purchased from Aladdin. SeMet
was purchased from Acros Organics. Crystallization screens were obtained from
Hampton Research. All other chemicals were obtained commercially.
Plasmid preparation, recombinant expression, and protein purication. The
DNA fragment encoding full-length WT P. putida KT2440 NicX was cloned into
the pET-24a (Novagen) vector between the NdeIHindIII sites using DNA primers
5-agtcatatgccggtgagcaatgcacaa-3and 5-tataagctttcgcgctcgcgactcct-3(bearing a
sequence encoding 6 C-terminal His-tags) (Supplementary Table 4). All mutants
were generated using a whole-plasmid PCR and DpnI digestion method, and the
sequences of the constructs were veried via Sanger sequencing, primer pairs used
for installing each mutation were shown in Supplementary Table 4. These plasmids
were transformed into E. coli BL21(DE3) cells. Cells possessing plasmids for the
wild type and the mutant variant of NicX were grown at 37 °C to an OD
600
of 0.8,
after which they were subjected to overnight induction at 16 °C with 0.4 mM
isopropyl β-D-1-thiogalactopyranoside. Centrifugation for 15 minutes at 4,000×g
was used to harvest the cells, with the resulting cell pellets resuspended in a binding
buffer (25 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 20 mM imidazole, 0.5 mM
PMSF, 1 mM FeCl
2,
10 mM β-Mercaptoethanol) and subsequently lysed using a
cell homogenizer. We then centrifuged the cell lysate and puried the supernatant
with Ni2+-NTA afnity chromatography (Qiagen) and Superdex200 gel ltration
chromatography (GE healthcare). The gel ltration buffer was comprised of 200
mM NaCl, 20 mM Tris-HCl (pH 8.0), and 2 mM dithiothreitol (DTT). Subse-
quently, fractions with bands putative recombinant NicX proteins (~39 kDA,
assessed via SDS-PAGE) were combined and concentrated to 25 mg ml1, and
were then ash-frozen in liquid nitrogen and stored at 80 °C.
Note that a SeMet-substituted NicX variant was expressed using the
methionine-autotrophic E. coli strain B834(DE3) cultured in M9 minimal media,
and was puried using the same procedure as for the native protein, except that
5 mM DTT was used in the Superdex200 gel ltration buffer. Purity was monitored
for all protein preparations based on SDS-PAGE, and protein concentrations were
determined using a NanoDrop2000 spectrophotometer.
Enzyme assay. Puried WT and NicX mutant variant proteins used in the in vitro
enzymatic activity assay. Activity assay was performed at 25 °C, monitoring the
absorbance at 320 nm using a UV-Vis 2550 spectrophotometer21. The reaction
mixture contained 20 mM Tris-HCl (pH 8.0), 50 µM FeCl
2
, as well as the DHP
substrate and the enzyme (concentrations of both depending on the nature of the
particular assay) in a total volume of 800 µl. The enzyme was incubated with FeCl
2
(1 mM) for 1min, and the reaction was initiated via the addition of DHP. Activity
was dened as the amount of enzyme that catalyzes the conversion of 1.0 µmol of
DHP in 1 min. The assay was performed independently three times, and data are
presented as means ± the standard deviations.
Crystallization, data collection, and structure determination. Crystals of NicX
were grown from a 1:1(v/v) mixture of a NicX protein solution (25 mg ml1), a
reservoir solution (0.2 M Succinic acid (pH 7.0) and 20% (w/v) PEG 3350), using the
hanging-drop vapor-diffusion method at 20 °C. The SeMet-NicX variant was crys-
tallized in the reservoir solution containing 0.2 M Sodium formate (pH 7.0) and 20%
(w/v) PEG 3350. The complexes were prepared by soaking NicX crystals in cryo-
protectant buffer supplemented with 5 mM Fe2+for about 5 min prior to soaking
crystals in cryoprotectant buffer supplemented with 5 mM DHP and 20 mM sodium
dithionite. After soaking for 3060 min, crystals were rapidly transferred into mother
liquor solutions containing 25% glycerol prior to cryocooling in liquid nitrogen.
Crystal diffraction data sets of the native and SeMet-NicX were collected at the
BL17U1 and BL19U1 beamlines of the Shanghai Synchrotron Radiation Facility by
using an ADSC Quantum 315r detector or a DECTRIS PILATUS3 6 M detector at
a wavelength of 0.97918 Å at 100 K. All data were processed and scaled using the
HKL3000 program41. The SAD phases were determined using the Autosol module
of PHENIX42. After the model-building with Coot43 and renement with
REFMAC44. The structures of NicX and complex NicXDHPNFM were solved
using coordinates of SeMet-NicX; the substrate/product molecules were placed in
the model based on the Fourier difference map, and rened using the geometric
restraints prepared using REEL in Phenix45. The gures were prepared using
PyMOL. Crystallographic statistics are listed in Table 1. The resulting coordinates
and structure factors have been deposited in the Protein Data Bank (Protein Data
Bank codes: 7CNT7CUP7CN3).
Inductively coupled plasma mass spectrometry. An inductively coupled plasma
mass spectrometer, iCAP Qc (Thermo Fisher scientic, USA), with KED (Kinetic
energy discrimination) system was used for detection. Before the ICP-MS assays,
the puried mutant protein was incubated with an appropriate amount of ferrous
ion for at least 30 min (the molar ratio of protein to ferrous ion was 1:5) and again
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subjected to a Superdex 200 column. 200 µl of protein sample was added 0.5 ml
HNO
3
and let it sit for half an hour prior to heat the water bath for 90 min. Before
use, sample volume was diluted to 5 ml by water. The blank sample with the same
amount of acid was prepared with the same procedure.
Secondary analysis of structure via circular dichroism spectra. We used
JASCO J-815 to obtain the circular dichroism spectra of NicX and its related
proteins, at 20 °C, while the samples were 0.2 mg ml1in 20 mM NaH
2
PO
4
-
Na
2
HPO
4
buffer (pH 7.4). We measured from 200 to 280 nm, with a cell length of
1.0 mm. We used the software CDPro to analyze the secondary structure com-
positions of the proteins.
Dynamic pH titration. The dynamic pH titrations were used to assess the dis-
sociation constants (pKa) of DHP, in aqueous solutions. An ionic strength (I) of
0.1 mol L1KCl was used to perform the titrations and a pH range of 4.012.0 was
used to perform the measurements.
MD simulation. All MD simulations were performed by using the Amber software
suite46. The initial protein structure used in the molecular dynamic simulation was
constructed with the crystal structures (PDB: 7CUP7CN3). The enzyme-
substrate complexation was referred with the catalytic mechanisms of the Catechol
dioxygenases and Rieske oxygenases13,14. The dioxygen tunnels toward the Fe
center were analyzed with CAVER 3.0 program47. Both two water-coordination
sites were tested for dioxygen displacement. In particular, the peroxide inter-
mediate was docked into the active site based on the crystal structures with
AutoDock 4 program48. Appropriate substrate conformations were selected for
multiple MD simulations. Protonation states of titratable residues were assigned at
pH 6.5 using the H ++web server49, and Fe/substrate-binding residues were
visually inspected then. The HF/6-31 G*//B3LYP/6-311 G*method was used to
generate the substrate molecule in Gaussian 09, while the antechamber program
was used to t the RESP charge (restrained electrostatic potential charge)50. The
python-based Metal Center Parameter Builder was used to build the force eld of
the iron center active site51. To prepare the topology and assess les of the enzyme-
substrate complex, we used a cubic TIP3P water box (10 Å thick) from the surface
of the complex, while sodium counter ion was used to neutralize the whole system.
The particle mesh Ewald (PME) method was used to calculate the long-range
electrostatic interactions in the MD simulation, while the SHAKE algorithm was
used to constrain the hydrogen-involving bond lengths. We performed two
minimization sequences, which relaxed the molecules of the solvents as well as the
whole system. The temperature of the system increased from 0 to 300 K during
100 ps, at a collision frequency of 2 ps1of Langevin dynamics. We calibrated the
system for 50 ps and collected the trajectory with constant pressure and tem-
perature (NPT). Finally, 25 ns production simulations without any restraint were
performed under NPT conditions. An integration time step of 2 fs was utilized with
structural snapshots being extracted every 1000 steps. The simulation trajectory
was analyzed by the cpptraj in Amber tools18.
QM calculations. Geometrical snapshots from the enzyme-peroxide MD cluster
were extracted as the pre-reaction states (PRS)52 for the O-O and C-C bond
breaking and forming, and were further subject to geometry optimization in
Gaussian 09 program53. The quantum mechanical cluster model consisted of side
chains of active site residues (His105, Glu177, His189, His265, Ser302, His318,and
Asp320) and the iron cation and peroxide intermediate, which added up to 99 atoms
and bore one positive charge. The optimization process was carried out at the level
of ωB97X-D functional and LANL2DZ (Fe) and 6-31G(d) basis sets in aqueous
solution with the steered molecular dynamics approach54. The triplet, quintet, and
septet potential energy surfaces were scanned along the C-C and O-O reaction
coordinates55. All stationary point structures were further optimized with the same
level of theory. Vibrational frequency analyzes were performed to ensure local
minima or rst-order saddle points, and the free energies were calculated at 298 K
(standard condition). In addition, the intrinsic reaction coordinates calculations
were carried out to identify transition states and immediate reactants and products.
Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article.
Data availability
The data supporting the ndings of this work are available within the paper and its
Supplementary Information les or from the corresponding authors on reasonable
request. Protein Data Bank (PDB): The coordinates and the structure factor amplitudes
for the Se-NicX, NicX, and NicX complexed with ligands were deposited in Protein Data
Bank under accession codes 7CNT,7CUP,7CN3; and have been released. Source data
are provided with this paper.
Received: 29 November 2019; Accepted: 11 January 2021;
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Acknowledgements
This work was supported by the grant of Ministry of Science and Technology of the
Peoples Republic of China (2018YFA0901200), by the grant from Science and Tech-
nology Commission of Shanghai Municipality (17JC1403300), by the Shuguang Pro-
gram(17SG09) supported by Shanghai Educational Development Foundation, by
Shanghai Excellent Academic Leaders Program (20XD1421900) from Science and
Technology Commission of Shanghai Municipality, and by the grants from National
Natural Science Foundation of China (31422004, 31970041, and 21377085). We thank
Dr. John H. Snyder, Prof. Jiahai Zhou, and Prof. Shuangjun Lin for assistance and
discussion regarding this manuscript.
Author contributions
H.T. and G.L. designed the experiments. G.L., F.H., and X.O. performed the experiments.
G.L., H.T., and Y-L.Z. wrote the manuscript. G.L., Y-L.Z., H.T., and P.X. revised the
manuscript. P.Z., G.L., and H.T. analyzed the data. H.T., Y-L.Z., and P.X. conceived the
project.
Competing interests
The authors declare no competing interests.
Additional information
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available at https://doi.org/10.1038/s41467-021-21567-9.
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... 34−36 Most recently, Xu and coworkers reported the X-ray crystal structures of NicX and the NicX−DHP−NFM complex. 37 As Figure 1 shows, the NicX enzyme from P. putida KT2440 is a homohexamer, in which the active site is composed of one non-heme iron ion coordinated by residues His265, His318, Asp320, and Ser302 and two water molecules, featured by the common moiety of the 2-His-1-carboxylate facial triad for dioxygen activation. 38−45 Note that the substrate (DHP) is not bound to the iron center but is stabilized in the active pocket by hydrogen bonding interactions with residues His105, Glu177, and His189. ...
... 38−45 Note that the substrate (DHP) is not bound to the iron center but is stabilized in the active pocket by hydrogen bonding interactions with residues His105, Glu177, and His189. According to the structural features, two possible binding models of dioxygen were proposed, 37 that is, the "equatorial position" and "apical position", located at the opposite positions of Asp320 and Ser302, respectively. ...
... Based on the characterized crystal structures, Xu and coworkers proposed the probable mechanism shown in Scheme 2a. 37 In this mechanism, the dioxygen is located at the apical position in the end-on mode. The reaction is initiated by the attack of the distal oxygen on the substrate, followed by the O−O bond cleavage, which is promoted by the proton transfer (PT) from the pyridine-ring N−H of DHP. ...
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2,5-Dihydroxypyridine dioxygenase (NicX) from Pseudomonas putida KT2440 is a mononuclear non-heme iron oxygenase responsible for the biodegradation of 2,5-dihydroxypyridine (DHP) to N-formylmaleamic acid (NFM). Here, extensive quantum mechanical-molecular mechanical (QM/MM) calculations and molecular dynamics (MD) simulations are used to elucidate the degradation mechanism of DHP by wild-type NicX and its H105F variant (NicXH105F) and the roles of key residues. In particular, NicX and NicXH105F can catalyze the ring opening degradation of DHP to NFM, but flexible mechanisms are adopted therein. Both reactions of NicX and NicXH105F are initiated by the attack of FeIII superoxide species onto the substrate, during which a proton-coupled electron transfer (PCET) process is involved. For wild-type NicX, the PCET reaction is mediated by the adjacent His105, while the further proton transfer from His105 to the peroxo species can remarkably enhance the following O-O cleavage. However, for the NicXH105F mutant, a water molecule replaces the role of residue His105, which not only stabilizes the substrate binding via a H bonding network but also functions as a base to mediate the PCET process. For the NicXH105A mutant, MD simulations show that the disruption of the H bonding network can displace the substrate binding, leading to the loss of enzyme activity. These findings can expand our understanding of the PCET-mediated O-O bond activation and the flexible catalytic routes in various mutants, which have general implications on enzyme catalysis.
... Early studies on ARHDs were mainly performed in culturable bacterial species (Mohammadi et al., 2011) and focused on catalytic mechanism (Liu et al., 2021;Qiu et al., 2022). Some important ARHDs have been characterized, and several primers that have been widely used were designed based on ARHD genes from only a few strains (Dhindwal et al., 2016;Furukawa et al., 2004;Iwai et al., 2011;Meynet et al., 2015). ...
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Biodegradation of aromatic compounds is ubiquitous in the environment and important for controlling organic pollutants. Aromatic ring-hydroxylating dioxygenases (ARHDs) are responsible for the first and rate-limiting step of aerobic biodegradation of aromatic compounds. The ARHD α subunit is a good biomarker for studying functional microorganisms in the environment, however their diversity and corresponding primer coverage are unclear, both of which require a comprehensive sequence database for the ARHD α subunit. Here amino acid sequences of the ARHD α subunit were collected, and a total of 103 sequences were selected as seed sequences that were distributed in 72 bacterial genera with 34 gene names. Based on both homolog search and key word confirmation against the GenBank, a sequence database of ARHD (DARHD) has been established and 6367 highly credible sequences were retrieved. DARHD contained 407 bacterial genera capable of degrading 38 aromatic substrates, and intricate relationships among the gene name, aromatic substrate and microbial taxa were observed. Thereafter, a total of 136 pairs of primers were collected and assessed. Results showed coverages of most published primers were low. Our research provides new insights for understanding the diversity of ARHD α subunit, and gives guidance on the design and application of primers in the future.
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Genome mining for the search and discovery of two new globin-like enzymes, TriB fromFusarium poae and TutaA from Schizophyllum commne, are involved in the synthesis of two linear terpenes tricinonoic acid (1) and 2-butenedioic acid (3). Both in vivo heterologous biosynthesis and in vitro biochemical assays showed that these two enzymes catalyzed the C-C double bond cleavage of a cyclic sesquiterpene precursor (−)-germacrene D (7) and a linear diterpene backbone schizostain (2), respectively. Our work presents an unusual formation mechanism of linear terpenes from fungi and expands the functional skills of globin-like enzymes in the synthesis of terpene compounds.
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Uridine diphosphate glucose dehydrogenase (UGDH) has a crucial role for the synthesis of UDP-glucuronic acid (UDPGA), which is the important sugar donor in the glucuronosylation of bioactive triterpene saponins in the traditional Chinese medicine Glycyrrhiza uralensis Fisch. UGDHs usually exist as isoforms in plants, but knowledge of their unique binding sites and patterns remain largely incomplete. Here, we mined five UGDH isoforms in G. uralensis transcriptome that catalyze the oxidation of UDP-glucose (UDPG) to UDPGA and had different catalytic efficiencies. Molecular modeling and molecular dynamics simulation revealed the potential catalytic residues for the catalytic efficiencies of the isoforms. Moreover, the UGDH isoforms exhibited substrate promiscuity by catalyzing two structurally diverse UDPG derivatives, which was further demonstrated by docking studies. This work gives new insights into the UGDH isoforms involved the biosynthesis of glucuronides in G. uralensis, and extends the understanding of the versatility of UGDHs.
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Dipicolinic acid (DPA) is a widely distributed pyridine derivative containing two carboxylate groups. However, the catabolism of DPA mediated by microorganisms are rarely reported. Alcaligenes faecalis strain JQ135, previously isolated in our lab, was able to degrade various pyridine derivatives, including nicotinic acid (NA). In this study, it was found that strain JQ135 could utilize DPA as the sole carbon and nitrogen source for growth and degrade 1.8 mM (300 mg/L) DPA to a non-detectable level within 12 h. The catabolism of DPA by strain JQ135 extracted two intermediates, i.e., 3-hydroxy-2,6-dipicolinic acid and 4-imino-pent-2-enedioic acid. Moreover, deletion of the key genes essential for NA catabolism (such as moaCDEBmoeA and maiA) did not affect DPA catabolism, indicating that the maleamate pathway and molybdenum cofactor are not involved in the DPA catabolism by strain JQ135. This study will provide insights into the bacterial catabolism of DPA.
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Purification and crystallization of an enzyme which catalyzes the oxidation of 2,5-dihydroxypyridine, an intermediate in nicotinic acid catabolism, are described. The labile enzyme is stabilized by dithiothreitol. Activity lost by dialysis or purification procedures is restored by incubation with dithiothreitol and ferrous sulfate. The enzyme is inhibited by sulfhydryl reagents and iron-chelating agents. The crystalline enzyme, on polyacrylamide electrophoresis with dithiothreitol, yields a single major band and a region of minor diffuse bands. In the absence of dithiothreitol the intensity of the minor bands is increased. On acrylamide gels containing sodium dodecyl sulfate, a single band with mobility corresponding to a molecular weight of 39,500 is obtained. A single region of enzyme activity corresponding to a molecular weight of 242,000 is obtained on sucrose gradients containing dithiothreitol. A portion of this activity is shifted to a region corresponding to lower molecular weight when dithiothreitol is omitted.
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Motivation Studying the transport paths of ligands, solvents, or ions in transmembrane proteins and proteins with buried binding sites is fundamental to the understanding of their biological function. A detailed analysis of the structural features influencing the transport paths is also important for engineering proteins for biomedical and biotechnological applications. Results CAVER Analyst 2.0 is a software tool for quantitative analysis and real-time visualization of tunnels and channels in static and dynamic structures. This version provides the users with many new functions, including advanced techniques for intuitive visual inspection of the spatiotemporal behavior of tunnels and channels. Novel integrated algorithms allow an efficient analysis and data reduction in large protein structures and molecular dynamic simulations. Availability CAVER Analyst 2.0 is a multi-platform standalone Java-based application. Binaries and documentation are freely available at www.caver.cz.
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Non-heme iron-dependent oxygenases catalyse the incorporation of O2 into a wide range of biological molecules and use diverse strategies to activate their substrates. Recent kinetic studies, including in crystallo, have provided experimental support for some of the intermediates used by different subclasses of this enzyme family. Plant non-heme iron-dependent oxygenases have diverse and important biological roles, including in growth signalling, stress responses and secondary metabolism. Recently identified roles include in strigolactone biosynthesis, O-demethylation in morphine biosynthesis and regulating the stability of hypoxia-responsive transcription factors. We discuss current structural and mechanistic understanding of plant non-heme iron oxygenases, and how their chemical/genetic manipulation could have agricultural benefit, for example, for improved yield, stress tolerance or herbicide development.
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Our review of the metabolic pathways of pyridines and aza-arenes showed that biodegradation of heterocyclic aromatic compounds occurs under both aerobic and anaerobic conditions. Depending upon the environmental conditions, different types of bacteria, fungi, and enzymes are involved in the degradation process of these compounds. Our review indicated that different organisms are using different pathways to biotransform a substrate. Our review also showed that the transformation rate of the pyridine derivatives is dependent on the substituents. For example, pyridine carboxylic acids have the highest transformation rate followed by mono-hydroxypyridines, methylpyridines, aminopyridines, and halogenated pyridines. Through the isolation of metabolites, it was possible to demonstrate the mineralization pathway of various heterocyclic aromatic compounds. By using 14C-labeled substrates, it was possible to show that ring fission of a specific heterocyclic compound occurs at a specific position of the ring. Furthermore, many researchers have been able to isolate and characterize the microorganisms or even the enzymes involved in the transformation of these compounds or their derivatives. In studies involving 18O labeling as well as the use of cofactors and coenzymes, it was possible to prove that specific enzymes (e.g., mono- or dioxygenases) are involved in a particular degradation step. By using H2 18O, it could be shown that in certain transformation reactions, the oxygen was derived from water and that therefore these reactions might also occur under anaerobic conditions.
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Chalcone isomerases are plant enzymes that perform enantioselective oxa-Michael cyclizations of 2'-hydroxychalcones into flavanones. An X-ray crystal structure of an enzyme-product complex and molecular dynamics simulations reveal an enzyme mechanism wherein the guanidinium ion of a conserved arginine positions the nucleophilic phenoxide and activates the electrophilic enone for cyclization through Brønsted and Lewis acid interactions. The reaction terminates by asymmetric protonation of the carbanion intermediate syn to the guanidinium. Interestingly, bifunctional guanidine- and urea-based chemical reagents, increasingly used for asymmetric organocatalytic applications, share mechanistic similarities with this natural system. Comparative protein crystal structures and molecular dynamics simulations further demonstrate how two active site water molecules coordinate a hydrogen bond network that enables expanded substrate reactivity for 6'-deoxychalcones in more recently evolved type-2 chalcone isomerases.
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We present the synthesis, properties, and characterization of [Fe(T1Et4iPrIP)(NO)(H2O)2](OTf)2 (1) (T1Et4iPrIP = Tris(1‐ethyl‐4‐isopropyl‐imidazolyl)phosphine) as a model for the nitrosyl adduct of gentisate 1,2‐dioxygenase (GDO). The further characterization of [Fe(T1Et4iPrIP)(THF)(NO)(OTf)](OTf) (2) which was previously communicated (Inorg. Chem. 2014, 53, 5414) is also presented. The weighted average Fe‐N‐O angle of 162° for 1 is very close to linear (> or = 165°) for these types of complexes. The coordinated water ligands participate in hydrogen bonding interactions. The spectral properties (EPR, UV‐vis, FTIR) for 1 are compared with 2 and found to be quite comparable. Complex 1 closely follows the relationship between the Fe‐N‐O angle and NO vibrational frequency which was previously identified for 6‐coordinate {FeNO}7 complexes. Liquid FTIR studies on 2 indicate that the v(NO) vibration position is sensitive to solvent shifting to lower energy (relative to the solid) in donor solvent THF and shifting to higher energy in dichloromethane. The basis for this behavior is discussed. The Keq for NO binding in 2 was calculated in THF and found to be 470 M‐1. Density functional theory (DFT) studies on 1 indicate donation of electron density to the iron center from the pi* orbitals of formally NO‐. Such a donation accounts for the near linearity of the Fe‐N‐O bond and the large v(NO) value of 1791 cm‐1.
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Polyketide synthases (PKSs) share a subset of biosynthetic steps in construction of a polyketide and the offload from PKS main module of specific product release is most often catalyzed by a thioesterase (TE). In spite of various PKS systems were discovered in polyketide biosynthsis, the molecular basis of TE-catalyzed macrocyclization remains challenging. In this study, MD simulations and QM/MM methods were combined to investigate the catalytic mechanism and substrate diversity of pikromycin (PIK) TE with two systems (PIK-TE-1 and PIK-TE-2), where substrates 1 and 2 are corresponding to TE-catalyzed precursors of 10-deoxymethynolide and narbonolide, respectively. The results showed that in comparison with PIK-TE-2, system PIK-TE-1 exhibited more tendency to form stable pre-reaction state, which is critical to macrocyclization. Besides, the structural characteristics of pre-reaction states were uncovered through analyses of hydrogen-bonding and hydrophobic interactions, which were found to play a key role in substrate recognition and product release. Furthermore, potential energy surfaces were calculated to study the molecular mechanism of macrocyclization including the formation of tetrahedral intermediates from si- and re-face nucleophilic attacks and the release of products. The energy barrier of macrocyclization from si-face attack was calculated to be 16.3 kcal/mol in PIK-TE-1, 3.6 kcal/mol lower than from re-face attack and 4.1 kcal/mol lower than si-face attack in PIK-TE-2. These results are in agreement with experimental observations that the yield of 10-deoxymethynolide is superior to narbonolide in PIK TE-catalyzed macrocyclization. Our findings elucidate the catalytic mechanism of PIK TE and provide a better understanding of type I PKS TEs in protein engineer.
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Density functional theory calculations have been performed to study the reaction mechanisms for the Rh(III)-catalyzed C-H functionalization versus deoxygenation of quinoline N-oxide (QNO) with diazo compounds, dimethyl diazomalonate and methyl phenyldiazoacetate. How different diazo compounds affect the reaction pathways has been analyzed and examined in detail. It was found that the stability of the Rh(III)-carbene intermediates derived from an N2 extrusion is crucial in determining the preferred reaction pathway.
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Many factors have been suggested to control the selectivity for extradiol or intradiol cleavage in catechol dioxygenases. The varied selectivity of model complexes and the ability to force an extradiol enzyme to do intradiol cleavage indicate that the problem may be complex. In this paper we focus on the regiospecificity of the proximal extradiol dioxygenase, homoprotocatechuate 2,3-dioxygenase (HPCD), for which considerable advances have been made in our understanding of the mechanism from an experimental and computational standpoint. Two key steps in the reaction mechanism were investigated: (1) attack of the substrate by the superoxide moiety and (2) attack of the substrate by the oxyl radical generated by O-O bond cleavage. The selectivity at both steps was investigated through a systematic study of the role of the substrate and the first and second coordination spheres. For the isolated native substrate, intradiol cleavage is calculated to be both kinetically and thermodynamically favored, therefore nature must use the enzyme environment to reverse this preference. Two second sphere residues were found to play key roles in controlling the regiospecificity of the reaction: Tyr257 and His200. Tyr257 controls the selectivity by modulating the electronic structure of the substrate, while His200 controls selectivity through steric effects and by preventing alternative pathways to intradiol cleavage.