Microbiological Research ] (]]]]) ]]]—]]]
Site-directed mutagenesis of gentisate
1,2-dioxygenases from Klebsiella pneumoniae
M5a1 and Ralstonia sp. strain U2
S. Luoa,b, D.Q. Liua,b, H. Liua, N.Y. Zhoua,?
aWuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
bGraduate School of Chinese Academy of Sciences, Beijing 100039, China
Accepted 19 July 2005
Gentisate 1,2-dioxygenase (GDO, EC 126.96.36.199) is the first enzyme in gentisate
pathway that catalyses the ring fission of gentisate to form maleylpyruvate.
Phylogenetic tree of amino acid sequences from 11 GDOs demonstrates that the
GDOs from different genus share identities between 12.1% and 64.8%. According to the
alignment result, four highly conserved histidine residues in GDO from Klebsiella
pneumoniae M5a1 and Ralstonia sp. strain U2 were chosen to be substituted with
aspartate residues. Enzyme analysis indicated that substitution of any of these four
histidine residues had resulted in the complete loss of its catalytic activity.
& 2005 Elsevier GmbH. All rights reserved.
Degradation of aromatic compounds by micro-
organisms is not only a basic step of carbon cycle in
the nature, but also plays a key role in the
detoxification of these compounds in environment.
Large amounts of polycyclic aromatic compounds
are aerobically transformed by monooxygenases or
dioxygenases to several central dihydroxylated
intermediates, such as catechol, protocatechuate,
and gentisate. Aromatic rings of these dihydroxy-
lated intermediates could be consequently cleaved
by dioxygenases, and the ring cleavage has been
classified into three pathways, ortho-, meta-, and
gentisate pathway according to the site of ring
fission (Werwath et al., 1998).
In the gentisate pathway, gentisate is cleaved by
gentisate 1,2-dioxygenase (GDO) to form maleylpyr-
uvate (Fig. 1). Maleylpyruvate can be converted to
fumarylpyruvate by isomerase and subsequently
hydrolysed to fumarate and pyruvate, both of which
are intermediates for the TCA cycle (Gao and Zhou,
ARTICLE IN PRESS
Ralstonia sp. strain
0944-5013/$-see front matter & 2005 Elsevier GmbH. All rights reserved.
?Corresponding author. Tel./fax: 862787197655.
E-mail address: firstname.lastname@example.org (N.Y. Zhou).
2003). The first report on an integrated gentisate
pathway at molecular level is from Ralstonia sp.
strain U2, which metabolises naphthalene via
gentisate (Zhou et al., 2001). In another example,
Klebsiella pneumoniae M5a1 has been demonstrated
to metabolise 3-hydroxybenzoate via gentisate, and
the catabolic genes have been cloned (Jones and
Cooper, 1990) and sequenced (Gao, 2003) (GenBank
accession number AY648560).
Up to now, the purification of GDO has been
et al., 1975; Harpel and Lipscomb, 1990a; Suarez
et al., 1996; Fu and Oriel, 1998; Werwath et al.,
1998; Feng et al., 1999), and the catalytic mechan-
isms of GDO are studied only in one report (Harpel
and Lipscomb, 1990b). GDO from Pseudomonas
alcaligenes NCIB 9867(P25X) was mutated through
random and site-directed mutagenesis, and nearly
300 mutants were obtained (Chua et al., 2001). The
results of enzyme assay of these mutants indicated
that the substitution of any of four highly conserved
His residues to Asp residues lead to the complete loss
of enzyme activity. However, we cannot yet make a
conclusion that the four His residues in other GDOs
are as essential as that in P25X since the crystal
structure of GDO has not yet been determined and
the identity values among the GDOs’ amino acid
sequences vary dramatically (12.1–64.8%). In this
study, through site-directed mutagenesis, we de-
monstrate that these four His residues in GDO from
K. pneumoniae M5a1 and Ralstonia sp. strain U2 are
also critical to the catalytic activity of GDO.
Materials and methods
Strains and plasmids
endA1 gyrA96 thi-1 hsdR17(rK
deoR D(lacZYA-argF)U169] (Life Technologies, UK),
E. coli Rosetta(DE3) pLysS[F ompT hsdSB(rBmBgal
dcm lacY1(DE3) pLysSRARE2(Cmr)] (Novagen, USA),
the plasmids used and constructed in this study are
listed in Table 1.
+) supE44 relA1
Enzymes and reagents
Restriction endonuclease and T4 ligase were
purchased from Takara (Dalian, China), Pfu DNA
polymerase and dNTPs were purchased from Shang-
hai Shenergy Biocolor Bioscience & Technology
Company (Shanghai, China), gentisate from Sigma
(Sigma, USA), PCR purification kit from V-gene
China). Nucleotide sequences were determined by
United Gene Holdings, Ltd. (Shanghai, China). PCR
primers were obtained from Shanghai Sangon
Biological Engineering Technology & Services Co.,
Ltd. (Shanghai, China).
Media and culture condition
Strains used in this study were grown overnight in
LB at 371C. When necessary 100mg/L ampicillin
and 34mg/L chloramphenicol were added.
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Figure 1. Gentisate 1,2-dioxygenase converts gentisate
to form maleylpyruvate.
Plasmids used and constructed in this study
Plasmid DescriptionSource or reference
1076bp EcoRI-NdeI-cut PCR fragment containing mhbD inserted into pET5a
8216bp SphI-cut fragment from Klebsiella pneumoniae M5a1 inserted into pUC18
1286bp EcoRI-NdeI-cut PCR fragment containing nagI inserted into pET5a
8.9kb EcoRI-cut fragment from Ralstonia sp. U2 inserted into pUC18
pZWGD5, in which CAT code for His108was changed to GAT
pZWGD5, in which CAC code for His110was changed to GAC
pZWGD5, in which CAC code for His149was changed to GAC
pZWGD5, in which CAC code for His151was changed to GAC
pWWF19-25, in which CAC code for His118was changed to GAC
pWWF19-25, in which CAC code for His120was changed to GAC
pWWF19-25, in which CAC code for His159was changed to GAC
pWWF19-25, in which CAC code for His161was changed to GAC
Studier and Moffatt, 1986
Zhou et al., 2001
Zhou et al., 2001
S. Luo et al.2
Site-directed mutagenesis of GDO
Site-directed mutagenesis of mhbD and nagI was
carried out by overlap-extension PCR (Pogulis et
al., 1996), and pBSI and pWWF60 were used as
templates for PCR, respectively. Primers used in
this study are listed in Table 2.
PCR fragments of GDO were digested with EcoRI
and NdeI, and then inserted into expression vector
pET5a. The recombinant plasmids were trans-
formed into E. coli DH5a and positive clones were
screened according to restriction enzyme analysis.
pZWLSD110, pZWLSD149, pZWLSD151, pZWLSI118,
pZWLSI120, pZWLSI159, and
sequenced to ensure the mutations occurred as
desired and no unintended mutation had been
incorporated during the PCR before they were
transformed into E. coli Rosetta for expression.
Expression of wild-type and mutant enzymes
in E. coli Rosetta
The GDO genes, cloned in vector pET5a, were
expressed in E. coli Rosetta cells which were grown
at 371C on LB media supplemented with ampicillin
and chloramphenicol. Isopropyl-b-D-thiogalactopyr-
anoside (IPTG) was added to the final concentration
of 0.4mM before the culture density, measured by
the absorbance at 600nm, reached 0.6. Cultures
were then grown for another 4h at 301C before the
cells were harvested by centrifugation.
Sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS–PAGE) and enzyme assay
The expression of GDO was analysed by SDS–PAGE
(Schaggfr and Jagow, 1987) with 12% acrylamide
separating gel and 5% stacking gel.
Cell extracts were prepared by resuspending the
bacterial pellets in ice-cold 50mM phosphate
buffer (pH 7.4) and lysed by sonication in an ice-
water bath, MhbD and its mutants for 15min, and
NagI and its mutants for 5min (disrupting for 6s
with 9s intervals). Cell debris was removed by
centrifugation at 15,000g for 30min at 41C.
GDO activity was assayed spectrophotometrically
at 301C by measuring the formation of maleylpyr-
uvate at 330nm (Zhou et al., 2001). Activity assay
was conducted in 500mL of reaction mixture
containing 0.2mM gentisate in 50mM phosphate
buffer (pH 7.4). The molar extinction coefficient of
13,000/Mcm was used. One unit of enzyme activity
is defined as the amount of enzyme required for
production of 1mmol maleylpyruvate per minute at
301C. Specific activities are expressed as units per
milligram of protein. Protein concentrations were
determined by Bradford kit according to the
manufacture’s instructions (Beyotime Institute of
Biotechnology, Haimen, China).
Sequence and phylogenetic analysis
The sequences were aligned by using CLUSTALX,
and phylogenetic analyses were performed using
ARTICLE IN PRESS
Oligonucleotides used for site-directed mutagenesis
mhbD H108D forward
mhbD H108D reverse
mhbD H110D forward
mhbD H110D reverse
mhbD H149D forward
mhbD H149D reverse
mhbD H151D forward
mhbD H151D reverse
nagI H118D forward
nagI H118D reverse
nagI H120D forward
nagI H120D reverse
nagI H159D forward
nagI H159D reverse
nagI H161D forward
nagI H161D reverse
The bases representing the restriction sites are in boldface. The bases changed are underlined.
Mutagenesis of gentisate 1,2-dioxygenases3
the neighbour-joining algorithm of PHYLIP (version
3.572c). BLASTP was used for the amino acid
Sequence and phylogenetic analysis
The GDO sequences, which share different
identities with MhbD and NagI in GenBank, were
selected to perform multiple sequences alignment
and phylogenetic tree. The phylogenetic tree is
shown in Fig. 2. The result of alignment indicates
that the amino acid sequences of GDO from
different organisms share different degrees of
identities, ranging from 12.1% to 64.8% (data not
shown). NagI demonstrates poor identities with
MhbD and XlnE, which are 32.9% and 25.6%,
respectively. The low identities among GDOs
indicate that the origins of gentisate pathway are
apparently diverse, which may be attributed to
adapt themselves to gentisate derivatives (Gao,
Site-directed mutagenesis of conserved His
According to the result of sequences alignment of
MhbD, NagI and XlnE, motif scanning (http://
hits.isb-sib.ch/cgi-bin/PFSCAN), and PHI-Blast in
found that the residues from position 101 to 152
in MhbD (from 110 to 163 in NagI and 102 to 153 in
XlnE) are highly conserved (shown in Fig. 3). It has
been predicted that GDO belongs to the cupin
superfamily, which refers to a b-barrel structural
domain, on the basis of primary sequence (Dunwell
et al., 2000). Figure 3 shows that these conserved
sequences seem to contain the characteristic cupin
domain, which comprises two histidine-containing
motifs, the conserved sequences of these motifs
are G-X(5)-H-X-H-X(3,4)-E-X(6)-G and G-X(5)-P-X-G-
X(2)-H-X(3)-N, respectively (Khuri et al., 2001).
The four highly conserved His residues in GDO are
located in these motifs and three of them are also
conserved in the cupin domain.
Expression of wild-type and mutant enzymes
and activity assay
Through overlap-extension PCR, eight mutant
plasmids were obtained (they are pZWLSD108,
pZWLSD110, pZWLSD149, and pZWLSD151 of MhbD,
pZWLSI118, pZWLSI120, pZWLSI159, and pZWLSI161
of NagI). Sequence determination confirmed that
each His residue at different positions had been
substituted by Asp residues in these mutants,
respectively. Expressions of wild-type and mutants
MhbD and NagI were performed in E. coli Rosetta
strain, and highly soluble form proteins were
obtained. Figure 4 shows the expressions of wild-
type and mutants MhbD and similar results were
obtained with the expressions of wild-type and
mutants NagI (data not shown). There is no
difference in the expression of GDO genes between
wild type and mutants, and it can be concluded
that the substitutions of these four conserved His
residues individually had no impact on the expres-
sions of GDO from the two different bacterial
Wild-type MhbD and NagI exhibited evident GDO
activity (the specific activities are 0.54 and 0.37U/
detected in any of the eight mutants of MhbD and
NagI. The comparison of GDO activities between
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Figure 2. Phylogram of consensus tree obtained from
neighbour-joining bootstrap analysis of 11 GDO amino
Bcep2722 is from Burkholderia fungorum Bcep_225
(ZP_00029914.1), MhbD from Klebsiella pneumoniae
M5a1 (AAW63413.1), UCBBP from Pseudomonas aerugi-
nosa UCBPP-PA14Paer_1 (ZP_00135722.1), DbdB from
Xanthobacter polyaromaticivorans (BAC98955.1), XlnE
(CAA12267.1), Blr3412 from Bradyrhizobium japonicum
USDA 110 DNA (BAC45375.1), BH2002 from Bacillus
halodurans (NP_242868.1), SdgD from Streptomyces sp.
(AAD12619.1). An unnamed GDO from Haloferax sp.
D1227 (AAC25761.1) was used as an outgroup. Bootstrap
values refer to which they are closest. The scale bar
represents a Jukes–Cantor distance.
S. Luo et al.4
wild-type and mutant MhbD is shown in Fig. 5 (the
corresponding comparison of NagI is similar to that
of MhbD, but the results were not shown).
The substitutions of any of the four highly
conserved histidine to aspartate residues in GDO
from strain M5a1 and strain U2 had resulted in the
complete loss of their catalytic activities. This is
consistent with the results of mutagenesis studies
of GDO in P. alcaligenes NCIB 9867(P25X) (Chua et
al., 2001). Furthermore, it should be noted that the
GDO genes of the above studies originated from
different genus and the identities of their amino
acid sequences is less than 33%. The result clearly
suggests that these four His residues are vital for
GDO catalytic activity.
It has been reported that iron in GDO is the
primary site for substrate interaction. Gentisate
binds directly to the iron through the carbon 1
carboxylate and carbon 2 hydroxyl substituents,
and it results in the active site iron coming close to
the site of ring cleavage (Harpel and Lipscomb,
1990b). Imidazole group in His residue is very
active, and it could rapidly offer or receive
electron at almost the same rate. It is proposed
that during the biological evolution imidazole group
might exist as a catalytic structure in an enzyme
rather than an ordinary part of the protein
structure (Yu, 1990). His residues are often found
to be coordinated with iron in active sites in
conserved motif in many dioxygenases whose
crystallographic data have been obtained. For
example, His335 and His371 in homogentisate
1995), His145 and His209 in 2,3-dihydroxybiphenyl
1,2-dioxygenase (BphC) (Han et al., 1995), His153
and His214 in catechol 2,3-dioxygenase (2,3-CTD)
(Kita et al., 1999), His12, His61 in protocatechuate
4,5-dioxygenase (Sugimoto et al., 1999), His 208,
His 213, and His 362 in naphthalene dioxygenase
(Carredano et al., 2000). Moreover, some dioxy-
genases that have not been crystallographically
characterised also exhibit similar motif with His
residues function as iron ligands. His 222 and His
228 in toluene dioxygenase is a good case in point
(Jiang et al., 1996).
On the other hand, it is found that within some
cupin superfamily members, the metal binding motif
is highly conserved, according to structure-based
sequence alignment (Pang et al., 2004). A cupin
protein, quercetin 2,3-dioxygenase from Bacillus
subtilis, whose crystal structure had been recently
determined, has irons in active sites and six conserved
His residues, His 62, His 64, His 103, His 234, His 236,
and His 275 in two cupin motifs, respectively. These
six His residues coordinate the Fe2+at the two active
sites (Gopal et al., 2005). A similar situation is also
found in another newly identified cupin nuclear
protein pirin, in which iron is in active site and three
His residues bind to the iron (Pang et al., 2004).
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Figure 3. Partial multiple sequence alignment of the amino acid sequences from three GDOs. Conserved His residues
are marked with ~ and amino acid residues conserved in all three GDO species are marked with *, the positions of the
two conserved cupin motifs are boxed.
Figure 4. SDS–PAGE of overexpressed MhbD and its
mutants in E. coli Rosetta on a 12% gel. Lane 1, molecular
weight markers; lane 2, blank control (pET5a was
transformed into E. coli Rosetta); lanes 3–7, cell extracts
containing H151D, H149D, H110D, H108D, and MhbD
obtained after induction with IPTG, respectively. The
molecular mass of the overexpressed polypeptide (in-
dicated by an arrow on the right) is ?35kDa.
Mutagenesis of gentisate 1,2-dioxygenases5
In addition, His residues not only act as the iron
binder, but also was found to play different roles in
extradiol-cleaving dioxygenases. A prominent ex-
ample is the 2,3-dihydroxybiphenyl 1,2-dioxygen-
ase (BphC) in Pseudomonas sp. KKS102. The His
residues in the enzyme seem to deprotonate the
hydroxyl group of the substrate, to stabilise a
negative charge on the O2 molecular, and to
function as a proton donor (Sato et al., 2002).
Nevertheless, only based on the above data, it
would be plausible to draw a conclusion that these
His residues in this study play the key role of iron
coordination as the same in other dioxygenases or
cupin proteins. The illustration of particular catalytic
mechanisms of GDO would not be clarified until the
resolution of GDO crystal structure was performed.
This work was supported by National Natural
Science Foundation of China (Grant No. 30170036)
and by the ‘‘Hundred Talents Program’’ from
Chinese Academy of Sciences to NYZ. We also thank
Mrs. S.F. Wang for her technical assistance.
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Figure 5. Time course of production of maleylpyruvate from gentisate by MhbD and its mutants. Cell extracts of E. coli
Rosetta containing pZWGD5 (MhbD), pZWLSD108 (H108D), pZWLSD110 (H110D), pZWLSD149 (H149D), and pZWLSD151
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Mutagenesis of gentisate 1,2-dioxygenases7