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Erb TJ, Berg IA, Brecht V, Muller M, Fuchs G, Alber BE.. Synthesis of C5-dicarboxylic acids from C2-units involving crotonyl-CoA carboxylase/reductase: the ethylmalonyl-CoA pathway. Proc Natl Acad Sci U S A 104: 10631-10636

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Fifty years ago, Kornberg and Krebs established the glyoxylate cycle as the pathway for the synthesis of cell constituents from C2-units. However, since then, many bacteria have been described that do not contain isocitrate lyase, the key enzyme of this pathway. Here, a pathway termed the ethylmalonyl-CoA pathway operating in such organisms is described. Isotopically labeled acetate and bicarbonate were transformed to ethylmalonyl-CoA by cell extracts of acetate-grown, isocitrate lyase-negative Rhodobacter sphaeroides as determined by NMR spectroscopy. Crotonyl-CoA carboxylase/reductase, catalyzing crotonyl-CoA + CO2 + NADPH → ethylmalonyl-CoA⁻ + NADP⁺ was identified as the key enzyme of the ethylmalonyl-CoA pathway. The reductive carboxylation of an enoyl-thioester is a unique biochemical reaction, unprecedented in biology. The enzyme from R. sphaeroides was heterologously produced in Escherichia coli and characterized. Crotonyl-CoA carboxylase/reductase (or its gene) can be used as a marker for the presence of the ethylmalonyl-CoA pathway, which functions not only in acetyl-CoA assimilation. In Streptomyces sp., it may also supply precursors (ethylmalonyl-CoA) for antibiotic biosynthesis. For methylotrophic bacteria such as Methylobacterium extorquens, extension of the serine cycle with reactions of the ethylmalonyl-CoA pathway leads to a simplified scheme for isocitrate lyase-independent C1 assimilation. • acetyl-CoA assimilation • glyoxylate cycle • methylotrophy • polyketide • serine cycle
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Synthesis of C
5
-dicarboxylic acids from C
2
-units
involving crotonyl-CoA carboxylase/reductase:
The ethylmalonyl-CoA pathway
Tobias J. Erb*, Ivan A. Berg*
, Volker Brecht
, Michael Mu
¨
ller
, Georg Fuchs*, and Birgit E. Alber*
§
*Mikrobiologie, Institut fu¨ r Biologie II and
Pharmazeutische und Medizinische Chemie, Fakulta¨t fu¨ r Chemie, Pharmazie und Geowissenschaften,
Albert-Ludwigs-Universita¨ t Freiburg, Scha¨ nzlestrasse 1, 79104 Freiburg, Germany
Edited by Hans Kornberg, Boston University, Boston, MA, and approved May 7, 2007 (received for review March 25, 2007)
Fifty years ago, Kornberg and Krebs established the glyoxylate
cycle as the pathway for the synthesis of cell constituents from
C
2
-units. However, since then, many bacteria have been described
that do not contain isocitrate lyase, the key enzyme of this
pathway. Here, a pathway termed the ethylmalonyl-CoA pathway
operating in such organisms is described. Isotopically labeled
acetate and bicarbonate were transformed to ethylmalonyl-CoA by
cell extracts of acetate-grown, isocitrate lyase-negative
Rhodobacter sphaeroides as determined by NMR spectroscopy.
Crotonyl-CoA carboxylase/reductase, catalyzing crotonyl-CoA
CO
2
NADPH 3 ethylmalonyl-CoA
NADP
was identified as
the key enzyme of the ethylmalonyl-CoA pathway. The reductive
carboxylation of an enoyl-thioester is a unique biochemical reac-
tion, unprecedented in biology. The enzyme from R. sphaeroides
was heterologously produced in Escherichia coli and characterized.
Crotonyl-CoA carboxylase/reductase (or its gene) can be used as a
marker for the presence of the ethylmalonyl-CoA pathway, which
functions not only in acetyl-CoA assimilation. In Streptomyces sp.,
it may also supply precursors (ethylmalonyl-CoA) for antibiotic
biosynthesis. For methylotrophic bacteria such as Methylobacte-
rium extorquens, extension of the serine cycle with reactions of the
ethylmalonyl-CoA pathway leads to a simplified scheme for iso-
citrate lyase-independent C
1
assimilation.
acetyl-CoA assimilation glyoxylate cycle methylotrophy polyketide
serine cycle
T
his year marks the 50th anniversary of the discovery of the
glyoxylate cycle by Kornberg and Krebs (1). This anaplerotic
reaction sequence enables an organism to use substrates, which
enter the central carbon metabolism on the level of acetyl-CoA,
as sole carbon source. Examples of such substrates are fatty acids,
alc ohols, and esters, including various fer mentation products,
but also waxes, alkenes, and methylated compounds. Originally
delineated for bacteria, the glyoxylate c ycle is also required for
the metabolism of storage oil by plants during germination of
seedlings (2) and for the conversion of triacylglycerols to car-
bohydrates in developing eggs of nematodes (3). Isocitrate lyase,
the first key enz yme of the glyoxylate cycle, together with
enz ymes of the citric acid cycle, is responsible for the oxidation
of acet yl-CoA to glyoxylate (Fig. 1). The sec ond key enzyme,
malate synthase, condenses glyoxylate and another molecule of
acet yl-CoA to malate. The subsequent oxidation of malate
regenerates the initial acetyl-CoA acceptor molecule oxaloac-
et ate in the citric acid cycle. Therefore, any intermediate of the
citric acid cycle can be withdrawn f rom the cycle and used for cell
carbon biosynthesis.
However, the glyoxylate cycle cannot be the sole solution for
acet yl-CoA assimilation, because several organisms that require
such an anaplerotic reaction sequence lack isocitrate lyase
activit y (48) or show a labeling pattern after growth on acetate
inc onsistent with the operation of the glyoxylate c ycle in acetate
assimilation (9, 10). These organ isms include many purple
nonsulfur bacteria, for example Rhodobacter sphaeroides and
Rhodospir illum rubrum (4, 5, 11), and other
-proteobacteria,
like the methylotroph Methylobacterium ex torquens (6, 12), the
facult ative denitrifier Paracoccus ver sutus (7), and, inferred by
genome sequence analysis, several aerobic anoxygenic pho-
totrophs (13). In addition, the diverse group of streptomycetes
uses an alternate, isocitrate lyase-independent route for acetyl-
CoA assimilation, and there appears to be a direct link to
antibiotic biosynthesis (8, 14).
We have recently shown that acetate assimilation by R.
sphaeroides requires the conversion of a C
4
-c ompound, aceto-
acet yl-CoA, derived from two acetyl-CoA molecules, to the
C
5
-c ompound mesaconyl-CoA (13).
-Methylmalyl-CoA,
for med by hydration of mesaconyl-CoA, is cleaved to glyoxylate
and propionyl-CoA. Condensation of glyoxylate and another
molecule of acet yl-CoA yields malate; propionyl-CoA is carbox-
ylated and yields suc cinate. The connecting steps between the
C
4
- and C
5
-branch of the postulated pathway have not been
elucidated but had been proposed to include a carboxylation step
(13). This study aimed at determining the substrate and product
of this carboxylation step. Much to our surprise, an enz yme was
present in cell extracts of R. sphaeroides, which catalyzed an
ATP-independent reductive carboxylation of the enoyl-CoA
ester, crotonyl-CoA. The gene encoding this enzyme, represent-
ing the key reaction in the proposed pathway for acet yl-CoA
assimilation, was identified and heterologously expressed, and
the enzy me was studied.
Results
Carboxylation Activities in Cell Extracts of
Rhodobacter sphaeroides
.
Inc orporation of [
14
C]bicarbonate by cell extracts of aerobically
acet ate-grown R. sphaeroides in the presence of different C
4
-
thioesters of CoA was examined. Given that mesaconyl-CoA, the
first identified C
5
-c ompound in the pathway, is expected to be
reductively formed from t wo molecules of acetyl-CoA and CO
2
,
NADPH was added as a source of reducing equivalents.
Acetoacet yl-CoA (0.08 U mg
1
, where U is
mol min
1
) as well
as (R)-3-hydroxybutyryl-CoA- (0.2 U mg
1
) dependent [
14
C]bi-
carbonate fixation was found, which required NADPH but,
surprisingly, was independent of ATP. The NADPH-dependent
carboxylation activ ity with crotonyl-CoA was severalfold higher
Authors contributions: T.J.E. and I.A.B. contributed equally to this work; T.J.E., I.A.B., V.B.,
M.M., G.F., and B.E.A. designed research; T.J.E., I.A.B., and V.B. performed research; T.J.E.,
I.A.B., M.M., G.F., and B.E.A. analyzed data; and T.J.E., I.A.B., and B.E.A. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviation: U,
mol min
1
.
On leavefrom the Department of Microbiology, Moscow State University, Moscow, Russia.
§
To whom correspondence should be sent at the present address: Department of Micro-
biology, Ohio State University, 484 West 12th Avenue, Columbus, OH 43210. E-mail:
alber.8@osu.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0702791104/DC1.
© 2007 by The National Academy of Sciences of the USA
www.pnas.orgcgidoi10.1073pnas.0702791104 PNAS
June 19, 2007
vol. 104
no. 25
10631–10636
MICROBIOLOGY
(1.1 U mg
1
). Butyryl-CoA was carboxylated only in the pres-
ence of ATP, albeit with very low rates (0.005 U mg
1
). The
but yryl-CoA carboxylase activity was inhibited by avidin and, by
partial purification of the enzyme, found to be a side activ ity of
the ATP- and biotin-dependent propionyl-CoA carboxylase,
which is needed in a later part of the pathway.
Determination of Products of the Carboxylation Reaction in Cell
Extracts of
R. sphaeroides
. Products of the carboxylation reaction
were analyzed by reversed-phase high-perfor mance liquid chro-
matography (HPLC) with
14
C monitoring (Fig. 2). [U-
14
C]acet yl-
CoA, NADPH, and bicarbonate were incubated w ith cell ex-
tracts of R. sphaeroides at 30°C. Af ter 45 min of incubation, a
prominent
14
C-labeled CoA-thioester was formed; this com-
pound was derived from a transient product formed after 5 min
of incubation (Fig. 2 B and C). Identification of the CoA-esters
was based on cochromatography w ith standards (acetoacet yl-
CoA, ethylmalonyl-CoA, and butyryl-CoA) and on the detection
of their molecular masses by using HPLC-MS. The transient
product was identified as acetoacetyl-CoA w ith a mass of 851 Da,
the second product was identified as ethylmalonyl-CoA with a
mass of 881 Da. When [
14
C]bicarbonate was incubated with
unlabeled acetyl-CoA, ethylmalonyl-CoA was the sole labeled
product formed (Fig. 2D). To confirm the identity of ethylma-
lonyl-CoA as the product of the reductive carboxylation reaction,
cell extract of R. sphaeroides was incubated with [
13
C]bicarbonate
and [U-
13
C]acet yl-CoA in the presence of NADPH for 30 min.
CoA-esters were isolated from the reaction mixture by using
solid-phase extraction and analyzed by 2D NMR spectroscopy
[supporting information (SI) Fig. 6]. The major c ompound
identified was characterized through
13
C NMR-signals (SI Fig. 6
A and B) at 198.7 ppm [C O, doublet (d), J 44.4 Hz], 174.7
(C O, d, J 48.8 Hz), 65.2 [CH, multiplet (m)], 24.6 [CH
2
,
doublet of doublets (dd), J 33.6 Hz], and 12.6 (CH
3
,d,J 35.2
Hz). The corresponding signals in the proton NMR (SI Fig. 6C)
at 4.3 ppm (CH, m), 1.9 (CH
2
, m), and 0.9 (CH
3
, m) were
identified through a gHSQC experiment (SI Fig. 6E). An
INADEQUATE experiment verified the proposed structure of
a 2-ethylmalonate-thioester (SI Fig. 6F). The inc orporation of
[
13
C]bicarbonate was 80%, deduced from additional
13
C NMR
signals at 174.7 ppm and 24.6 (d, J 34.6 Hz) (SI Fig. 6B). Minor
amounts of butyryl-CoA and 3-hydroxybutyryl-CoA were identi-
fied through
13
C NMR. Moreover, at least three more com-
pounds showing the characteristic doublet signal (J 50 Hz) in
the range of 173–179 ppm gave a hint for carboxylated deriva-
tives. All of these gave a cross signal to signals at 40 ppm,
suggesting the presence of acetyl-CoA and derivatives.
Identification of Crotonyl-CoA Carboxylase/Reductase. Crotonyl-
CoA was the best substrate for NADPH-dependent carboxyla-
tion in cell extracts of R. sphaeroides, suggesting that it might be
the direct carboxylation substrate. Crotonyl-CoA reductase (en-
c oded by the gene ccr) has been previously purified from
Streptomyces collinus and shown to be involved in assimilation of
C
1
- and C
2
-c ompounds (8, 15). Mutation of the ccr-like gene in
M. extorquens resulted in the inability to oxidize acetyl-CoA to
glyoxylate (16). To test whether crotonyl-CoA reduct ase not only
cat alyzes the reduction of crotonyl-CoA to butyryl-CoA as
suggested (15) but, rather, the reductive carboxylation of
crotonyl-CoA to ethylmalonyl-CoA, the ccr-like gene of R.
sphaeroides was heterologously expressed in Escherichia coli.
Cell extract of recombinant E. coli catalyzed the crotonyl-CoA-
dependent oxidation of NADPH in the presence of bicarbonate
(Fig. 3B). No activity was found in the presence of either
acetoacet yl-CoA or (R)-3-hydroxybutyryl-CoA. The enzyme
(120 mg) was purified from9gofE. coli cells in two steps by
using DEAE-Sepharose and Cibacron Blue chromatography
(Fig. 3A). The purified protein catalyzed the fixation of [
14
C]-
bicarbonate in the presence of NADPH and crotonyl-CoA with
a specific activity of 103 units mg
1
and was named crotonyl-CoA
carboxylase/reduct ase; its properties are summarized in SI Table
1. Gel-filtration chromatography of the native enz yme gave a
Fig. 1. The glyoxylate cycle as proposed by Kornberg and Krebs 50 years ago.
The citric acid cycle is modified to bypass the two decarboxylation steps by the
action of its two key enzymes isocitrate lyase and malate synthase. This allows
the net synthesis of malate from two molecules of acetyl-CoA.
Fig. 2. HPLC analysis of CoA thioesters formed from acetyl-CoA and NaHCO
3
by cell extracts of R. sphaeroides in the presence of NADPH at 30°C.
[U-
14
C]acetyl-CoA was synthesized by incubation of 1 mM [U-
14
C]acetate (320
kBq ml
1
), 4 mM ATP, 1 mM CoA, 5 mM KCl, and 4 mM MgCl
2
with 6 Uml
1
of acetyl-CoA synthetase in 70 mM TrisHCl buffer (pH 7.9). (A) The reaction
was started by addition of 0.5 mM 5–5-dithiobis(2-nitrobenzoic acid), 35 mM
NaHCO
3
, 6.5 mM NADPH, and 0.8 mg of cell-extract protein. (B and C) After 5
min (B) and 45 min (C), incubation samples were withdrawn from the assay
mixture and analyzed by reverse-phase HPLC. (D) Products formed in the
presence of NaH
14
CO
3
(480 kBqml
1
) after 45 min incubation; unlabeled
acetate was used. Products were identified by MS-HPLC, and the elution times
were as follows: free CoA, 6.2 min; ethylmalonyl-CoA, 10.6 min; acetyl-CoA,
12.6 min; acetoacetyl-CoA, 14.6 min; crotonyl-CoA, 23 min; and butyryl-CoA,
25 min.
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www.pnas.orgcgidoi10.1073pnas.0702791104 Erb et al.
molecular mass of 105 kDa, suggesting a homodimeric str ucture.
Comprehensive metal analysis (30 elements) of crotonyl-CoA
carboxylase/reduct ase by plasma emission spectroscopy indi-
cated the absence of any metals.
The product of the reductive carboxylation of crotonyl-CoA
was identified as ethylmalonyl-CoA by HPLC-MS. The reaction
followed Michaelis–Menten kinetics with apparent K
m
values of
0.4 mM for crotonyl-CoA, 0.7 mM for NADPH, and 14 mM for
NaHCO
3
.CO
2
and not bicarbonate was determined to be the
reactive carbon species by using a modified method by Thauer et
al . (ref. 17 and data not shown). The reaction is reversible, w ith
an apparent rate of 12 U mg
1
. Crotonyl-CoA carboxylase/
reduct ase also catalyzed the reduction of crotonyl-CoA to
but yryl-CoA (the product was identified by HPLC-MS) at low
rate (12 U mg
1
), but only in the absence of bicarbonate/CO
2
;
in the presence of bicarbonate/CO
2
ethylmalonyl-CoA was
for med exclusively. This observation c ould be explained by the
following mechanism: first, the
-carbon of crotonyl-CoA ac-
cepts a hydride from NADPH, then the
-carbon is either
carboxylated to yield ethylmalonyl-CoA or, in the absence of
CO
2
, a proton replaces the electrophilic substrate, forming
but yryl-CoA. According to biochemical data, we suggest the
following enzy matic overall reaction:
crotonyl-CoA NADPH CO
2
3
ethylmalonyl-CoA
NADP
.
Crotonyl-CoA Carboxylase/Reductase Activity in Cell Extracts of Iso-
citrate Lyase-Negative Bacteria.
The unique reductive carboxyla-
tion of crotonyl-CoA serves as a key reaction of the pathway,
which is named after its characteristic intermediate ethylmalonyl-
CoA. Extracts of R. sphaeroides grown photoheterotrophically
with acetate catalyzed the crotonyl-CoA- and CO
2
-dependent
oxidation of NADPH (0.7 U mg
1
); this activity was down-
regulated at least 60-fold in cells grown photoheterotrophically
with suc cinate ( 0.01 U mg
1
), giving support to the suggested
function ing of the ethylmalonyl-CoA pathway in acetate assim-
ilation. High crotonyl-CoA carboxylase/reductase activity (0.8 U
mg
1
) was present in extracts of M. extorquens g rown with
methanol, suggesting that the key enz yme of the ethylmalonyl-
CoA pathway is participating in C
1
assimilation. Extracts of
Streptomyces coelicolor grown with butyrate also catalyzed the
crotonyl-CoA- and CO
2
-dependent oxidation of NADPH (0.4 U
mg
1
). Inc orporation of [
14
C]bicarbonate into ethylmalonyl-
CoA by extracts of butyrate-g rown cells (0.4 U mg
1
) was
down-regulated 20-fold in extracts of suc cinate-grown cells (0.02
Umg
1
). These results suggest that crotonyl-CoA reductase of
Streptomyces (15) catalyses not only the reduction but also the
c oncurrent carboxylation of crotonyl-CoA and that the ethyl-
malonyl-CoA pathway is functioning in these organisms.
Discussion
Crotonyl-CoA carboxylase/reductase cat alyzes the central reac-
tion in an acetyl-CoA assimilation pathway, which is distinct
f rom the glyoxylate cycle (Fig. 1). The pathway, shown in Fig. 4,
was named the ethylmalonyl-CoA pathway after its characteristic
inter mediate and product of its key enzyme, crotonyl-CoA
carboxylase/reduct ase. This enz yme catalyzes the reductive car-
boxylation of an enoyl-CoA ester, a reaction unprecedented in
biology. Therefore, the presence of its unique activity in cell
extracts of organ isms utilizing substrates as sole carbon source,
which are metabolized by means of acetyl-CoA, can be used as
an indication for the presence of the ethylmalonyl-CoA pathway.
The gene encoding crotonyl-CoA carboxylase/reductase (ccr)is
present in the genomes of all organisms proposed to use an
acet yl-CoA assimilation pathway distinct from the glyoxylate
c ycle (13). We could recently show that meaA, which is found
clustered with ccr in genomes, encodes coenzy me B
12
-dependent
ethylmalonyl-CoA mutase forming methylsuc cinyl-CoA (T.J.B.,
G.F., and B.E.A., unpublished results). An acyl-CoA dehydro-
genase up-regulated in acetate- versus glucose-grown R. spha-
eroides may catalyze the oxidation of methylsuc cinyl-CoA to
mesaconyl-CoA (13), thereby closing the remain ing gap in the
pathway (Fig. 4). A homologous gene is part of the same gene
cluster encoding ccr and meaA in Streptomyces species, and
mut ation of the corresponding gene in M. extorquens results in
a methanol-minus phenotype (12). Whether this acyl-CoA de-
hydrogenase indeed encodes for methylsuccinyl-CoA dehydro-
genase remains to be shown.
A ll genes implicated in the ethylmalonyl-CoA pathway for
acet ate assimilation of R. sphaeroides are conserved for M.
e xtorquens (13). This isocitrate lyase-negative methylotroph uses
the serine cycle for C
1
-assimilation (t ype II methylotroph) and
for this requires a pathway for the oxidation of acetyl-CoA to
glyoxylate (6, 19). Here, we have shown high crotonyl-CoA
carboxylase/reduct ase activity in cell extracts of methanol-grown
M. ex torquens. The presence of the ethylmalonyl-CoA pathway
(excluding the condensation of acetyl-CoA and glyoxylate) in M.
Fig. 3. Recombinant crotonyl-CoA carboxylase/reductase. (A) Denaturing
PAGE of various steps during purification. Lane 1, 25
gofE. coli cell-extract
protein before induction; lane 2, 25
gofE. coli cell-extract protein after 4 h
of induction; lane 3, 20
g of protein after ultracentrifugation; lane 4, 15
g
of protein from the DEAE column step; lane 5, 8
g of protein from the
Cibacron Blue column step; lane 6, molecular mass marker (97 kDa, phosphor-
ylase B; 67 kDa, BSA; 45 kDa, ovalbumin; 34 kDa, lactate dehydrogenase;
29 kDa, carbonic anhydrase; 14 kDa, lysozyme). (B) Specific activity of the
crotonyl-CoA carboxylase/reductase at various steps during purification. The
specific activity was determined spectrophotometrically by the crotonyl-CoA-
dependent oxidation of NADPH at 360 nm. Numbering is according to A.
Erb et al. PNAS
June 19, 2007
vol. 104
no. 25
10633
MICROBIOLOGY
e xtorquens simplifies the scheme for isocitrate lyase-negative C
1
-
assimilation (Fig. 5) compared with former proposals (12).
Genes of the ethylmalonyl-CoA pathway, including ccr, were
af fected in mutants of M. extorquens unable to form glyoxylate
f rom acetyl-CoA. However, different cataly tic roles were ini-
tially assigned to the corresponding gene products (12, 16, 20).
It has been reported that marine specie s of the Roseobacter clade,
which constitute a substantial fraction of ocean bacterioplankton
involved in sulfur cycling, are capable of demethylation reactions
and therefore are likely to be facultative methylotrophs (21).
However, the use of C
1
-c ompounds by these aerobic phototrophs
has not been explicitly shown. All genes implicated in the
herein-described ethylmalonyl-CoA pathway, including ccr, now
k nown to encode the key enzyme crotonyl-CoA carboxylase/
reduct ase, are conserved in sequenced representatives of this
phylogenetic group (13), suggesting that the extended serine
cycle (Fig. 5) may be involved in metabolism of further ecologically
important compounds such as dimethylsulfoniopropionate.
For S. coelicolor, the gene encoding crotonyl-CoA carbox y-
lase/reduct ase (ccr) is part of a gene cluster contain ing additional
genes involved in acetyl-CoA assimilation (8, 13). Similar gene
clusters are found in other Streptomyces species (22), suggesting
that the ethylmalonyl-CoA pathway functions in several strep-
tomycetes during grow th on compounds like butyrate, requiring
a pathway for acet yl-CoA assimilation. Interestingly, homo-
logues of ccr are present in other actinomycetes (SI Fig. 7) and
are part of gene clusters encoding polyketide synthases. This
finding provides support for the observed involvement of ccr in
antibiotic biosynthesis by supplying ethylmalonyl-CoA as an
extender unit (14, 23).
We conclude that the ethylmalonyl-CoA pathway is not a
‘‘st and-alone’’ pathway but, instead, integrates dif ferent meta-
bolic routes such as assimilation of compounds, which would
require the glyoxylate cycle, methylotrophy, antibiotic biosyn-
thesis, and also synthesis and utilization of polyhydroxybutyrate,
a major storage compound of all organisms mentioned here.
Materials and Methods
Bacterial Strains and Growth Conditions. R. sphaeroides 2.4.1
(DSMZ 158) and M. extorquens AM1 (DSMZ 1338) were grown
aerobically in the dark at 30°C and pH 6.7 in a 200 l fermenter
(Bioengineering, Wald, Switzerland; air flow, 2060 liters
min
1
; 150–220 rpm) on a defined minimal medium (13) sup-
plemented with 10 mM sodium acetate (R. sphaeroides) or 0.5%
methanol (M. extorquens). For regulatory studies, R. sphaeroides
was also g rown anaerobically in incandescent light (8,000 lux) in
2-liter bottles in minimal medium supplemented with 10 mM
acet ate or 10 mM succinate. S. coelicolor A3 (2) (DSMZ 40783)
was g rown aerobically on minimal medium supplemented with
10 mM succinate or 10 mM but yrate in 2-liter Erlenmeyer flasks
with baffles, filled with 500 ml of medium cont aining a met al
Fig. 4. The ethylmalonyl-CoA pathway as studied in isocitrate lyase-negative
R. sphaeroides. Crotonyl-CoA carboxylase/reductase was identified as the key
enzyme of the herein-described acetyl-CoA assimilation pathway distinct from
the glyoxylate cycle. Mutations in the genes encoding
-ketothiolase and
mesaconyl-CoA hydratase were previously shown to result in an acetate-minus
phenotype (13). The bifunctional
-methylmalyl-CoA/malyl-CoA lyase cataly-
ses the cleavage of
-methylmalyl-CoA as well as the condensation of acetyl-
CoA and glyoxylate to form malyl-CoA (18). Dotted lines indicate steps that
have not been elucidated so far. Exogenous CO
2
is not required for growth
with acetate as sole carbon source, indicating that the two molecules of CO
2
fixed in the ethylmalonyl-CoA pathway are derived from the oxidation of
acetyl-CoA.
Fig. 5. Proposed pathway for the assimilation of C
1
compounds by isocitrate
lyase-negative type II methylotrophs by using the serine cycle for formalde-
hyde fixation. The ethylmalonyl-CoA pathway described here (excluding the
condensation of acetyl-CoA and glyoxylate) is integrated in the serine cycle
(upper part) and is involved in assimilation of acetyl-CoA and regeneration of
glyoxylate during growth on C
1
compounds. It is assumed that during growth
on C
2
compounds the ethylmalonyl-CoA pathway (Fig. 4) is used exclusively.
Dotted lines indicate more than one reaction step.
10634
www.pnas.orgcgidoi10.1073pnas.0702791104 Erb et al.
spiral spring to break up cell clumps. Cells were harvested at
midex ponential growth phase at an OD
578
0.5 1.0 (R.
sphaeroides and S. coelicolor)or2.5(M. extorquens). Cells were
stored at 80°C until use.
Syntheses. Acetoacetyl-CoA was synthesized from diketene as
described (24). Butyryl-CoA and crotonyl-CoA were synthesized
f rom their anhydrides (24). (R)-3-hydrox ybutyryl-CoA and ( R/
S)-ethylmalonyl-CoA were synthesized by the mixed-anhydride
method (25).
Cell Extracts and Enzyme Measurement. Frozen cells (300400 mg)
were resuspended in 0.5 ml of 100 mM TrisHCl (pH 7.9) and 50
gml
1
of DNase I. Af ter addition of 1.1 g of glass beads
(diameter 0.1–0.25 mm), the cell suspension was treated in a
mixer mill (type MM2; Retsch, Haare, Germany) for 9 min at 30
Hz. The supernatant obtained after centrifugation (10 min,
14,000 g, 4°C) was used for assays. The protein c ontent of the
cell extracts was determined by the Bradford method (26) by
using BSA as a standard and was 10 mg ml
1
for R. sphaeroides
and M. extorquens or2mgml
1
for S. coelicolor. For specific
activities, one unit (1 U) corresponds to one
mol product
for med per min.
Incorporation of [
14
C]bicarbonate into acid-stable compounds.
Carbox-
ylation reaction in the presence of different CoA-thioesters and
NADPH was tested radiochemically. The reaction mixture (0.35
ml) c ontained 80 mM TrisHCl buffer (pH 7.9), 33 mM
NaHCO
3
, 1 MBq ml
1
NaH
14
CO
3
(Amersham, Braunschweig,
Ger many), 5 mM NADPH, 2 mM CoA-ester, and cell extract
(0.3–0.5 mg of protein per ml
1
). To study ATP-dependence, 5
mM ATP and 5 mM MgCl
2
were added to the reaction mixture.
The reaction was stopped at different time points by transferring
100
l of the reaction mixture to 500
l of 5% trichloroacetic
acid. The samples were shaken overnight to remove noninc or-
porated
14
CO
2
and the amount of remaining
14
C was determined
by liquid scintillation c ounting by using 3 ml of Rotiszint 2200
scintillation mixture (Roth).
Crotonyl-CoA carboxylase/reductase.
The crotonyl-CoA-dependent
oxidation of NADPH was followed spectrophotometrically at
360 nm (
NADPH
3,400 M
1
cm
1
) by using a cuvette with a
path length of 0.1 cm. The reaction mixture (0.2 ml) contained
100 mM TrisHCl buffer (pH 7.9), 4 mM NADPH, 2 mM
crotonyl-CoA, and 0.040.8 mg of cell extract protein or 1–5
g
of purified crotonyl-CoA carboxylase/reductase. The reaction
was started by the addition of 33 mM KHCO
3
or NaHCO
3
. The
apparent K
m
values of crotonyl-CoA and NaHCO
3
were deter-
mined by varying the c oncentration of NaHCO
3
(0.466.6 mM)
or crotonyl-CoA (0.125–2.0 mM), while keeping the other sub-
strates at saturating concentrations. The apparent K
m
value of
NADPH was determined by incorporation of [
14
C]bicarbonate
into (acid stable) ethylmalonyl-CoA. The reaction mixture (0.33
ml) contained 100 mM TrisHCl buffer (pH 7.9), 3 mM crotonyl-
CoA, 3 mM NaHCO
3
, 64 kBq ml
1
NaH
14
CO
3
, and 7
gof
purified crotonyl-CoA carboxylase. The reaction was started by
adding NADPH (0.125–5 mM) to the assay mixture. The reac-
tion was stopped at dif ferent time points by transferring 50
lof
the reaction mixture to 50
l of 1.5 M HClO
4
. The samples were
shaken overn ight to remove nonincorporated
14
CO
2
, and the
amount of remaining
14
C was determined by liquid scintillation.
Backreaction.
The ethylmalonyl-CoA-dependent reduction of
NADP
was followed spectrophotometrically at 360 nm by using
a cuvette with a path length of 1 cm. The reaction mixture (0.4
ml) contained 90 mM TriHCl buf fer (pH 7.9), 5.7 mM NADP
,
and 23
g of purified crotonyl-CoA carboxylase/reduct ase. The
reaction was started by the addition of 1.5 mM ethylmalonyl-
CoA. The apparent K
m
value of ethylmalonyl-CoA was deter-
mined by varying the c oncentration of ethylmalonyl-CoA
(0.038–1.5 mM) while keeping the concentration of NADP
c onstant.
HPLC Analysis. At different time points, the conversion of acetyl-
CoA and bicarbonate in cell extracts of R. sphaeroides was
stopped by transferring 100
l of the reaction mixture to 400
l
of methanol. Protein was removed by centrifugation, methanol
was evaporated in a Speedvac concentrator, and samples were
analyzed for CoA-thioesters by reversed-phase HPLC on a
Waters 2690 separation module (Waters, Eschborn, Ger many)
by using a RP-C
18
c olumn (LiChrospher 100, end-capped, 5
m,
125 4 mm; Merck, Darmstadt, Germany); 100
l of centri-
fuged sample were injected onto the column. A 30-min gradient
f rom 2% to 10% (vol/vol) aceton itrile in 50 mM potassium
phosphate buffer (pH 6.7) at a flow rate of 1 ml min
1
was used.
Simult aneous detection of UV absorbance and radioactivity in
st andard compounds and reaction products was done with a
Waters 996 photodiode array detector and a Ramona 2000
radioactive monitor (Raytest, Straubenhardt, Ger many) in se-
ries.
HPLC-MS was performed on an Agilent 1100 system (Agilent
Technologies, Waldbronn, Germany) interfaced with an Ap-
plied Biosystems API 2000 triple-quadr upole mass spectrometer
by using the same separation conditions as on the Waters system
but with 40 mM ammonium acetate (pH 6.7) instead of phos-
phate buffer. The temperature of the Turbo-Ionspray auxiliary
gas was 400°C, and the ionization voltage was 4,500 V. The
samples were analyzed with a mass range of 100–1,600 Da.
NMR Spectroscopy. The
13
C-enriched products of the conversion
of [U-
13
C]acet yl-CoA (Cambridge Isotope Laboratories An-
dover, MA) and [
13
C]bicarbonate (Spectra Stable Isotopes,
Columbia, MD) in cell extracts of R. sphaeroides were analyzed
by NMR spectroscopy. [U-
13
C]acet yl-CoA was synthesized by
incubation of 1.1 mM [U-
13
C]acet ate, 4.4 mM ATP, 1.1 mM
CoA, 4.4 mM KCl, and 4.4 mM MgCl
2
with 0.15 U ml
1
of
acet yl-CoA synthet ase (Sigma–A ldrich, Deisenhofen, Germany)
in 22.4 ml of 78 mM Tris HCl buffer (pH 7.9). After 60 min of
incubation at 37°C, the reaction mixture was transferred to 37°C
and supplemented with 0.5 mM 5–5-dithiobis(2-nitrobenzoic
acid), 31.6 mM NaH
13
CO
3
, and 5.9 mM NADPH. The reaction
was started by the addition of 21 mg of cell-extract protein of R.
sphaeroides to a final volume of 31.6 ml. The reaction was
stopped after 30 min by transferring the whole mixture to 160 ml
of methanol. Protein was removed by centrifugation, and the
supernat ant was concentrated by flash evaporation at 40°C (150
mbar) to a volume of 20 ml. The pH of the yellow ish solution was
adjusted with formic acid to 3.0, and the solution was centrifuged
to remove the precipitate. The supernatant was applied onto a
25-ml (2 mg) ISOLUTE C
18
(EC) solid phase extraction column
(Separtis, Grenzach Whylen, Germany) which had been acti-
vated with 20 ml of methanol and equilibrated with 40 ml of
ammon ium formate buffer (100 mM, pH 4.0). The column was
washed with 20 ml of ammonium formate buf fer, followed by
elution of the products with 60 ml of methanol. The organic
solvent was concentrated again by flash evaporation, followed by
lyophilization of the remaining liquid. NMR spectra were re-
c orded with an Avance DRX-400 spectrometer (Bruker, Rhein-
stetten, Germany) at 27°C. Chemical shifts were rec orded and
reported in ppm relative to MeOH-d4 (
1
H:
3.31,
13
C:
49.15) as internal standard. INADEQUATE measurements
were performed w ith SF 100.624 MHz, D1 6 sec, Aq 0.1
sec, PW90 8.8
s 3db, NS 160, TD F2 4096 and TD
–F1 128.
Heterologous Expression of the
ccr
Gene from
R. sphaeroides
and
Production of the Protein in
E. coli
. The gene encoding crotonyl-
CoA carbox ylase/reductase was amplified by PCR from R.
Erb et al. PNAS
June 19, 2007
vol. 104
no. 25
10635
MICROBIOLOGY
sphaeroides chromosomal DNA by using the forward primer
(5-GGAGGCAACCATGGCCCTCGACGTGCAGAG-3) in-
troducing a NcoI site (italicized) at the initiation codon and
reverse primer (5-GAGACTTGCGGATCCCTCCGATCAG-
GCCTTGC-3) introducing a BamHI site (italicized) after the
stop codon. The PCR product was isolated and cloned into the
pET3d expression vector (Merck), generating pTE13. Compe-
tent E. coli BL21(DE3) cells (27) were transformed with pTE13
and grown at 37°C in a 200-liter fermenter (air flow, 80 liter
min
1
; 300 rpm) cont aining Luria-Bertani broth and 100
g
ml
1
ampicillin. Expression was induced at OD
578
0.75 with 0.5
mM isopropylthio-galactopyranoside. After additional grow th
for 3.5 h, the cells were harvested and stored in liquid nitrogen
until use.
Purification of Heterologously Produced Crotonyl-CoA Carboxylase/
Reductase.
The purification was performed at 4°C. Crotonyl-CoA
carboxylase/reduct ase activity was measured by using the spec-
trophotometric assay described above.
Preparation of cell extracts.
Frozen cells were suspended in the
double volume of buffer A (20 mM TrisHCl, pH 7.9) c ontaining
0.1 mg of DNase I per ml. The suspension was passed twice
through a French pressure cell at 137 MPa and then centrifuged
(100,000 g)at4°Cfor1h.
DEAE chromatography.
Supernat ants [15 ml (1.6 g of protein)] after
the centrifugation step were applied at a flow rate of 2.5 ml
min
1
onto a 30-ml DEAE-Sepharose Fast Flow column (Am-
ersham Biosciences), which had been equilibrated w ith 60 ml of
buf fer A. The column was washed with 90 ml of buffer A and
thereaf ter with 135 ml of buf fer A containing 50 mM KCl.
Activit y was eluted with 100 mM KCl in buffer A in a volume of
195 ml. Active fractions were pooled, desalted, and concentrated
to a final volume of 20 ml by ultrafiltration (Amicon YM 10
membrane; Millipore, Bedford, MA).
Affinity chromatography.
Concentrated protein solution [1.5 ml (17
mg of protein)] obtained by DEAE chromatog raphy were ap-
plied at a flow of 0.5 ml min
1
onto a 10-ml Cibacron blue 3GA
agarose 3000 CL c olumn (Sigma–Aldrich), which had been
equilibrated with 20 ml of buffer A. The column was washed with
22 ml of buffer A, followed by 37 ml of buffer A contain ing 100
mM KCl and 37 ml of buffer A c ontaining 200 ml of KCl. Activit y
was eluted with 500 mM KCl in buffer A in a volume of 30 ml.
Active f ractions were pooled, desalted, and c oncentrated to a
final volume of 1.5 ml by ultrafiltration (Amicon YM 10 mem-
brane; Millipore). The protein (7.5 mg) was stored at 20°C with
50% glycerol to avoid precipitation. The affin ity chromatogra-
phy step was scaled up to purif y 15 ml (153 mg of protein) of the
c oncentrated DEAE fractions by using a 60-ml Cibacron Blue
3GA agarose 3000 CL column (Sigma–Aldrich) and a flow of 2.5
ml min
1
.
Protein Analyzing Methods.
Standard techniques.
Protein fractions were
analyzed by SDS-12.5% polyacrylamide gel electrophore sis (28).
Determination of native molecular mass.
The purified protein was
applied at a flow rate of 1 ml min
1
onto a 120-ml Highload
Superdex 200 16/60 column (Amersham Biosciences) equili-
brated with buffer A cont aining 125 mM KCl. Activ ity eluted
with a retention volume of 18.2–18.8 ml. The native molecular
mass of the enzyme was estimated by using the same gel-
filtration column, calibrated with thy roglobulin (660 kDa), fer-
ritin (450 kDa), catalase (240 kDa), aldolase (150 kDa), BSA (67
kDa), ovalbumin (45 kDa), chymotrypsinogen A (25 kDa), and
RNaseA (13.7 kDa) as molecular mass standards.
Metal analysis.
Purified rec ombinant crotonyl-CoA carboxylase/
reduct ase (4.0 mg) was analyzed for metals by inductively
c oupled plasma emission spectroscopy (ICP-OES) in the Chem-
ical Analysis Laboratory of R. Auxier (University of Georgia,
Athens, GA).
We thank Chris Anthony for fruitful discussions concerning C
1
assim-
ilation. This work was supported by Deutsche Forschungsgemeinschaft
Grant AL677/1-1 and the Graduiertenkolleg ‘‘Biochemie der Enzyme’’
(fellowship to B.E.A.) as well as Degussa AG.
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www.pnas.orgcgidoi10.1073pnas.0702791104 Erb et al.
Rhodobacter sphaeroides 2.4.1
Methylobacterium extorquens AM1
Streptomyces coelicolor A3(2)
Xanthobacter autotrophicus
0.1
Stappia aggregata
Burkholderia cepacia
Streptomyces sp. HK803
Streptomyces mycarofaciens
Salinispora tropica B
Frankia sp. CcI3 B
Frankia sp. EAN1 D
Frankia alni ACN14 B
Frankia sp. CcI3 A
Frankia sp. EAN1 C
Frankia sp. EAN1 B
Nocardioides sp.
Streptomyces fradiae
Streptomyces diastaticus
Streptomyces kanamyceticus C
Streptomyces kanamyceticus A
Streptomyces kanamyceticus B
Streptomyces collinus
Streptomyces cinnamonensis
Streptomyces avermitilis A
Streptomyces neyagawaensis
Frankia alni ACN14 A
Streptomyces avermitilis B
Streptomyces nanchangensis
Streptomyces hygroscopicus
Salinispora tropica A
Frankia sp. EAN1 A
Janibacter sp.
Streptomyces lavendulae
Streptomyces avermitilis C
Erwinia carotovora
Pseudomonas syringae pv tomato
Pseudomonas syringae pv glycinea
Leptospira interrogans
Leptospira borgpetersenii sv L550
Leptospira borgpetersenii sv JB197
Rhodospirillum rubrum
Magnetospirillum magneticum
Magnetospirillum magnetotacticum B
Hyphomonas neptunium
Caulobacter crescentus
Caulobacter sp.
Magnetospirillum magnetotacticum A
Loktanella vestfoldensis
Roseovarius sp. HTCC2601
Oceanicola batsensis
Roseovarius sp. 217
Rhodobacterales bacterium
Roseovarius nubinhibens
Jannaschia sp.
Roseobacter denitrificans
Silicibacter pomeroyi
Silicibacter sp. TM1040
Roseobacter sp.
Sulfitobacter sp. EE-36
Sulfitobacter sp. NAS-14.1
Paracoccus denitrificans
Rhodobacter sphaeroides ATCC 17025
Rhodobacter sphaeroides ATCC 17029
Rhodobacter sphaeroides 2.4.1
Methylobacterium extorquens AM1
Streptomyces coelicolor A3(2)
Xanthobacter autotrophicus
0.1
Stappia aggregata
Burkholderia cepacia
Streptomyces sp. HK803
Streptomyces mycarofaciens
Salinispora tropica B
Frankia sp. CcI3 B
Frankia sp. EAN1 D
Frankia alni ACN14 B
Frankia sp. CcI3 A
Frankia sp. EAN1 C
Frankia sp. EAN1 B
Nocardioides sp.
Streptomyces fradiae
Streptomyces diastaticus
Streptomyces kanamyceticus C
Streptomyces kanamyceticus A
Streptomyces kanamyceticus B
Streptomyces collinus
Streptomyces cinnamonensis
Streptomyces avermitilis A
Streptomyces neyagawaensis
Frankia alni ACN14 A
Streptomyces avermitilis B
Streptomyces nanchangensis
Streptomyces hygroscopicus
Salinispora tropica A
Frankia sp. EAN1 A
Janibacter sp.
Streptomyces lavendulae
Streptomyces avermitilis C
Erwinia carotovora
Pseudomonas syringae pv tomato
Pseudomonas syringae pv glycinea
Leptospira interrogans
Leptospira borgpetersenii sv L550
Leptospira borgpetersenii sv JB197
Rhodospirillum rubrum
Magnetospirillum magneticum
Magnetospirillum magnetotacticum B
Hyphomonas neptunium
Caulobacter crescentus
Caulobacter sp.
Magnetospirillum magnetotacticum A
Loktanella vestfoldensis
Roseovarius sp. HTCC2601
Oceanicola batsensis
Roseovarius sp. 217
Rhodobacterales bacterium
Roseovarius nubinhibens
Jannaschia sp.
Roseobacter denitrificans
Silicibacter pomeroyi
Silicibacter sp. TM1040
Roseobacter sp.
Sulfitobacter sp. EE-36
Sulfitobacter sp. NAS-14.1
Paracoccus denitrificans
Rhodobacter sphaeroides ATCC 17025
Rhodobacter sphaeroides ATCC 17029
Abbreviation Organism gi number
Burkholderia cepacia Burkholderia cepacia AMMD 115360962
Caulobacter crescentus Caulobacter crescentus CB15 16127315
Caulobacter sp
. Caulobacter sp. K31 113932713
Erwinia carotovora Erwinia carotovora ssp.
atroseptica SCRI1043 50119550
Frankia alni A Frankia alni ACN14a 111221964
Frankia alni B Frankia alni ACN14a 111225263
Frankia sp
. CcI3 A Frankia sp. CcI3 86742371
Frankia sp. CcI3
B Frankia sp. CcI3 86739711
Frankia sp. EAN1
pec A Frankia sp. EAN1pec 68234930
Frankia sp
. EAN1pec B Frankia sp. EAN1pec 68229972
Frankia sp
. EAN1pec C Frankia sp. EAN1pec 68234827
Frankia sp. EAN1
pec D Frankia sp. EAN1pec 68228771
Hyphomonas neptunium Hyphomonas ne
ptunium ATCC 15444 114798785
Janibacter sp
. Janibacter sp. HTCC2649 84494559
Jannaschia sp. Jannaschia sp. CCS1 89053284
Leptospira borgpetersenii sv JB197
Leptospira borgpetersenii serovar Hardjo-bovis JB197 116332072
Leptospira bo
rgpetersenii sv L550 Leptospira borgpetersenii serovar Hardjo-bovis L550 116327313
Leptospira interrogans Leptospira interrogans ser
ovar Lai str. 56601 24212996
Loktanella vestfoldensis Loktanella vestfoldensis SKA53 84515894
Magnetospirillum magneticum Magnetospirillum magneticum M
B-1 83309408
Magnetospirillum magnetotacticum A Magnetospirillum magnetotacticum M
S-1 46206255
Magnetospirillum magnetotacticum B Magnetospirillum magnetotacticum M
S-1 46201505
Methylobacterium extorquens AM1 Methylobacterium extorquens AM
1 1020391
Nocardioides sp. Nocardioides sp.
JS614 71366311
Oceanicola batsensis Oceanicola batsensis HT
CC2597 84500164
Paracoccus denitrificans Paracoccus denitri
ficans PD1222 69933269
Pseudomonas syringae pv lycinea Pseudomonas syringae pv.
glycinea 3114701
Pseudomonas syringae pv to
mato Pseudomonas syringae pv. tomato str. DC3000 28871819
Rhodobacter sphaeroides AT
CC 17025 Rhodobacter sphaeroides ATCC 17025 83369517
Rhodobacter sphaeroides 1702
9 Rhodobacter sphaeroides ATCC 17029 83372577
Rhodobacter sphaeroides
2.
4.1
Rhodobacter sphaeroides 2.
4.1 77464540
Rhodobacterales bacterium Rhodobacterales bacterium HT
CC2654 84684998
Rhodospirillum rubrum Rhodospirillum rubrum ATCC 11170 83594393
Roseobacter denitrificans Roseobacter denitrificans OCh 114 110678943
Roseobacter sp. Roseobacter sp. MED193 86138964
Roseovarius nubinhibens Roseovarius nubinhibens I
SM 83952461
Roseovarius sp.
217 Roseovarius sp. 217 85705862
Roseovarius sp.
HTCC2601 Roseovarius sp. HTCC2601 114764853
Salinispora tropica A Salinispora tropica CNB-
440 113945786
Salinispora tropica B Salinispora tropica CNB-
440 113944816
Silicibacter pomeroyi Silicibacter pomeroyi DSS-
3 56695285
Silicibacter sp.
TM1040 Silicibacter sp. TM1040 99078082
Stappia aggregata Stappia aggregata
IAM 12614 118435348
Streptomyces avermitilis A Streptomyces avermitilis MA-
4680 29828453
Streptomyces avermitilis B Streptomyces avermitilis MA-
4680 29829432
Streptomyces avermitilis C Streptomyces avermitilis MA-
4680 29826955
Streptomyces cinnamonensis Streptomyces cinnamonensis 5834348
Streptomyces coelicolor A3(2) Streptomyces coelicolor A3(2) 21224777
Streptomyces collinus Streptomyces collinus 1046371
Streptomyces diastaticus Streptomyces diastaticus 38324684
Streptomyces fradiae Streptomyces fradiae 5139641
Streptomyces hygroscopicus Streptomyces hygroscopicus 39932998
Streptomyces kanamyceticus A Streptomyces kanamyceticus 51981328
Streptomyces kanamyceticus B Streptomyces kanamyceticus 85813980
Streptomyces kanamyceticus C Streptomyces kanamyceticus 108743504
Streptomyces lavendulae Streptomyces lavendulae 4731329
Streptomyces mycarofaciens Streptomyces mycarofaciens 110333715
Streptomyces nanchangensis Streptomyces nanchangensis 43336426
Streptomyces neyagawaensis Streptomyces neyagawaensis 74026500
Streptomyces sp.
HK803 Streptomyces sp. HK803 35186974
Sulfitobacter sp. EE-36 Sulfitobacter sp. EE-36 83943861
Sulfitobacter sp. NAS-14.1 Sulfitobacter sp. NAS-14.1 83953502
Xanthobacter autotrophicus Xanthobacter autotrophicus Py2 89358761
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Cobamides (Cbas) are cobalt-containing cyclic tetrapyrroles used by cells from all domains of life as co-catalyst of diverse reactions. There are several structural features that distinguish Cbas from one another. The most relevant of those features discussed in this review is the lower ligand, which is the nucleobase of a ribotide located in the lower face of the cyclic tetrapyrrole ring. The above-mentioned ribotide is known as the nucleotide loop, which is attached to the ring by a short linker. In Cbas, the nucleobase of the ribotide can be benzimidazole or derivatives of it, purine or derivatives of it, or phenolic compounds. Given the importance of Cbas in prokaryotic metabolism, it is not surprising that prokaryotes have evolved enzymes that cleave part or the entire nucleotide loop. This function is advantageous when Cbas contain nucleobases that somehow interfere with the function of Cba-dependent enzymes in the organism. After cleavage, Cbas are rebuilt via the nucleotide loop assembly (NLA) pathway, which includes enzymes that activate the nucleobase and the ring intermediate, followed by condensation of activated intermediates and a final dephosphorylation reaction. This exchange of nucleobases is known as Cba remodeling. The NLA pathway is used to salvage Cba precursors from the environment.
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This review comprehensively discusses microbial conversion of CO 2 to organic compounds. The efficiency of CO 2 fixation can be improved by mining CO 2 -fixing enzymes, developing CO 2 -fixing pathways and optimizing CO 2 -fixing microbial cell factories.
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The conversion of CO2 by enzymes such as carbonic anhydrase or carboxylases plays a crucial role in many biological processes. However, in situ methods following the microscopic details of CO2 conversion at the active site are limited. Here, we used infrared spectroscopy to study the interaction of CO2, water, bicarbonate, and other reactants with β-carbonic anhydrase from Escherichia coli (EcCA) and crotonyl-CoA carboxylase/reductase from Kitasatospora setae (KsCcr), two of the fastest CO2-converting enzymes in nature. Our data reveal that KsCcr possesses a so far unknown metal-independent CA-like activity. Site-directed mutagenesis of conserved active site residues combined with molecular dynamics simulations tracing CO2 distributions in the active site of KsCCr identify an ‘activated’ water molecule forming the hydroxyl anion that attacks CO2 and yields bicarbonate (HCO3⁻). Computer simulations also explain why substrate binding inhibits the anhydrase activity. Altogether, we demonstrate how in situ infrared spectroscopy combined with molecular dynamics simulations provides a simple yet powerful new approach to investigate the atomistic reaction mechanisms of different enzymes with CO2.
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Two general methods for the synthesis of acyl coenzyme A and other thiol esters are described in this chapter. The first procedure an acid anhydride is allowed to react with the mercaptan in cold aqueous solution at pH 7.5. The second method makes use of thiol ester interchange reactions of the type. In the synthesis of simple thiol esters by the anhydride and mixed anhydride procedures, the yields are normally 80 to 100%, based on the free —SH. The formation of thiol ester derivatives of α, β-unsaturated fatty acid is much poorer—15 to 50%. These low yields are because of the occurrence of side reactions in which the –SH groups add to the double bonds of. the anhydride and thiol esters. In addition to the general methods, thiol esters have been prepared by reaction of mereaptans with acyl chlorides and thiol acids. A method for the synthesis of acetoacetyl thiol esters by reaction of mercaptans with ketene has been presented. In enzymatic assay of Ac-SCoA measurement is made of the decrease in optical density at 232 to 240 mμ that is associated with the hydrolysis of Ac-SCoA. The complete hydrolysis of Ac-SCoA is brought about in a few seconds by incubation in the presence of phosphotransacetylase and arsenate.
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A crotonyl-CoA reductase (EC 1.3.1.38, acyl-CoA:NADP+trans -2-oxidoreductase) catalyzing the conversion of crotonyl-CoA to butyryl-CoA has been purified and characterized from Streptomyces collinus. This enzyme, a dimer with subunits of identical mass (48 kDa), exhibits a Km, = 18 μM for crotonyl-CoA and 15 μM for NADPH. The enzyme was unable to catalyze the reduction of any other enoyl-CoA thioesters or to utilize NADH as an electron donor. A highly effective inhibition by straight-chain fatty acids (Ki=9.5 μM for palmitoyl-CoA) compared with branched-chain fatty acids (Ki>400 μM for isopalmitoyl-CoA) was observed. All of these properties are consistent with a proposed role of the enzyme in providing butyryl-CoA as a starter unit for straight-chain fatty acid biosynthesis. The crotonyl-CoA reductase gene was cloned in Escherichia coli. This gene, with a proposed designation of ccr, is encoded in a 1344-bp open reading frame which predicts a primary translation product of 448 amino acids with a calculated molecular mass of 49.4 kDa. Several dispersed regions of highly significant sequence similarity were noted between the deduced amino acid sequence and various alcohol dehydrogenases and fatty acid synthases, including one region that contains a putative NADPH binding site. The ccr gene product was expressed in E. coli and the induced crotonyl-CoA reductase was purified tenfold and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate/polyacrylamide to the native protein.
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It is well known that proteolysis often occurs after rupture of metazoan cells. Thus proteins isolated from extracts may not be representative of their native cellular counterparts. In the present research, extensive proteolysis was observed in crude extracts of the freeliving soil nematode Caenorhabditis elegans and the parasitic nematode Ascaris suum. Phenylmethylsulfonyl fluoride (PMSF) reduced the loss in activity of isocitrate lyase (EC 4.1.3.1), fumarase (EC 4.2.1.2), and citrate synthase (EC 4.1.3.7) in extracts of C. elegans but had little or no effect upon loss of malate synthase (EC 4.1.3.2). Catalase (EC 1.11.1.6) was stable. The loss of isocitrate lyase and citrate synthase was less pronounced in extracts of 22-day-old embryos of A. suum. Catalase decayed in these extracts. The addition of PMSF reduced the loss in all three of these activities. Fumarase was stable. The number of active fragments of isocitrate lyase recovered after filtration on Sephadex G-200 increased with the length of storage of crude extracts in the absence of PMSF at 4 C. Even in the presence of PMSF five activity peaks were observed after storage of extracts of C. elegans at 4 C for 72 hr. The molecular weights of active species ranged between 549,000 and 128,000 for isocitrate lyase in extracts of either C. elegans or A. suum. The 549,000- and 214,000-dalton species of isocitrate lyase from A. suum were much more labile at 50 C than the 543,000- and 195,000-dalton species from C. elegans.
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In cell-free extracts of Thiobacillus uersutus, an organism which has been reported to be isocitrate lyase negative, an isocitrate lyase activity of 52 & 18 nmol min-' (mg protein)-' was observed after anaerobic growth in a chemostat on acetate plus nitrate, i.e. during denitrification. Following growth on succinate plus nitrate, isocitrate lyase activity was only 1 & 2 nmol min-l (mg protein)-'. In cell-free extracts derived from aerobic chemostat cultures isocitrate lyase activity was always nil. The identity of the enzyme was analysed using a number of different methods, namely (a) three different enzyme assays, (b) l3C-NMR spectroscopy of the reaction products, (c) HPLC analysis of the reaction products, (d) mass spectrometry of derivatized glyoxylate enzymically produced from isocitrate and (e) radiography of derivatized glyoxylate enzymically produced from ( I4C)citrate. All these methods gave results consistent with the enzyme-catalysed conversion of isocitrate to glyoxylate and succinate. The metabolism of acetate or substrates which are converted to acetyl-CoA moieties without the intermediary generation of pyruvate proceeds through the tricarboxylic acid cycle (TCA cycle) and the glyoxylate cycle (Doelle, 1975; Lehninger, 1975; Schlegel, 1981). Dixon & Kornberg (1959) were the first to demonstrate the occurrence of the two key glyoxylate cycle enzymes, isocitrate lyase and malate synthase. However, bacteria have been isolated which are able to grow on acetate or substrates such as ethanol or 3-hydroxybutyrate and which do not have measurable isocitrate lyase activity. Most if not all of these organisms belong to the group of facultative methylotrophs using the isocitrate lyase negative serine pathway during methylotrophic growth (Anthony, 1982). As methylotrophic or heterotrophic growth on C2 substrates apparently can occur without the presence of isocitrate lyase activity, these organisms were thought to possess an alternative metabolic route to generate glyoxylate. To obtain a better insight into the metabolism of acetate in an isocitrate lyase negative organism we chose Thiobacillus versutus, reported to lack isocitrate lyase (Gottschal & Kuenen, 1980), for our experiments. The intermediary metabolism of acetate was analysed in vivo by 3C-NMR spectroscopy. T. versutus was adapted to anaerobic growth on acetate in the presence of nitrate for the preparation of NMR samples. This was done in order to facilitate the NMR experiments. Moreover, we assumed that by replacing oxygen by nitrate as electron acceptor the metabolism of the carbon and energy source would not be altered. Very surprisingly, however, T. versutus was found to possess isocitrate lyase activity during denitrifying growth on acetate, whereas during aerobic growth on the same substrate no activity was detected. In view of this finding we felt obliged to confirm the nature of the observed enzyme activity by identifying the reaction products.
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Reduced ferredoxin: CO2 oxidoreductase (CO2 reductase) from Clostridium pasteurianum catalyzes the reduction of ‘CO2’ to formate with reduced ferredoxin, an isotopic exchange between ‘CO2’ and formate in the absence of ferredoxin, and the oxidation of formate to ‘CO2’ with oxidized ferredoxin. The active species of ‘CO2’, i.e. CO2 or HCO3− (H2CO3), utilized by the enzyme was determined. The method employed for the species identification was that of Cooper et al. (1968). Both ‘CO2’ reduction to formate and the exchange reaction were studied. Data were obtained which are compatible with those expected if CO2 is the active species. The V and the dissociation constant Ks of the enzyme · CO2 complex in dependence of pH were determined from initial velocity studies of the exchange reaction. V was found to be only slightly affected by pH between 5.5 and 7.5. Ks was markedly dependent on pH; the constant increased with decreasing pH from 0.2 mM at pH 7.5 to 3 mM at pH 5.5.
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A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
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When Rhodopseudomonas gelatinosa was grown on acetate aerobically in the dark both enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase, could be detected. However, under anaerobic conditions in the light only isocitrate lyase, but not malate synthase, could be found. The reactions, which bypass the malate synthase reaction are those catalyzed by alanine glyoxylate aminotransferase and the enzymes of the serine pathway. Other Rhodospirillaceae were tested for isocitrate lyase and malate synthase activity after growth with acetate; they could be divided into three groups: 1. organisms possessing both enzymes; 2. organisms containing malate synthase only; 3. R. gelatinosa containing only isocitrate lyase when grown anaerobically in the light.