Streptomyces virginiae PPDC Is a New Type of Phenylpyruvate Decarboxylase Composed of Two Subunits

Article abstract of DOI:10.1021/acschembio.7b00307

Streptomyces virginiae phenylpyruvate decarboxylase (PPDC) has not been identified before. Two putative branched-chain α-keto acid dehydrogenase subunit genes bkdC and bkdD from S. virginiae are similar to halves of other PPDC coding sequences. We cloned and characterized them biochemically in this work. The two proteins formed a stable complex attested by pull-down assay, consistent with the finding that their soluble expression was obtained only when they were coexpressed in Escherichia coli. The subunits were redesignated as SvPPDCα and SvPPDCβ, because the SvPPDCα/β complex catalyzed the conversion of phenylpyruvate to phenylacetaldehyde, reflecting the nature of the enzyme. Moreover, mutations of conserved residues in either of the two subunits led to inactivation or decreased specific activity of the enzymatic reaction. All previously identified PPDCs are encoded by a single gene. Here, we identified a new type of PPDC that contains two subunits, which gives new insights into the PPDC family.

Full text of DOI:10.1021/acschembio.7b00307

Letters
pubs.acs.org/acschemicalbiology
Streptomyces virginiae PPDC Is a New Type of Phenylpyruvate
Decarboxylase Composed of Two Subunits
†
,§
‡
†
,†,∥
Xiaoshu Xu, Chao Wang, Jun Chen, and Sheng Yang*
†
‡
Key Laboratory of Synthetic Biology and National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and
Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China
§
University of Chinese Academy of Sciences, Beijing 100049, China
∥
Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing 210009, China
*
S Supporting Information
ABSTRACT: Streptomyces virginiae phenylpyruvate decarbox-
ylase (PPDC) has not been identified before. Two putative
branched-chain α-keto acid dehydrogenase subunit genes bkdC
and bkdD from S. virginiae are similar to halves of other PPDC
coding sequences. We cloned and characterized them biochem-
ically in this work. The two proteins formed a stable complex
attested by pull-down assay, consistent with the finding that
their soluble expression was obtained only when they were
coexpressed in Escherichia coli. The subunits were redesignated
as SvPPDCα and SvPPDCβ, because the SvPPDCα/β complex
catalyzed the conversion of phenylpyruvate to phenylacetalde-
hyde, reflecting the nature of the enzyme. Moreover, mutations of conserved residues in either of the two subunits led to
inactivation or decreased specific activity of the enzymatic reaction. All previously identified PPDCs are encoded by a single gene.
Here, we identified a new type of PPDC that contains two subunits, which gives new insights into the PPDC family.
hiamine diphosphate (ThDP)-dependent enzymes belong
to a number of different protein families and are involved in
The aminopyrimidine and diphosphate moieties of ThDP
interact with the PYR domain of one subunit and the PP domain
T
15,17
almost all major metabolic pathways. These enzymes catalyze
various biochemical reactions that include, mainly, the cleav-
age and making of carbon−carbon bonds neighboring keto
of the other subunit, respectively.
Phenylpyruvate interacts
with the thiazolium ring of ThDP to catalyze the decarboxylation.
In ThDP-dependent enzymes, both the PYR and PP domains are
conserved while the R domain is diverse. The R domain was
shown to be a regulatory domain involved in the allosteric sub-
strate activation process, which was characterized in AbPPDC
1
−3
groups.
A typical reaction is the nonoxidative decarboxyla-
tion of α-keto acids. In the past few years, a number of ThDP-
dependent decarboxylases have been characterized to catalyze
α-keto acids to aldehydes by nonoxidative decarboxylation.
Phenylpyruvate decarboxylase (PPDC, EC 4.1.1.43) is a ThDP-
dependent enzyme that catalyzes the nonoxidative decarbox4-
ylation of phenylpyruvate to phenylacetaldehyde and CO2.
PPDC activity has been reported in a few microorganisms using
4
when indolepyruvate was used as the substrate. A similar
substrate activation mode has also been observed in some
19,20
pyruvate decarboxylases (PDC, EC 4.1.1.1).
bkdC and bkdD
are two adjacent genes screened from a cosmid library of the
21
Streptomyces virginiae genome. The possible functions are
predicted by sequence similarities with other proteins and are
annotated as E1α and E1β subunits of the branched-chain α-keto
5
crude cell lysate, including Acinetobacter calcoaceticus, Nocardia
6
6,7
8
sp. 239, Thauera aromatica, Pseudomonas putida, Achromo-
21
9
4,10,11
acid dehydrogenase complex in public databases. However, the
biochemical function has not been experimentally characterized.
Here, we characterized the S. virginiae BkdC and BkdD pro-
teins in an enzyme complex that catalyzes the decarboxylation
bacter eurydice, Azospirillum brasilense,
cerevisiae,
Saccharomyces
1
2,13
14
and Dekkera bruxellensis. However, only the
PPDCs from S. cerevisiae (ScPPDC) and A. brasilense (AbPPDC)
have been biochemically characterized in detail. Crystal struc-
4
,11
of phenylpyruvate to phenylacetaldehyde and CO . On the basis
tures of AbPPDC have been published, which has helped in
establishing the catalytic mechanism of the PPDCs.
2
of this characterization, we redesignated BkdC and BkdD as
PPDCα and PPDCβ subunits, respectively. Sequence com-
parisons with AbPPDC and ScPPDC and the nature of the
enzymatic reaction indicated that the SvPPDCα/β complex was a
AbPPDC has canonical subunit architecture just as other
ThDP-dependent family members. Each subunit contains three
domains, including an N-terminal pyrimidine (PYR) domain,
a regulatory (R) domain, and a C-terminal pyrophosphate
1
0,15,16
2+
(
PP) domain.
The cofactor ThDP and Mg are bound
Received: April 8, 2017
Accepted: July 12, 2017
to the interface formed exclusively by interactions between
17,18
residues in the PYR and PP domains from the two subunits.
©
XXXX American Chemical Society
A
DOI: 10.1021/acschembio.7b00307
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Letters
Figure 1. Formation of an enzyme complex by SvPPDCα and SvPPDCβ. (a) SDS-PAGE analysis of the soluble and insoluble fractions of the Escherichia
coli lysate expressing SvPPDCα or SvPPDCβ, or coexpressing them. S and P indicate the soluble and pellet (insoluble) fractions, respectively. Ev-S:
soluble fractions for empty vector. Ev-P: insoluble fractions for empty vector. The SvPPDCα or SvPPDCβ protein bands are indicated by black and red
arrows, respectively. (b) His-tagged SvPPDCα and non-His-tagged SvPPDCβ were coexpressed and purified on a Ni-NTA agarose column. His-tagged
SvPPDCα was detected by immunoblotting using anti-His antibody. CBB: Coomassie brilliant blue. WB: Western blot. Ev: empty vector.
ThDP-dependent enzyme that contains the typical ThDP
binding motifs.
To explore the catalytic characters of SvPPDC, we first cloned
Svppdcα and Svppdcβ and expressed them in Escherichia coli. The
SDS-PAGE analysis of the two individual recombinant proteins
showed that they had molecular weights of about 41 kDa and
subunits, enzyme activity was detected using crude lysates of
E. coli cells expressing the recombinant SvPPDCα, SvPPDCβ,
or SvPPDCα/β complex as the catalysts for utilizing phe-
nylpyruvate. The results showed that enzyme activity was
detected only when SvPPDCα and SvPPDCβ were both present
(Figure 2c). Taken together, our results unambiguously suggest
that SvPPDCα/β is an active enzyme complex that catalyzes
phenylpyruvate to phenylacetaldehyde.
3
8 kDa, respectively. SvPPDCα and SvPPDCβ were found to
form insoluble inclusive bodies (Figure 1a). When we coexpre-
ssed the two proteins, they showed marked solubility (Figure 1a).
In many instances, proteins that form complexes are not able to
fold correctly and be expressed in a soluble state without their
The reaction kinetics of SvPPDCα/β were determined using
phenylpyruvate as the substrate at different concentrations.
As shown in Figure 3a, the SvPPDCα/β complex displayed
sigmoidal plots of the reaction velocity versus phenylpyruvate
concentrations, indicating that SvPPDCα/β is probably an
allosteric enzyme, with a Hill coefficient of 4.7 (4.71 ± 0.64). The
binding of phenylpyruvate to one site in the complex may affect
the properties of other active sites, which is described as a
substrate activation mode that has been observed in other
22
interacting partners. Therefore, this observation suggested that
SvPPDCα and SvPPDCβ may interact, and the interaction was
required for correct protein folding and assembly. To further
validate this, we coexpressed His-tagged SvPPDCα and non-His-
tagged SvPPDCβ in E. coli. When SvPPDCα was purified on a
Ni-nitrilotriacetic acid (NTA) agarose column, SvPPDCβ was
copurified with a nearly equal intensity (Figure 1b), indicating
that they formed a stable enzyme complex.
1
9,20
PDCs.
The K value of SvPPDCα/β was 1.13 mM, and the
m
−1
kcat value was 0.33 min (Figure 3b). To compare these kinetic
characteristics with a well-identified PPDC under similar con-
ditions, we cloned ScPPDC and measured its reaction kinetics
(Figure S1a). The ScPPDC protein exhibited little evidence of
allosteric activation by phenylpyruvate, with a Hill coefficient
On the basis of sequence analysis, we found that SvPPDCα/β
shared approximately 70% amino acid similarity with two pro-
teins in Streptomyces pristinaespiralis, which are putative subunits
of a pyruvate dehydrogenase-like complex and are predicted to
catalyze the conversion of phenylpyruvate to phenylacetyl-
close to 1 (1.28 ± 0.17), which is in accordance with the char-
2
3
13
CoA. We then ask if SvPPDCα/β could utilize phenylpyruvate
or pyruvate. The two subunits of the active enzyme complex
SvPPDCα/β were copurified and used to incubate the potential
substrates phenylpyruvate or pyruvate. The extracts were anal-
yzed with gas chromatography−mass spectrometry (GC-MS).
No new product was detected in the reaction mixture containing
pyruvate. A new product in the reaction mixture containing
phenylpyruvate was detected. The retention time of the GC
spectrum and the molecular ions in the MS spectrum of the
product were identical to the authentic standard phenyl-
acetaldehyde (Figure 2a).
acteristics of ScPPDC reported previously. The K value of
m
−
1
ScPPDC was 2.70 mM, and the k value was 538 min , which is
cat
13
similar to previously reported values. The catalytic efficiency of
ScPPDC was higher than that of SvPPDCα/β, possibly because
of its key roles in phenylalanine and tryptophan catabolism in the
Ehrlich pathway, which permits the use of these amino acids as a
13
nitrogen source for yeast growth.
ScPPDC and AbPPDC contain PYR, R, and PP domains in a
single unit and are encoded by a single gene. PYR and PP are the
ThDP-binding domains, as described above. In contrast,
SvPPDCα contains a PP domain but no PYR domain, and
SvPPDCβ contains a PYR domain and a transketolase C-terminal
(TKC)-like domain (Figure 3c). This subunit architecture, which
is unusual among previously identified PPDC-like enzymes, is
identical to the E1α and E1β subunits of a 2-oxoisovalerate
The purified SvPPDCα/β was able to convert phenylpyruvate
to phenylacetaldehyde in the presence of ThDP and Mg2+
(
Figure 2b). However, the enzyme activity cannot be detected
2
+
in the absence of ThDP or Mg (Figure 2b), indicating that the
SvPPDCα/β activity depended on ThDP and Mg as cofactors.
To validate the SvPPDCα/β activity depends on either of the
2
+
17
dehydrogenase-like enzyme complex. The 2-oxoisovalerate
dehydrogenase-like enzyme is a large multisubunit complex that
B
DOI: 10.1021/acschembio.7b00307
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Letters
Figure 2. Catalyzation of phenylpyruvate to phenylacetaldehyde by SvPPDCα/β. Values represent means ± standard deviation of four replicated
experiments. (a) GC-MS analysis confirms that the product is phenylacetaldehyde. The sample was the reaction products produced by the substrate plus
the purified SvPPDCα/β (phenylpyruvate + SvPPDCα/β), or crude fractions of cell lysate expressing the empty vector (phenylpyruvate + Ev, negative
control). (b) Cofactor and substrate analyses of purified SvPPDCα/β. (c) The enzyme activity analyzed by phenylacetaldehyde production by 100 μg of
soluble fractions of Esherichia coli lysate expressing SvPPDCα, SvPPDCβ, or SvPPDCα/β. The concentration of phenylacetaldehyde accumulated in 6 h
was quantified by HPLC. n.d., not detected.
catalyzes the oxidative decarboxylation reactions, which is
may act as the decarboxylases catalyzing nonoxidative decarbox-
ylation.
24
typically identified in the pyruvate dehydrogenase complex.
This may be the reason that the previously annotated SvBkdC
SvPPDCα) and SvBkdD (SvPPDCβ) are predicted as the
E1α and E1β subunits of the branched-chain α-keto acid (a
-oxoisovalerate) dehydrogenase complex in public databases.
However, our biochemical analysis characterized SvBkdC/D
SvPPDCα/β) as an atypical PPDC to catalyze the nonoxida-
According to the available crystal structures of AbPPDC and the
catalytic nature of this enzyme, we propose that the PYR domain of
SvPPDCβ and PP domains of SvPPDCα are responsible for
(
2+
2
binding ThDP, Mg , and thesubstratephenylpyruvate. The TKC-
like domain was reported previously to be recruited by several
members of the ThDP-dependent family but formed no part of the
(
17
tive decarboxylation of phenylpyruvate. It is also possible that
other SvPPDCα/β-like proteins, which are annotated as subunits
of the 2-oxoisovalerate dehydrogenase-like enzyme complex,
active site, suggesting that the TKC-like domain of SvPPDCβ
may act as a regulatory domain associated with substrate activation,
as is the case for the R domain in AbPPDC and ScPPDC.
C
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Letters
SvPPDCα/β was affected by mutations in either SvPPDCα or
SvPPDCβ, further supporting the idea that the activity of
SvPPDCα/β requires both SvPPDCα and SvPPDCβ.
In conclusion, we identified a new type of PPDC composed of
two subunits from S. virginiae that was different from previously
4
,11,13
reported PPDCs, which are encoded by a single gene,
giving new insights into the PPDC family. Another reported
ThDP-dependent decarboxylase, sulfopyruvate decarboxylase,
also has two subunits corresponding to the PYR and PP
25
domains. It catalyzes the decarboxylation of sulfopyruvate to
sulfoacetaldehyde and CO . Coexpression can promote cofold-
2
ing of protein partners, which is an effective way of expressing
2
2
proteins composed of multisubunits. In this study, we
confirmed that SvPPDCα and SvPPDCβ interacted and had
synergistic functions for soluble expression and catalysis. These
two proteins were identified from S. virginiae, a strain that
21,26
produces antibiotic virginiamycin.
Up to now, enzymes
involved in the virginiamycin biosynthesis pathway have not
been identified biochemically. The svppdcα/β genes are located
adjacent to the visG gene, which is indicated to be involved in
the biosynthesis of virginiamycin S, possibly to provide an
21
L-phenylglycine molecule. Phenylpyruvate is an important
precursor for L-phenylglycine biosynthesis. Therefore, the spe-
cific activity of SvPPDCα/β for phenylpyruvate may relate to the
physiological function in virginiamycin biosynthesis, which
should be investigated in future work.
METHODS
■
Plasmids, Cloning, and Reagents. We cloned Svppdcα and
Svppdcβ genes from the S. virginiae (ATCC19817) genome and
submitted the sequences to the GenBank database (accession number:
MF179145 and MF179146). The gene encoding ScPPDC
Figure 3. Kinetics and illustration of the functional domains of
SvPPDCα/β. (a) Dependence of the reaction rate on phenylpyruvate
concentrations for SvPPDCα/β. The solid line is a representative fitted
curve of four independent replicates. (b) Kinetic parameters of
SvPPDCα/β. Values represent the means ± standard deviation of four
independent replicates. (c) Putative domains of AbPPDC, ScPPDC,
SvPPDCα, and SvPPDCβ.
(
NP_010668.3) was cloned from the S.cerevisiae genome. The frag-
ments were subcloned into pET28a for protein expression, respectively.
For coexpression of SvPPDCα and SvPPDCβ, fragments were
subcloned into pETDuet-1, generating pETDuet-NT-SvPPDCα/β, in
which each gene was expressed as N-terminal fusions with a 6 × His-tag.
Then, the plasmid was used for whole plasmid PCR to generate
pETDuet-SNT-SvPPDCα/β, which removed the His-tag from
SvPPDCβ. The primers and plasmids used in this study are listed in
Table S1. Reagents used are described in the Supporting Information.
Protein Expression and Preparation of E. coli Crude Lysate.
Detailed procedures are described in the Supporting Information.
Pulldown Assay. The crude lysate expressing His-tagged SvPPDCα
and non-His-tagged SvPPDCβ with pETDuet-SNT-SvPPDCα/β was
prepared as described in the Supporting Information. The interactions
The catalytic collaboration of SvPPDCα and SvPPDCβ was
further validated by mutating functional residues in either of the
two subunits. When the amino acid sequences of the PYR and PP
domains from SvPPDC, AbPPDC, and ScPPDC were aligned
(
Figure 4a and b), we found that although they shared very low
sequence identities, the ThDP binding motif (GDGX24−27NN)
was conserved in the three PP domains. In addition, there was a
conserved glutamate (Glu47) in the PYR domain. Glu47 of
SvPPDCβ matched Glu48 of AbPPDC, which was proposed to
be a key residue in promoting the protonation and deprotona-
27
of them were verified by pulldown assay, as reported previously.
Briefly, 1 mL of the proteins from the supernatant was incubated with
0 μL of prewashed Ni-NTA agarose (GE Healthcare) for 1 h at 4 °C
4
5
tion process of the catalytic reaction. These conserved residues,
with gentle shaking. The protein bound agarose was then washed once
with buffer B (50 mM Tris-HCl, 1 M NaCl, 2 mM DTT, pH 8.0) and
three times with buffer A (50 mM Tris-HCl, 200 mM NaCl, 2 mM DTT,
pH 8.0) to remove unbound proteins. Bound proteins were eluted by
boiling in 1× SDS loading buffer and separated by SDS-PAGE.
Gly167, Asp168, and Gly169 of SvPPDCα and Glu47 of
SvPPDCβ, were chosen for mutagenesis.
Three vectors were constructed to coexpress the following
G167A
/β, SvPPDCαG167AD168AG169A/
mutant complexes: SvPPDCα
E47A
β, and SvPPDCα/β . The purified complexes were assessed by
SDS-PAGE analyses (Figure 4c), which indicated that the
mutations had no effect on the solubility and stability of both
subunits. Mutations of the conserved residues involved in
cofactor binding may result in low catalytic efficiencies. We there-
fore measured the specific activities of the mutant complexes
with the wild-type enzyme as a control. The catalytic activities of
Protein Purification. Recombinant SvPPDCα and SvPPDCβ were
co-overexpressed each with N-terminal His-tag fusions, in E. coli BL21
(DE3) transformed with pETDuet-NT-SvPPDCα/β. Protein purifica-
tion was conducted with a rapid Ni-NTA agarose minicolumn as
2
8
previously described.
Enzyme Activity Assay. The enzyme assays were performed as
2
9
described previously, with some modifications. Briefly, enzyme activity
was measured in assays (100 μL) containing 50 mM Tris-HCl (pH 7.5),
G167A
G167AD168AG169A
SvPPDCα
/β and SvPPDCα
/β were not
5
mM phenylpyruvate (or pyruvate), 0.5 mM ThDP, 5 mM MgCl , and
2
detectable, indicating the indispensable roles of the conserved
appropriately diluted purified PPDC (40 μL). The reaction was initiated
by the addition of the enzyme and quenched by the addition of 50 μL of
acetonitrile after setting intervals. A total of 30 μg SvPPDCα/β was added
to give a linear time course at 10 min intervals for 100 min of the assay.
ThDP binding motif (Figure 4d). The catalytic activity of
E47A
SvPPDCα/β
was decreased compared with the wild-type
(Figure 4d). These data indicated that the catalytic activity of
D
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Figure 4. Conserved functional amino acids in SvPPDCα/β. (a, b) Alignment of the conserved PP (a) and PYR domains (b) of SvPPDC, ScPPDC, and
AbPPDC. Asterisks indicate the conserved residues for ThDP-binding and catalysis. Pink arrows indicate the chosen mutation sites of SvPPDC. (c)
E47A
G167A
SDS-PAGE analyses of purified wild type and mutant SvPPDCα/β. (d) Specific activities of SvPPDCα/β, SvPPDCα/β , SvPPDCα
SvPPDCα
Asterisks indicate statistically significant differences (P < 0.001) as determined by Student’s t test.
/β, and
G167AD168AG169A
/β with a phenylpyruvate concentration of 5 mM. Values represent the means ± standard deviation of triplicated experiments.
The generated products were quantitated by HPLC. Enzyme activity
was defined as the amount of enzyme necessary to produce 1 μmol of
phenylacetaldehyde (or acetaldehyde) per minute at 30 °C, at pH 7.5.
The Km and kcat values were determined by varying the phenyl-
pyruvate concentration. Kinetic data were acquired using Graphpad
Prism 5.0 software. Protein concentration was calculated using the
Bradford assay (Sangong Corp., Shanghai, China) with bovine serum
albumin as the standard.
HPLC Analysis. The quantitative analyses of phenylacetaldehyde (or
29
acetaldehyde) were performed as reported previously. The elution was
analyzed using a UV detector at 213 nm or a refractive index detector.
The peak corresponding to phenylacetaldehyde (or acetaldehyde) was
confirmed by alignment with standard samples.
Qualitative Detection by GC-MS. The phenylacetaldehyde
produced by the decarboxylation of phenylpyruvate was identified by
GC-MS. Products of the enzyme reaction were extracted by isooctane.
E
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Letters
A standard phenylacetaldehyde aqueous solution was processed in
an identical manner to create the identity of the generated compound.
The chromatograms and mass spectra of the samples were compared
with standards and the National Institute of Standards and Technology
(7) Schneider, S., Mohamed, M. E., and Fuchs, G. (1997) Anaerobic
metabolism of L-phenylalanine via benzoyl-CoA in the denitrifying
bacterium Thauera aromatica. Arch. Microbiol. 168, 310−320.
(8) Guo, Z. W., Goswami, A., Mirfakhrae, K. D., and Patel, R. N. (1999)
Asymmetric acyloin condensation catalyzed by phenylpyruvate
decarboxylase. Tetrahedron: Asymmetry 10, 4667−4675.
(NIST) libraries to confirm the production of phenylacetaldehyde.
Detailed methodsofGC-MS are described inthe SupportingInformation.
Computational Sequence Analyses and Mutagenesis Assays.
The conserved domains were analyzed using the Conserved Domain
(9) Asakawa, T., Wada, H., and Yamano, T. (1968) Enzymatic
conversion of phenylpyruvate to phenylacetate. Biochim. Biophys. Acta,
Gen. Subj. 170, 375−391.
30
Database (https://www.ncbi.nlm.nih.gov/cdd/) version 3.15. The
sequences of the conserved domains were aligned using CLUSTAL W.
The chosen conserved residues were mutated to alanine by site-directed
mutagenesis of the plasmid pETDuet-NT-SvPPDCα/β. The mutations
were generated by whole-plasmid PCR with primers containing an
alanine codon at the target sites. The resulting mutant complexes of
SvPPDCα/β were expressed and purified, followed by determination of
phenylacetaldehyde production as described.
(10) Spaepen, S., Versees, W., Gocke, D., Pohl, M., Steyaert, J., and
Vanderleyden, J. (2007) Characterization of phenylpyruvate decarbox-
ylase, involved in auxin production of Azospirillum brasilense. J. Bacteriol.
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89, 7626−7633.
11) Versees, W., Spaepen, S., Wood, M. D. H., Leeper, F. J.,
Vanderleyden, J., and Steyaert, J. (2007) Molecular mechanism of
allosteric substrate activation in a thiamine diphosphate-dependent
decarboxylase. J. Biol. Chem. 282, 35269−35278.
(12) Vuralhan, Z., Morais, M. A., Tai, S. L., Piper, M. D. W., and Pronk,
J. T. (2003) Identification and characterization of phenylpyruvate
decarboxylase genes in Saccharomyces cerevisiae. Appl. Environ. Microbiol.
ASSOCIATED CONTENT
Supporting Information
■
*
S
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acschembio.7b00307.
6
(
9, 4534−4541.
13) Kneen, M. M., Stan, R., Yep, A., Tyler, R. P., Saehuan, C., and
Supporting methods, Table S1, and Figure S1 (PDF)
McLeish, M. J. (2011) Characterization of a thiamin diphosphate-
dependent phenylpyruvate decarboxylase from Saccharomyces cerevisiae.
FEBS J. 278, 1842−1853.
AUTHOR INFORMATION
Corresponding Author
Address: Institute of Plant Physiology and Ecology, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences,
■
*
(14) de Souza Liberal, A. T., Carazzolle, M. F., Pereira, G. A., Simoes,
D. A., and de Morais, M. A. (2012) The yeast Dekkera bruxellensis
genome contains two orthologs of the ARO10 gene encoding for
phenylpyruvate decarboxylase. World J. Microbiol. Biotechnol. 28, 2473−
2478.
3
5
00 Fenglin Road, Shanghai 200032, China. Phone: +86 21
4924173. Fax: +86 21 54924015. E-mail: syang@sibs.ac.cn.
(15) Duggleby, R. G. (2006) Domain relationships in thiamine
diphosphate-dependent enzymes. Acc. Chem. Res. 39, 550−557.
ORCID
(16) Candy, J. M., and Duggleby, R. G. (1998) Structure and properties
Sheng Yang: 0000-0003-3742-9989
Notes
The authors declare no competing financial interest.
of pyruvate decarboxylase and site-directed mutagenesis of the
Zymomonas mobilis enzyme. Biochim. Biophys. Acta, Protein Struct. Mol.
Enzymol. 1385, 323−338.
(17) Costelloe, S. J., Ward, J. M., and Dalby, P. A. (2008) Evolutionary
analysis of the TPP-dependent enzyme family. J. Mol. Evol. 66, 36−49.
ACKNOWLEDGMENTS
■
(18) Muller, Y. A., Lindqvist, Y., Furey, W., Schulz, G. E., Jordan, F., and
This work was supported by the National Basic Research
Program of China (973:2014CB745101), the National Natural
Science Foundation of China (31170102), Shanghai scientific
research project (14XD1424900), and Derivative Bank of
Chinese Biological Resources (ZSYS-014). We thank W. Hu
for technical support for mass spectral analysis.
Schneider, G. (1993) A thiamin diphosphate binding fold revealed by
comparison of the crystal structures of transketolase, pyruvate oxidase
and pyruvate decarboxylase. Structure 1, 95−103.
(19) Krieger, F., Spinka, M., Golbik, R., Hubner, G., and Konig, S.
(2002) Pyruvate decarboxylase from Kluyveromyces lactis. An enzyme
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20) Sergienko, E. A., and Jordan, F. (2002) New model for activation
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