Download Cloning and characterization of CmGPD1, the Candida magnoliae

RESEARCH ARTICLE
Cloning and characterization of CmGPD1, the Candida magnoliae
homologue of glycerol-3-phosphate dehydrogenase
Dae-Hee Lee1,2, Myoung-Dong Kim3, Yeon-Woo Ryu4 & Jin-Ho Seo1
1
Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea; 2Research Institute for Agriculture and Life Sciences, Seoul National
University, Seoul, Korea; 3School of Bioscience and Biotechnology and Institute of Bioscience and Biotechnology, Kangwon National University,
Chuncheon, Korea; and 4Department of Molecular Science and Technology, Ajou University, Suwon, Korea
Correspondence: Jin-Ho Seo, Department of
Agricultural Biotechnology, Seoul National
University, Seoul 151-921, Korea. Tel.: 182 2
880 4855; fax: 182 2 873 5095; e-mail:
[email protected]
Present address: Dae-Hee Lee, Department
of Bioengineering, University of California,
San Diego, La Jolla, CA 92093, USA.
Received 10 January 2008; revised 25 August
2008; accepted 26 August 2008.
First published online 2 October 2008.
DOI:10.1111/j.1567-1364.2008.00446.x
Editor: Hyun Kang
Abstract
Glycerol-3-phosphate dehydrogenase (GPDH) plays a central role in glycerol
metabolism. A genomic CmGPD1 gene encoding NADH-dependent GPDH was
isolated from Candida magnoliae producing a significant amount of glycerol. The
gene encodes a polypeptide of 360 amino acids, which shows high homology with
known NADH-dependent GPDHs of other species. The CmGPD1 gene was
expressed in recombinant Escherichia coli with the maltose-binding protein
(MBP) fusion system and purified to homogeneity using simple affinity chromatography. The purified CmGpd1p without the MBP fusion displayed an apparent
molecular mass of 40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The CmGpd1p enzyme exhibited a Kcat/Km value of 195
min1 mM1 for dihydroxyacetone phosphate whereas Kcat/Km for glycerol3-phosphate is 0.385 min1 mM1. In a complementation study, CmGpd1p
rescued the ability of glycerol synthesis and salt tolerance in a Saccharomyces
cerevisiae GPD1DGPD2D mutant strain. The overall results indicated that
CmGPD1 encodes a functional homologue of S. cerevisiae GPDH.
Keywords
Candida magnoliae ; glycerol-3-phosphate
dehydrogenase; glycerol; complementation
study; salt tolerance.
Introduction
Glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the
reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G-3-P), which is subsequently dephosphorylated to glycerol by the action of a glycerol
phosphatase (GPP). Glycerol is a key metabolic intermediate
in the carbon flow between glycolytic catabolism and the
synthesis of fatty acids in prokaryotes and eukaryotes
(Rognstad et al., 1974). Glycerol metabolism is important
in biotechnology for ethanol production or wine smoothness (Remize et al., 2003). Hence, the metabolic pathways
for glycerol biosynthesis as well as its mechanism for
intracellular accumulation have attracted attention. In Saccharomyces cerevisiae, this three-carbon polyol plays a major
role in the physiological processes including biosynthesis of
phospholipid and triacylglycerol, redox balance (Ansell
et al., 1997) and osmoadaptation (Hohmann, 2002). It is
c 2008 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd.
No claim to original Korean government works
well known that polyols are crucial to the osmoregulation of
yeast as compatible solutes. When yeast cells are exposed to
hyperosmotic stress, they accumulate one or more protective solutes such as glycerol, D-arabitol and mannitol. These
polyols prevent the rapid diffusion of water from the cell
into the surrounding medium to compensate for the loss of
turgor pressure (Brown, 1978; Yancey et al., 1982). Among
them, glycerol is the most prominent compatible solute in
S. cerevisiae as in many other types of yeast. Two isogenes
(GPD1 and GPD2) encoding different GPDH enzymes
involved in the first step of glycerol production were
identified in S. cerevisiae (Larsson et al., 1993; Eriksson
et al., 1995). The expression of the GPD1 gene is induced by
osmotic stress, whereas the GPD2 gene is expressed under
anaerobic conditions (Eriksson et al., 1995). The expression
of the GPD1 gene is also partly regulated by the high
osmolarity glycerol pathway (Albertyn et al., 1994). Heterologous expression of the GPD genes in yeast increased
FEMS Yeast Res 8 (2008) 1324–1333
1325
Molecular cloning and characterization of CmGPD1
glycerol production (Watanabe et al., 2004), suggesting that
the production of glycerol is mainly dependent on the
activity of GPDH enzyme (Nevoigt & Stahl, 1996). However,
the regulatory mechanism of intracellular glycerol synthesis
has not been well clarified in other yeasts such as Candida
magnoliae.
The osmotolerant yeast C. magnoliae isolated from honeycomb is known as an erythritol producer (Kim et al., 1996).
Candida magnoliae is able to grow in the presence of up to
50% (w/v) sugars and produces erythritol, mannitol and
glycerol as compatible solutes in response to high sugar
concentrations (Yu et al., 2006). When C. magnoliae was
grown in high concentrations of fructose, it produced a
significant amount of glycerol that was almost the same
amount of erythritol (Yu et al., 2006). Similarly, intracellular
glycerol accumulation is also critical for C. magnoliae to
maintain osmolarity like S. cerevisiae. As mentioned above,
GPDH plays a key role in glycerol metabolism. Consequently, cloning and characterization of GPDH enzyme is
an important step in the study of the mechanisms regulating
glycerol biosynthesis in C. magnoliae. The present study
describes the cloning and sequence analysis of the GPD1
gene of C. magnoliae. The functionality of the gene was
demonstrated by its heterologous expression in the S.
cerevisiae mutant lacking the ability of glycerol synthesis
and was supported by its homology with other eukaryotic
GPDHs and enzymatic properties.
Materials and methods
Strains, plasmids and culture conditions
Candida magnoliae JH110 (KFCC deposit number: 10 900)
was used for the preparation of genomic DNA. All PCR
products intended for sequence analysis were cloned into
the pGEM-T Easy vector (Promega) to facilitate DNA
sequencing. Escherichia coli DH5a and BL21 cells were used
for plasmid preparation and expression host, respectively.
Bacterial cells were grown at 37 1C in Luria–Bertani (LB)
medium (0.5% yeast extract, 1% tryptone and 1% NaCl)
supplemented with 100 mg mL1 ampicillin. The vector
pMAL-TEV derived from plasmid pMAL-c2X (New England Biolabs) was used for bacterial expression of CmGPD1
as a maltose-binding protein (MBP)-tagged fusion protein
to the N-terminus. Plasmid pMAL-TEV was constructed by
replacing the cleavage site of Factor Xa protease with the
cleavage sequence specific for tobacco etch virus (TEV)
protease. Saccharomyces cerevisiae YSH6-142-3D (MATa
GPD1D<TRP1 GPD2D<URA3, derived from a strain
W303-1A) was kindly donated by Professor Lennart Adler
(Gothenburg University, Germany) (Ansell et al., 1997),
which is unable to synthesize glycerol. This deletion mutant
was used for a complementation study of CmGPD1 as
FEMS Yeast Res 8 (2008) 1324–1333
described before (Thome, 2004) and grown routinely on
YPD medium (1% yeast extract, 2% Bacto peptone and 2%
glucose) or complete minimal medium [0.67% yeast nitrogen base (YNB) with 2% glucose] lacking the appropriate
requirements for selection at 30 1C. When required, 2% agar
was added to the media. Osmotic sensitivity was assessed by
preparation of YPD drop-plates spotted in 20-fold serial
dilutions of the mid-log phase cultures. Plates were adjusted
with 0, 0.8 and 1.2 M NaCl and incubated at 30 1C for 4 days.
DNA isolation and sequencing
Yeast genomic DNA was isolated with the DNeasy Blood &
Tissue Kit (Qiagen), but cell lysis was performed by incubation at 30 1C for 90 min with zymolase (Sigma). Plasmid
DNA was isolated using the AccuPrep Plasmid Mini Extraction Kit (Bioneer). All DNA sequences were determined at
the National Instrumentation Center for Environmental
Management (Korea).
Isolation of CmGPD1
The schematic isolation steps of the genomic CmGPD1 gene
are described in Fig. 1. The cDNA library was constructed as
part of a C. magnoliae expressed sequenced tag (EST)
sequencing project that contributed to comprehensive characterization of gene expression when C. magnoliae was
exposed to the external osmotic stresses (unpublished data).
The cDNA clone containing the putative CmGPD1 sequence
lacking the 5 0 -upstream region was 990 bp long when it was
isolated by random sequencing of clones from the cDNA
library of C. magnoliae. The deduced amino acid sequence
of the partial CmGPD1 cDNA, was highly homologous to
those of the previously reported NADH-dependent GPDH
Fig. 1. Schematic isolation steps of CmGPD1 from Candida magnoliae
EST library. The cDNA library was constructed as part of a C. magnoliae
EST sequencing project. The unknown genomic DNA sequence in the 5 0 upstream region of the partial genomic CmGPD1 gene was identified by
genome walking.
c 2008 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd.
No claim to original Korean government works
1326
enzymes of other yeasts. Two primers GPD1 (5 0 -GCACTG
CCGTCGCGAAGCTCG-3 0 ) and GPD2 (5 0 -CCTCAACG
GCGAGGCCGTTCT-3 0 ) were designed based on the
sequence of the partial CmGPD1 cDNA and PCR was
performed using C. magnoliae genomic DNA as a template.
The PCR product of c. 990 bp was amplified, purified by gel
extraction, cloned into the pGEM-T vector and sequenced.
The PCR product (partial genomic CmGPD1 fragment) has
exactly the same sequence of the partial CmGPD1 EST.
Thereafter, the subsequent experiments were carried out
with the partial genomic CmGPD1 sequence. The unknown
genomic DNA sequence in the 5 0 -upstream region of the
partial genomic CmGPD1 gene was identified by genome
walking, performed by following the manufacturer’s protocols of the DNA Walking SpeedUp Kit (Seegene). For the
upstream sequences, two PCR amplifications – a primary
amplification, followed by a nested PCR – were carried out.
The complete nucleotide sequences of genomic CmGPD1
were obtained after the 5 0 -flanking regions of the partial
genomic CmGPD1 gene were cloned, sequenced and
assembled.
Sequence analysis
Searches for nucleotide and protein sequence similarities
were conducted using the BLAST algorithm at the National
Center for Biotechnology Information (NCBI, http://
www.ncbi.nlm.nih.gov/blast). The deduced amino acid sequences were obtained using the web-based translation tool
of the Expert Protein Analysis System (ExPASy, http://kr.
expasy.org/tools/dna.html). Multiple sequence alignment of
the deduced amino acid sequence of CmGPD1 was performed with the corresponding sequences from various
organisms using the GeneDoc (Nicholas et al., 1997). Based
on this alignment, a phylogenetic tree was constructed with
MEGA 3.1 software (Kumar et al., 2004) using the neighborjoining method (Saitou & Nei, 1987). Boot-strap analysis
(Felsenstein, 2001) was used with 1000 replicates to test the
relative support for the branches produced by the neighborjoining analysis. All the analyzed sequences of GPDH
enzymes were retrieved from GenBank (http://www.ncbi.
nlm.nih.gov/Genbank/index.html) database.
D.-H. Lee et al.
(5 0 -CTATTTACAAGCTATCCTCGAGC-3 0 ) was used as a
probe and labeled by random priming in accordance with
the manufacturer’s manuals (DIG Labeling, Roche Applied
Science). Hybridizations were allowed to proceed as instructed by the supplier at 42 1C overnight using the DIG
Easy Hyb (Roche Applied Science). Posthybridization
washes were performed at room temperature and development of the blots was according to the manufacturer’s
protocols.
Expression of recombinant CmGPD1 in E. coli
For expression of the CmGPD1 gene in E. coli, a primer pair,
N- and C-termini-specific GPD5 (5 0 -GGACTAGTATGAGT
TACGCTAAGAAGTTCAAG-3 0 , SpeI site is underlined) and
GPD6 (5 0 -CCCAAGCTTCTATTTACAAGCTATCCTCGA
GC-3 0 , HindIII site is underlined) was designed based on
the full-length genomic CmGPD1 sequence and used in PCR
to amplify the CmGPD1 ORF from C. magnoliae genomic
DNA. The amplified DNA fragment was digested with SpeI/
HindIII and cloned into a SpeI/HindIII-treated pMAL-TEV
vector. The resulting plasmid, pGPDMBP, was transformed
into E. coli BL21 cells for expression of the fusion protein.
Escherichia coli BL21 cells harboring the expression vector
were cultured in LB media containing 100 mg mL1 ampicillin at 30 1C with vigorous shaking until the OD600 nm
reached 0.7. Protein expression was induced by adding
isopropyl-1-thio-b-D-galactopyranoside (IPTG) to the final
concentration of 0.05 mM and growth continued for 6 h.
The cells were then harvested by centrifugation at 6000 g for
20 min at 4 1C and resuspended in 50 mM sodium phosphate buffer, pH 6.0, containing the protease inhibitor
cocktail (Sigma) for disruption by sonication. The crude
extract was fractionated into soluble and insoluble fractions
by centrifugation at 20 000 g for 30 min at 4 1C. These
fractions were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to
Laemmli (1970) with 12% polyacrylamide gel. The gels were
visualized by staining using Coomassie brilliant blue R-250.
The soluble fraction was used in the subsequent purification
of CmGpd1p.
Purification of the CmGpd1p enzyme
Southern blot analysis
For Southern hybridizations, a nonradioactive labeling and
detection system were used (Roche Applied Science). Total
C. magnoliae genomic DNA was isolated and digested with
different restriction enzymes. The products were separated
on 0.8% (w/v) agarose gels and transferred to a positively
charged nylon membrane (Roche Applied Science) following the standard methods (Sambrook et al., 1989). A
CmGPD1 fragment amplified by PCR using primers GPD3
(5 0 -ATGAGTTACGCTAAGAAGTTCAAG-3 0 ) and GPD4
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Journal compilation Published by Blackwell Publishing Ltd.
No claim to original Korean government works
The soluble cell fraction was directly applied to a column
manually packed with Amylose Resin High Flow (New
England Biolabs). The column was washed with washing
buffer [20 mM Tris-HCl buffer, pH 7.4, 200 mM NaCl and
10% (w/v) glycerol] containing 1 mM EDTA. The bound
protein was eluted using 10 mM maltose in washing buffer.
The peak fractions showing the enzyme activity were
pooled, concentrated and dialyzed against 20 mM Tris-HCl
buffer, pH 7.4, containing 10 mM Mg21 and 100 mM NaCl
at 4 1C. In order to remove MBP from the purified fusion
FEMS Yeast Res 8 (2008) 1324–1333
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Molecular cloning and characterization of CmGPD1
protein, the eluate was subjected to the MBP cleavage
reaction with AcTEV protease (Invitrogen) at 30 1C for 6 h.
MBP was removed from the reaction mixture by rebinding
MBP to an amylose-coupled resin. AcTEV protease contains
a polyhistidine tag at the N-terminus. After removal of MBP,
AcTEV protease was eliminated using affinity chromatography on a nickel-chelating resin.
CmGpd1p enzyme assay
GPDH activity was measured as described by Gancedo et al.
(1968) with some modifications. The activity for DHAP
reduction was measured at 340 nm and 30 1C using a spectrophotometer (UltroSpec 4000, GE Healthcare Bio-Sciences).
The assay mixture was 1 mL of a solution containing
13.4 mM DHAP, 0.2 mM NADH, 20 mM triethanolamine
buffer, pH 8.0, 1 mM b-mercaptoethanol and purified enzyme sufficient to produce changes in absorbance. Assays in
the direction of G-3-P oxidation were performed in a mixture
containing 0.67 mM G-3-P and 2.5 mM NAD1. Absorbance
changes were monitored for 5 min after addition of the
desired coenzyme. All assays were carried out in duplicate or
in triplicate. One unit of enzyme activity was defined as
the amount of enzyme that produces 1 mmol of NADH
(or NAD1) per min under the assay conditions. Specific
activity was calculated as a unit per milligram of protein
(U mg1 protein). Protein content was determined using a
protein assay kit (Bio-Rad) with bovine serum albumin as
the standard.
Steady-state kinetics
The kinetic parameters of purified CmGpd1p were determined by measurement of the initial rates using the GPDH
assay. The enzyme reaction for DHAP reduction was performed at optimal pH and temperature by varying concentrations of one substrate (DHAP or NADH) while the other
was maintained constant. The assay of G-3-P oxidation was
carried out under same conditions except for varying the
concentrations of a substrate (G-3-P or NAD1). The kinetic
parameters were calculated by plotting the initial rates
against substrate concentrations to fit the Michaelis–Menten
equation.
letters indicate the restriction enzyme sites, BamHI and
HindIII, respectively, for recombination into pRS425. The
resulting plasmid was named as pCmGPD1. Transformation
of S. cerevisiae GPD1DGPD2D was performed using the
Alkali Cation Yeast Transformation kit (BIO 101) according
to the manufacturer’s protocol. The transformants were
cultivated in Leu-dropout medium [0.67% (w/v) YNB
without amino acids with addition of 0.16% (w/v) yeast
synthetic dropout medium supplement without leucine]
containing 2% (w/v) glucose supplemented with or without
0.67 M NaCl at 30 1C with shaking.
Measurement of glycerol content
Extracellular and intracellular glycerol content was measured as described previously (Watanabe et al., 2004).
Samples were taken from cultures grown in complete
minimal media with or without 0.67 M NaCl and centrifuged at 8000 g for 5 min. The supernatant was collected
and filtered for determination of extracellular glycerol
content. The cell pellet was washed twice with the
previous culture medium. Cells were resuspended in
2 mL distilled cold water and boiled for 10 min. After
centrifugation at 10 000 g for 10 min, the supernatant
was used to measure the intracellular glycerol content.
Glycerol content was determined from filtered supernatants
using a HPLC (Agilent 1100 series system, Agilent Technologies). Samples were injected on an Aminex HPX-87 H
column (Bio-Rad) connected to a cation-H guard column
(Bio-Rad) at 65 1C. Sugars were eluted with 5 mM sulfuric
acid at a flow rate of 0.6 mL min1 for 25 min. Detection was
carried out using a differential refractive index detector
at 35 1C and the compounds were compared with the
standards.
Nucleotide sequence accession number
The nucleotide sequence of genomic the CmGPD1 gene has
been submitted to the GenBank database under accession
number DQ294292.
Results and discussion
Isolation of CmGPD1 and sequence analysis
Functional complementation in yeast
Plasmid pRS425 containing the yeast GAPDH promoter and
the CYC1 terminator (Mumberg et al., 1995) was used as an
expression vector in the GPD gene deletion mutant yeast
(GPD1DGPD2D). The CmGPD1 ORF was prepared using
PCR with the GPD7 primer (5 0 -TTGCGCGGATCCAT
GAGTTACGCTAAGAAGTTCAAGG-3 0 ), GPD8 primer
(5 0 -CCCAAGCTTTTACAAGCTATCCTCGAGCAGG-3 0 ) and
the genomic DNA of C. magnoliae as a template. Underlined
FEMS Yeast Res 8 (2008) 1324–1333
A 1602-bp CmGPD1 with 5 0 - and 3 0 -untranslated regions
was obtained from C. magnoliae genomic DNA using
SeeGene DNA walking (Hwang et al., 2003). The fullsequenced DNA contained an ORF of 1083 bp with an ATG
initiation codon and a TAG termination codon. This gene
encoded a polypeptide of 360 amino acid residues with a
predicted molecular mass of 39.3 kDa and an isoelectric
point of 5.59. The deduced amino acid sequence of the ORF
was used for a similarity search with published GPDHs of
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Journal compilation Published by Blackwell Publishing Ltd.
No claim to original Korean government works
1328
D.-H. Lee et al.
Fig. 2. Multiple alignment of the deduced amino acid sequence for the GPD gene from Candida magnoliae with other GPDHs. GPDHs are identified by
their GenBank accession numbers: Zygosaccharomyces rouxii (Q9HGY2); Saccharomyces cerevisiae (NP_010262); Candida albicans (XP_715512);
Candida tropicalis (Q4PNS1); and Debaryomyces hansenii (AAF33211). The putative NADH-binding domain is indicated by a box (GXGXXG). Grayshaded amino acids are conserved in at least four or five of the six Gpd1ps shown. Black-shaded amino acids are conserved in all sequences.
other yeasts. The results are summarized in Fig. 2. The
CmGpd1p exhibited the highest identity to recently isolated
Gpd1p of Zygosaccharomyces rouxii (Q9HGY2, 58% identity) followed by that of S. cerevisiae (NP_010262, 57%
identity). When compared with the Gpd1ps of Candida
albicans (XP_715512), Candida tropicalis (Q4PNS1) and
Debaryomyces hansenii (AAF33211), the percentage identities of the CmGpd1p were 54%, 55% and 55%, respectively.
There was, however, no similarity to NAD(P)H-dependent
GPDHs that provide phospholipid backbones for bacteria.
The NADH-dependent dehydrogenase consists of two functional domains: a coenzyme-binding domain in the Nterminal half and a catalytic domain (Otto et al., 1980).
The NADH-binding sites of dehydrogenases have a highly
conserved Gly–X–Gly–X–X–Gly sequence, where X is any
amino acid (Wierenga et al., 1986; Nagy et al., 2000). In
contrast, some NADPH-binding sites have an alanine at the
c 2008 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd.
No claim to original Korean government works
position corresponding to the third glycine residue of the
conserved trio (Scrutton et al., 1990). The consensus sequence Gly–Ser–Gly–Asn–Trp–Gly (GSGNWG) in the deduced amino acid sequence of the CmGPD1 gene was
identified at position 13–18 (Fig. 2). These six amino acids
forming this motif have also been reported to remain
conserved from yeast to humans (Ohmiya et al., 1995).
Based on sequence alignment, the relative positions of the
conserved sequences are the same in the Gpd1p families,
suggesting a similar NADH-binding domain structure.
Therefore, the gene product of C. magnoliae was classified
as Gpd1p with a closer structural relationship with the
Gpd1p family. To investigate the structural specificity of
CmGpd1p within the Gpd1p family, a phylogenetic tree was
constructed based on the full-length amino acid sequences
of Gpd1ps from various organisms using the neighborjoining method (Saitou & Nei, 1987) (Fig. 3). The
FEMS Yeast Res 8 (2008) 1324–1333
1329
Molecular cloning and characterization of CmGPD1
C. albicans (XP 715512)
D. hansenii (AAF33211)
C. tropicalis (Q4PNS1)
71
Z. rouxii (Q9HGY2)
A. fumigatus (XP 749965)
98
S. pombe (Q09845)
92
C. magnoliae (DQ294292)
T. tengcongensis (NP 623215)
95
E. coli (YP 671582)
B. longum (NP 695549)
100
S. coelicolor (NP 629694)
85
95
C. diphtheriae (CAE49650)
B. licheniformis (YP 078199)
Homo sapiens (AAB50200)
100
100
A. thaliana (NP 187648)
CmGpd1p alone forms a family in the phylogenetic tree in
the yeast Gpd1p clade separate from other known Gpd1p
clades. This result indicates that C. magnoliae has evolved
differently from the other family members.
100
85
80
Southern blot analysis
There are at least two isogenes coding for Gpds in
S. cerevisiae, only one of which is osmosensitive (Ohmiya
et al., 1995). In contrast, in Drosophila melanogaster a single
gene is translated into different isoenzymes, whose expression is related to development (Bewley et al., 1989). To
determine the genetic arrangement of the GPD gene in the
genome of C. magnoliae, Southern hybridizations performed using genomic DNA restricted with BamHI, EcoRI,
HindIII and SalI showed a single fragment with homology
to the entire CmGPD1 ORF-derived probe (Fig. 4a). This
0.1
Fig. 3. A phylogenetic tree of CmGpd1p. This phylogenetic tree was
made based on the full-length deduced amino acid sequences of GPD
genes using the neighbor-joining method. The branch length indicates
the evolutionary distance between family members. Gpd1ps are identified by their GenBank accession number.
(a)
kbp
M
1
2
3
4
8576
(b)
kDa
7427
200
150
6106
120
4899
M
1
2
3
4
5
100
85
3639
2799
70
MBP-CmGpd1p
1953
1882
60
1515
50
1482
1164
40
CmGpd1p
992
718
Sacl Bgll
30
Scal Bgll Sphl
Bgll Sacl
xhol
Sphl
CmGPD1 ORF
Probe
Fig. 4. (a) Southern gel-blot analysis of Candida magnoliae genomic DNA and the CmGPD1 restriction map. It shows a single restriction fragment of C.
magnoliae-digested DNA hybridizing to a probe of the entire CmGPD1-coding sequence. The restriction fragments detected by the probe have
approximate sizes of 7.4 kb (BamHI, lane 1), 2.3 kb (EcoRI, lane 2), 4.5 kb (HindIII, lane 3) and 2.7 kb (SalI, lane 4). Lane M indicates the DNA size marker.
The map below describes the relevant restriction sites on the CmGPD1 gene sequence. Noncoding regions are in gray and the ORF of CmGPD1 (1.08 kb)
is represented by a solid black arrow. (b) Expression and purification of recombinant MBP-CmGpd1p fusion protein. Proteins were separated using SDSPAGE. Lane M, molecular mass markers; lane 1, IPTG-induced total fraction; lane 2, soluble fraction; lane 3, insoluble fraction; lane 4, the eluate from
the amylose-coupled column; and lane 5, the purified cmGpd1p after cleavage of the MBP-CmGpd1p fusion protein by TEV protease.
FEMS Yeast Res 8 (2008) 1324–1333
c 2008 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd.
No claim to original Korean government works
1330
D.-H. Lee et al.
result suggests that GPD may exist as a single gene in
C. magnoliae, similar to the genetic arrangement of GPD
in the genome of D. hansenii. However, it is possible that in
C. magnoliae a single gene codes for two isoenzymes or that
the probe derived from one gene does not hybridize to
a second gene, as occurs in Schizosaccharomyces pombe
(Ohmiya et al., 1995). More studies are needed for a better
understanding of the genetic arrangement of the GPD gene
in C. magnoliae.
Expression and purification of CmGpd1p
In order to verify the functionality of the proposed
CmGPD1 gene product and to produce the recombinant
enzyme in high yield, the pGPDMBP vector harboring the
coding region of the CmGPD1 was constructed as described
in Materials and methods. As shown in Fig. 4b, the
expressed protein band was enriched in the soluble fraction,
which corresponds to about 75% of the expressed proteins,
the remainder being in the insoluble fraction. Generally,
expression of heterologous genes in recombinant E. coli
results in the formation of insoluble and inactive aggregates
known as inclusion bodies. It is necessary to recover the
active proteins from the inclusion bodies using an appropriate renaturation method. Fusion with MBP at the
N-terminus produced highly soluble CmGpd1p in recombinant E. coli. It is well established that MBP has the ability to
enhance the solubility of its fusion partners and facilitate
one-step purification of the fusion protein to homogeneity
(Nallamsetty & Waugh, 2006). The apparent molecular mass
of the overexpressed fusion protein was about 82 kDa, which
is in good agreement with the expected molecular mass of
CmGpd1p (39.3 kDa) incorporated with the 42.5 kDa MBP
moiety. The MBP-fused CmGpd1p was purified to homogeneity using one-step affinity chromatography on the
amylose-coupled column (Fig. 4b, lane 4). Because of the
presence of a highly specific cleavage site of TEV protease
located between the MBP tag and CmGpd1p, intact
CmGpd1p can easily be generated by incubating with TEV
protease and subsequent removal of MBP and TEV protease
by rebinding MBP or TEV protease to the amylose-coupled
column or nickel-chelating resin, respectively. The purity of
the recovered intact CmGpd1p protein was verified using
SDS-PAGE and Coomassie staining, which showed a single
band at around 40 kDa (Fig. 4b, lane 5).
Substrate specificity and kinetic analysis
Table 1 summarizes the kinetic parameters for different
substrates. No activity was observed for glycerol, glycerol-1phosphate, glycerol-2-phosphate or glyceraldehyde phosphate (data not shown), suggesting that the CmGpd1p has
a substrate range similar to GPDHs of S. cerevisiae and D.
hansenii. It is known that this enzyme catalyzes the NADHdependent DHAP reduction and also the NAD1-dependent
G-3-P oxidation. The NADPH-dependent DHAP reduction
and NADP1-dependent G-3-P oxidation were not detected,
indicating that the enzyme has no or very low affinity for
NADP(H) as a coenzyme in contrast to the enzyme from
Saccharomyces carlsbergensis where NADH could be replaced
by NADPH (Nader et al., 1979). However, this result is in
good accordance with the observations described previously
for Gpd1ps from baker’s yeast (Albertyn et al., 1992) and
D. hansenii (Nilsson & Adler, 1990). The CmGpd1p exhibited much higher catalytic efficiency with DHAP
(Kcat/Km = 195 min1 mM1) than with G-3-P (Kcat/Km =
0.385 min1 mM1), suggesting that under the physiological
conditions the formation of G-3-P is thermodynamically
favorable in the cell and hence CmGpd1p has been better
adapted to glycerol biosynthesis but not to the utilization of
glycerol in C. magnoliae. The Km values for NADH and
DHAP of CmGpd1p were about 85% and 36%, respectively,
lower than those reported for S. cerevisiae (Km values for
NADH and DHAP: 0.13 0.003 and 1.6 0.04 mM, respectively, Jingmin et al., 1996), indicating higher affinities of
CmGpd1p for NADH and DHAP. However, affinities of
CmGpd1p for NADH and DHAP were lower compared with
those known for GPDH of D. hansenii (Km values for NADH
and DHAP: 6.6 3 and 130 30 mM, respectively, Nilsson
& Adler, 1990). Kcat of the DHAP reduction with NADH as a
cofactor was higher than that of G-3-P oxidation with
NAD1. The lower Km observed for NADH in comparison
with Km for DHAP could be explained by the fact that the
binding of NADH induced a conformational change that
increased the affinity of the enzyme for the other substrates.
Generally, the ‘ordered Bi–Bi’ mechanism occurs in the
Table 1. Substrate specificity of GPDH from Candida magnoliae
Substrate varied
DHAP reduction
DHAP
NADH
G-3-P oxidation
G-3-P
NAD1
Fixed second substrate
Km (mM)
Kcat (min1)
Kcat/Km (min1 mM1)
0.2 mM NADH
13.4 mM DHAP
1.03 0.02
0.02 0.005
201 11
191 14
195
9550
2.5 mM NAD1
0.67 mM G-3-P
11.4 0.4
3.18 0.6
4.39 0.31
5.12 0.42
0.385
1.61
Values are means SD from three independent experiments.
c 2008 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd.
No claim to original Korean government works
FEMS Yeast Res 8 (2008) 1324–1333
1331
Molecular cloning and characterization of CmGPD1
Table 2. Total glycerol content of the parental strain and mutants transformed with pCmGPD1 or with pRS425
Intracellular glycerol content (mmol g1 DCW)
Extracellular glycerol content (g L1)
Strains
NaCl
1NaCl
NaCl
1NaCl
S. cerevisiae W303-1A
GPD1DGPD2D/pRS425
GPD1DGPD2D/pCmGPD1
24.1 2.4
12.1 1.3
19.3 1.1
148 5.2
21.7 2.4
126 4.8
1.10 0.3
0.13 0.02
1.32 0.4
3.87 0.5
ND
3.25 0.7
Reproducibility was confirmed by duplicate independent experiments. DCW, dry cell weight. ND, not detected.
YPD
YPD + 0.8 M NaCl
YPD + 1.2 M NaCl
WT (W303-1A)
GPD1∆GPD2∆/pRS425
GPD1∆GPD2∆/pCmGPD1
Fig. 5. Osmotic sensitivity of transformant cells harboring pCmGPD1. Saccharomyces cerevisiae W303-1A wild-type (WT) strain, GPD1DGPD2D
mutant cells containing the pRS425 vector and GPD1DGPD2D mutant cells harboring pCmGPD1 were serially diluted 20-fold, spotted onto YPD plates
with 0, 0.8 and 1.2 M NaCl and grown at 30 1C for 4 days.
reaction of NAD1-linked dehydrogenases, with the
coenzyme binding first (Figueroa-Soto & Valenzuela-Soto,
2000; Ozer et al., 2001).
Functional complementation in yeast mutant
To verify the physiological function of cloned CmGPD1,
complementation analysis was performed. The ORF of the
CmGPD1 gene was expressed heterologously in S. cerevisiae
YSH6-142-3D (Ansell et al., 1997). This yeast lacks an ability
to synthesize glycerol, due to disruption of two GPD genes
(GPD1DGPD2D) (Albertyn et al., 1994, Ansell et al., 1997).
The analysis of deduced amino acid sequence indicates that
these two yeasts are related species and that their basic
metabolic functions are similar (Fig. 2). This suggests that
the GPD1DGPD2D strain is suitable for analysis of the
C. magnoliae glycerol metabolism genes examined in the
present study. As described in the experimental section,
the S. cerevisiae GPD1DGPD2D mutant was transformed
with plasmid pCmGPD1. The transformant harboring the
empty vector was used as a control strain. In S. cerevisiae,
increase of the osmolarity in the growth medium induces the
production of glycerol. Therefore, all transformants were
cultivated in a growth medium with or without supplementation of 0.67 M NaCl. When cultivated in medium without
NaCl, the intracellular and extracellular glycerol concentration of the mutant yeast transformed with plasmid
pCmGPD1 were much higher than the GPD1DGPD2D
strain containing the plasmid without the CmGPD1 gene
FEMS Yeast Res 8 (2008) 1324–1333
(Table 2). Total glycerol production of the mutant harboring
pCmGPD1 was increased by supplementation of 0.67 M
NaCl in the medium, in a manner similar to that observed
for the wild-type strain (S. cerevisiae W303-1A). Glycerol
production in this mutant was slightly lower than that of
the positive control stain (S. cerevisiae W303-1A). However, the biosynthetic ability to produce glycerol in the
GPD1DGPD2D mutant was restored by heterologous expression of the CmGPD1 gene. Although S. cerevisiae possesses
two GPD genes, GPD1 is responsible for the majority of the
glycerol production during conditions of elevated osmolarity
(Ansell et al., 1997). The S. cerevisiae GPD1DGPD2D harboring the CmGPD1 gene was also examined for salt tolerance
by plating them on YPD plates with increasing NaCl
concentration. The growth patterns of transformant are very
similar to those of S. cerevisiae W303-1A wild type, which
suggests that the transformant expressing the CmGPD1 gene
restored the wild-type tolerance to NaCl (Fig. 5).
In conclusion, this study has revealed that C. magnoliae
has a CmGPD1 similar to GPD genes in other yeasts based
on their homologies of deduced amino acid sequences and
enzymatic properties. Additionally, the complementation
study indicated that heterologous expression of the
CmGPD1 gene restored the ability of glycerol synthesis and
salt tolerance in the GPD1DGPD2D mutant yeast. These
overall results indicated that CmGPD1 encodes a functional
homologue of S. cerevisiae GPDH. More investigations of
CmGPD1 regulation and its deletion for functional analysis
in C. magnoliae may yield fruitful information.
c 2008 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd.
No claim to original Korean government works
1332
Acknowledgements
We are grateful to Professor Lennart Adler for generously
providing S. cerevisiae strains W303-1A and the
GPD1DGPD2D mutant. This study was supported by Korea
Science and Engineering Foundation (R01-2004-000-10221-0).
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c 2008 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd.
No claim to original Korean government works