From the Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Received for publication, May 24, 2002, and in revised form, October 21, 2002
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ABSTRACT |
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Although the presence of an enzyme that catalyzes
Among Conversion of homoisocitrate to 2-oxoadipate is supposed to occur in
lysine biosynthesis through -decarboxylating dehydrogenation of homoisocitrate to synthesize
2-oxoadipate has been postulated in the lysine biosynthesis pathway
through
-aminoadipate (AAA), the enzyme has not yet been analyzed at all, because no gene encoding the enzyme has been identified until recently. A gene encoding a protein with a significant amino acid sequence identity to both isocitrate dehydrogenase and
3-isopropylmalate dehydrogenase was cloned from Thermus
thermophilus HB27. The gene product produced in recombinant
Escherichia coli cells demonstrated homoisocitrate
dehydrogenase (HICDH) activity. A knockout mutant of the gene showed an
AAA-auxotrophic phenotype, indicating that the gene product is involved
in lysine biosynthesis through AAA. We therefore named this gene
hicdh. HICDH, the gene product, did not catalyze the
conversion of 3-isopropylmalate to 2-oxoisocaproate, a leucine
biosynthetic reaction, but it did recognize isocitrate, a related
compound in the tricarboxylic acid cycle, as well as homoisocitrate as
a substrate. It is of interest that HICDH catalyzes the reaction with
isocitrate about 20 times more efficiently than the reaction with the
putative native substrate, homoisocitrate. The broad specificity and
possible dual function suggest that this enzyme represents a key link
in the evolution of the pathways utilizing citrate derivatives.
Site-directed mutagenesis study reveals that replacement of
Arg85 with Val in HICDH causes complete loss of activity
with isocitrate but significant activity with 3-isopropylmalate and
retains activity with homoisocitrate. These results indicate that
Arg85 is a key residue for both substrate specificity and
evolution of
-decarboxylating dehydrogenases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-decarboxylating dehydrogenases, 3-isopropylmalate
dehydrogenase (IPMDH1; EC
1.1.1.85), an enzyme catalyzing the fourth reaction in leucine
biosynthesis, and isocitrate dehydrogenase (ICDH; EC 1.1.1.42), one of
the enzymes in the tricarboxylic acid cycle, catalyze similar
-decarboxylating dehydrogenation of structurally similar compounds, 3-isopropylmalate and isocitrate, respectively (Fig. 1). These enzymes
share structural and functional similarities and are therefore thought
to have diverged from a common ancestral enzyme (1-4). Many studies
have been done to elucidate the structural and functional basis for
substrate specificity, heat stability, and evolution of this protein
family (5-9).
-aminoadipate (AAA), a pathway that has
been believed to be present only in fungi and yeast (Fig. 1) (10-13). The enzyme containing
-decarboxylating dehydrogenation activity involved in this process
is homoisocitrate dehydrogenase (HICDH; EC 1.1.1.115). The reactions
catalyzed by HICDH proceed in a manner similar to those by IPMDH and
ICDH. In addition, IPMDH and ICDH show distinct identity in amino acid
sequence to each other. Until recently, no genes have been identified
and characterized as hicdh even in Saccharomyces
cerevisiae, although its whole genome sequence has been determined
(14). Recently, Chen and Jeong (3) reported that the gene named
YIL094C of S. cerevisiae and the gene with
the accession number T38612 of Schizosaccharomyces pombe
could be likely candidates encoding HICDHs based on the amino acid
sequence alignment for this protein family and the three-dimensional
structures of IPMDH and ICDH. They further showed that the T38612
product actually had HICDH activity but did not characterize it
further. This new member of
-decarboxylating dehydrogenases is
expected to lead us not only to elucidation of the substrate
recognition but also to understanding evolution of the protein family
as well as cellular metabolism including amino acid biosynthesis.
However, unlike with ICDH and IPMDH, there is little information on
properties of HICDH.
View larger version (55K):
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Fig. 1.
Relationship of catalytic reactions of HICDH,
ICDH, and IPMDH.
Thermus thermophilus HB27, an extremely thermophilic
bacterium, optimally grows at 70 °C. We recently found that this
bacterium synthesizes lysine through AAA as an intermediate (15) like fungi and yeast, although all bacteria and plants are believed to
synthesize lysine through diaminopimelate. Sequence analysis of the
cloned genes that were shown to be involved in the lysine biosynthesis
suggested that the first half of the biosynthesis (from 2-oxoglutarate
to AAA) proceeded in the same manner as that of fungi and yeast. The
reactions were also suggested to be similar to those of leucine
biosynthesis and a part of the tricarboxylic acid cycle (16). The
second half of the biosynthesis from AAA to lysine is, on the other
hand, similar to the conversion of glutamate to ornithine in arginine
biosynthesis (17). We have succeeded in cloning of most of the genes
involved in lysine AAA biosynthesis in T. thermophilus HB27
(15-18). However, the gene encoding HICDH that converts homoisocitrate
to 2-oxoadipate was not contained in the cloned DNA fragments. In this
paper, we describe cloning of the gene encoding HICDH from T. thermophilus HB27 and, for the first time, detailed analysis of
properties for HICDH including site-directed mutagenesis study. The
evolutionary relationship among leucine biosynthesis, the tricarboxylic
acid cycle, and newly identified lysine biosynthesis are also discussed.
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MATERIALS AND METHODS |
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Strains, Media, and Chemicals--
T.
thermophilus HB27 was cultivated as described previously
(15, 19, 20). Escherichia coli DH5 and JM109 (21) were used for DNA manipulation, and E. coli BL21-CodonPlus
(DE3)-RIL [F
, ompT, hsdS
(rB
, mB
),
dcm+, Tetr, gal,
(DE3), endA, Hte, [argU, ileY,
leuW, Camr]] (Stratagene, La Jolla, CA) and
JM109 were used as the host for gene expression. 2× YT medium (21) was
generally used for cultivation of E. coli cells. Antibiotics
and isopropyl-
-D-thiogalactopyranoside were added to the
medium when required. All chemicals were purchased from Sigma, Wako
Pure Chemical (Osaka, Japan), and Kanto Chemicals (Tokyo, Japan).
Enzymes for DNA manipulation were purchased from Takara Shuzo (Kyoto,
Japan) and TOYOBO (Osaka, Japan).
Molecular Cloning and Sequencing of hicdh
Gene--
Oligonucleotides used in this study are listed in Table
I. Based on amino acid sequence alignment
among IPMDHs, ICDHs, and putative HICDHs from various sources,
oligonucleotides (HICD-N1 and HICD-C1) were designed and used as
degenerate primers for PCR for obtaining a DNA fragment corresponding
to the hicdh gene of T. thermophilus HB27. The
following thermal cycle was used: step 1, 95 °C, 5 min; step 2, 95 °C, 1 min; step 3, 68 °C, 1 min; step 4, 75 °C, 1 min; step
5, 75 °C, 7 min. Steps 2-4 were repeated 25 cycles. An amplified
635-bp fragment was blunted, phosphorylated, and ligated into the
EcoRV site of pBluescript II KS (+) (Stratagene). The
resulting plasmid was digested with EcoRI and
HindIII, and the smaller fragment containing the amplified
DNA fragment was used as the probe for the following Southern
hybridization. Southern hybridization was carried out with a Random
Primer Fluorescein Labeling kit (PerkinElmer Life Sciences)
against chromosomal DNA of T. thermophilus HB27 digested
with several restriction enzymes. An approximately 2.6-kb
SacI fragment showing positive hybridization with the probe
was cloned into pUC18 and introduced into E. coli DH5.
Colony hybridization was carried out using the same probe for Southern
hybridization. A plasmid contained in a hybridization-positive colony
was named pHICD-Sac2600. The nucleotide sequence was determined by the
method of Sanger et al. (22).
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Knockout of hicdh in T. thermophilus HB27 and Auxotrophic Complementation Test-- The plasmid for knockout of hicdh was constructed as follows. Two independent PCRs using DFHICN1/DFHICN2 for amplifying 590 bp of a 5'-portion of the hicdh gene and DFHICC1/DFHICC2 for amplifying 619 bp of a 3'-portion of the hicdh gene as primers were performed with pHICD-Sac2600 as the template. PCR conditions were as follows: step 1, 94 °C for 2 min; step 2, 94 °C for 15 s; step 3, 68 °C for 30 s; step 4, 68 °C for 1 min; step 5, 68 °C for 7 min. Steps 2-4 were repeated 30 times using a thermostable DNA polymerase, KOD-plus (TOYOBO). After the amplified 5'- and 3'-fragments were digested with EcoRI/XbaI and XbaI/HindIII, respectively, both the resulting fragments were ligated into pUC18 previously digested with EcoRI/HindIII. The resulting plasmid was named p18DFHIC. The next PCR for amplifying the heat-stable kanamycin nucleotidyltransferase gene (23) was performed using KmR-Nt/KmR-Ct as the primers and pUC39-442KmR (24) as the template. PCR conditions were as follows: step 1, 94 °C for 2 min; step 2, 94 °C for 15 s; step 3, 55 °C for 30 s; step 4, 68 °C for 2 min; step 5, 68 °C for 7 min. Steps 2-4 were repeated 30 times using KOD-plus. An amplified fragment was digested with XbaI and cloned into p18DFHIC previously digested with the same enzyme. The resulting plasmid named p18DFHICKmR was used for knockout of the hicdh gene of T. thermophilus HB27 by the method described previously (17). Colonies growing on TM plate (20) supplemented with 50 µg/ml of kanamycin were picked, and disruption of the genes was confirmed by Southern hybridization. For the auxotrophic complementation test, the knockout mutant, named MJ101, was inoculated on the minimal medium (MM plate) (21) and the MM plate supplemented with 0.1 mM AAA or 0.1 mM lysine.
Phylogenetic Analysis-- A phylogenetic tree was constructed by using the neighbor joining method. Sequence data for the analysis were obtained from the GenBankTM and PIR data bases. The amino acid sequences were aligned by using ClustalW (25) on the DDBJ. Using this aligned data, a phylogenetic tree was constructed by a computer program, Mega 2 (26).
Expression of hicdh, icd, and leuB from T. thermophilus HB27 in
E. coli and Preparation of the Crude Extract--
NdeI and
EcoRI recognition sites were introduced around the start
codon and the termination codon of hicdh from T. thermophilus HB27, respectively, by PCR using synthetic
oligonucleotides, 19HICN and HICDH-Ct. PCR conditions were as follows:
step 1, 94 °C for 2 min; step 2, 94 °C for 1 min; step 3, 65 °C for 1 min; step 4, 72 °C for 2 min; step 5, 72 °C for 7 min. Steps 2-4 were repeated 30 times using Ex-taq (Takara Shuzo). The
amplified fragment was digested with HindIII and
EcoRI and cloned into pUC18. After the nucleotide sequence
was verified, an NdeI- and EcoRI-digested fragment was cloned into pET26b (+) (Novagen, Darmstadt, Germany). The
resulting plasmid, pET-tHICDH101, was used for expression of
hicdh. To construct the plasmids for overexpression of
leuB and icd genes from T. thermophilus HB27, PCRs with the chromosomal DNA were performed by
using primers 19leuBN1/19leuBC2 and ICN/ICC, respectively. PCRs were
performed under the same conditions used for amplifying the
hicdh gene. Amplified fragments were digested with
HindIII and EcoRI, subcloned into pUC119 for
leuB and pUC18 for icd, to yield pUC119-sleuB and
pUC18-ICD, respectively. For overproduction of HICDH, E. coli BL21-Codon-Plus (DE3)-RIL harboring pET-tHICDH101 was
cultured in 2× YT medium containing 50 µg/ml kanamycin and 30 µg/ml chloramphenicol. When the E. coli cells were grown
to give an A600 of 0.5, isopropyl--D-thiogalactopyranoside (final concentration
1 mM) was added. The culture was continued for an
additional 12 h after the induction. For overexpression of
leuB and icd, E. coli JM109 harboring
pUC119-sleuB or pUC18-ICD was cultured in the same way for the
hicdh overexpression.
E. coli cells that overexpressed hicdh, icd, or leuB from T. thermophilus HB27 were suspended in 5 ml of buffer IV (20 mM potassium phosphate, pH 7.5, 0.5 mM EDTA) and disrupted by sonication. The supernatant prepared by centrifugation at 40,000 × g for 20 min was heated at 70 °C for 20 min, and denatured proteins from E. coli cells were removed by centrifugation.
Purification of Recombinant HICDH-- E. coli cells of 10 g (wet weight) harboring pET-tHICDH101 were suspended in 20 ml of buffer IV and disrupted by sonication. The supernatant prepared by centrifugation at 40,000 × g for 20 min was heated at 85 °C for 20 min, and denatured proteins from E. coli cells were removed by centrifugation. Supernatant fractions were applied onto an anion exchange column, DE-52 (Whatman Japan, Tokyo, Japan), pre-equilibrated with buffer IV, washed with buffer IV containing 0.1 M NaCl, and eluted with buffer IV containing 0.2 M NaCl. Ammonium sulfate was added to the eluted fractions at a final concentration of 45%, and the resultant precipitate was collected by centrifugation at 40,000 × g for 30 min, dissolved in buffer IV, and applied onto a Hi-load 26/60 Superdex200 prep grade column (Amersham Biosciences) pre-equilibrated with buffer IV containing 0.2 M NaCl. ICDH from T. thermophilus HB27 was also purified by the method described previously (27).
Purity of the recombinant enzyme was verified by SDS-12% PAGE. Proteins were determined by the method of Bradford (28) by using a Bio-Rad protein assay kit (Nippon Bio-Rad, Tokyo, Japan). Sedimentation equilibrium analysis of the purified HICDH was carried out with a Beckman Optima XL-A analytical ultracentrifuge fitted with a Beckman An-60Ti analytical rotor. The subunit organization of the enzyme was analyzed according to the procedure of Van Holde and Baldwin (29).
Heat Stability of HICDH and ICDH of T. thermophilus HB27-- Heat stability of HICDH and ICDH was analyzed as follows. For calculating the melting temperature (Tm) of the enzymes, purified enzyme dissolved in buffer IV (0.5 mg/ml) was incubated at various temperatures for 20 min and immediately chilled in ice water. Then aggregated proteins were removed by centrifugation (23,500 × g for 5 min), and the remaining activity in the supernatant was measured. For determining the half-life (t1/2) of the enzymes, the enzyme dissolved in buffer IV (0.5 mg/ml) was incubated at 90 °C, and an aliquot was taken at an appropriate interval followed by immediate cooling in ice water. After centrifugation to remove denatured proteins, the remaining activity of the supernatant was measured.
Enzyme Assays-- Ten microliters of the enzyme solution (0.1 mg/ml for isocitrate and 0.2 mg/ml for homoisocitrate) was added to the reaction mixture (50 mM HEPES, pH 8.0, 200 mM KCl, 5 mM MgCl2, 1 mM NAD+, and 25-5000 µM homoisocitrate) that was preincubated at 60 °C for 5 min. For determination of the kinetic constants for isocitrate, 4-400 µM isocitrate was added to the reaction mixture in place of homoisocitrate. The reaction was monitored at 60 °C by following the increase in absorbance at 340 nm. Specific activity was determined by using a 400 µM concentration of each substrate, and 1 unit of the enzymes was defined as the amount of enzyme that produced 1 µmol of NADH or NADPH/min at 60 °C in the enzymatic reaction.
Kinetic parameters were calculated by using an initial velocity program, HYPER, of Cleland (30).
Construction of Altered HICDHs--
Site-directed mutagenesis
was carried out by PCR. To construct an HICDH mutant, HICDH-7Sc, two
independent PCRs were performed using 19HICN/HICD7Sc-1 and
HICD7Sc-2/HICDHCt as primers. PCR conditions were as follows: step 1, 94 °C, 2 min; step 2, 94 °C, 1 min; step 3, 65 °C, 1 min; step
4, 72 °C, 1 min; step 5, 72 °C, 7 min. Steps 2-4 were repeated
30 times. A portion of each reaction product was mixed with each other
and subjected to additional PCR using 19HICN/HCIDHCt as primers. PCR
conditions were as follows: step 1, 94 °C, 10 min; step 2, 94 °C,
1 min; step 3, 65 °C, 1 min; step 4, 72 °C, 2 min; step 5, 72 °C, 7 min. Steps 2-4 were repeated 30 times. An amplified
fragment was digested with HindIII and EcoRI and
subcloned into pUC18 previously digested with the same enzymes.
Construction of the expression plasmid for the altered hicdh
was carried out under the same strategy used for wild-type hicdh. Construction of other altered HICDHs (HICDH/4Sc,
HICDH/5Sc, HICDH/3Sc, HICDH/2Sc, and HICDH/R85V) was carried out in the
same way by using a different pair of primers.
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RESULTS |
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Cloning and Sequence Analysis of the hicdh Gene-- Putative candidates for HICDHs have been proposed in two yeasts (3). In addition, the whole genome sequence has been determined (31) for a radiation-resistant bacterium, Deinococcus radiodurans, that is closely related to T. thermophilus in taxonomy and suggested to possess the lysine biosynthetic system through AAA found in T. thermophilus (32). D. radiodurans possesses a homolog of yeast HICDH genes in addition to putative leuB and icd. Based on the amino acid sequences of the putative HICDHs from yeast and the Deinococcus strain and Blast analysis against the incomplete T. thermophilus HB27 genome sequence (available on the World Wide Web at www.g2l.bio.uni-goettingen.de/blast/blast_search.html), we designed a pair of primers (HICD-N1 and HICD-C1) to amplify a portion of hicdh of T. thermophilus HB27 by PCR. The resulting amplified DNA fragment of about 640 bp carried a sequence that was similar to those of the hicdh/leuB/icd family but obviously different from those of leuB and icd from T. thermophilus (33-35). We then tried to clone the DNA fragment of T. thermophilus HB27 that hybridized against the amplified DNA fragment.
When the chromosomal DNA was digested with several restriction enzymes and subjected to Southern hybridization using the amplified DNA fragment as the probe, SacI digestion gave a positive signal of ~2.6 kb. The DNA fragment was recovered and inserted into pUC18. A clone positive in the colony hybridization assay was picked, and the plasmid contained in the transformant was named pHICD-Sac2600. In this SacI fragment, three open reading frames were found. One of the open reading frames encoded a protein showing 44 and 45% identity in amino acid sequence to IPMDH and ICDH from T. thermophilus HB8, respectively (33-35). The other two open reading frames did not show amino acid sequence similarity to other proteins whose functions were identified.
Based on the crystal structures of IPMDH and ICDH, amino acid residues
responsible for the recognition of a common portion, the malate moiety,
of the substrates and amino acid residues determining the substrate
specificity can be easily predicted (36-38). As expected, amino acid
residues involved in the recognition of the malate moiety of the
substrate are all conserved even in the putative Thermus
HICDH (Fig. 2). Furthermore, by
comparison of the amino acid residues at positions 255 and 266, the
enzyme is suggested to utilize NAD+ as the coenzyme. On the
other hand, the amino acid residues responsible for the recognition of
the -moieties in IPMDH and ICDH are different in HICDH. These
observations suggest that this homolog does not serve as IPMDH and
ICDH, but HICDH. Phylogenetic analysis of this protein family also
supports this hypothesis because the homolog forms one group with
putative HICDHs that separated from groups of IPMDHs and ICDHs (Fig.
3).
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Knockout of hicdh in T. thermophilus HB27--
We performed a
knockout experiment of the homolog to examine whether or not this gene
is related to lysine biosynthesis in T. thermophilus HB27. A
knockout mutant, MJ101, did not grow on minimal medium (Fig.
4). However, growth of the mutant was
restored by the addition of AAA or lysine, indicating that the homolog is related to lysine biosynthesis of the microorganism and has a role
in a reaction step before AAA synthesis as expected. We hereafter call
this homolog hicdh.
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Specific Activities of HICDH, ICDH, and IPMDH from T. thermophilus HB27-- We next constructed plasmids for expression in E. coli of hicdh, icd, and leuB from T. thermophilus HB27 and determined the specific activities of the gene products after removal of most of the proteins in the E. coli crude extract by heat treatment and successive centrifugation. Thermus HICDH gave a specific activity of 564 units/mg with homoisocitrate as the substrate in an NAD+-dependent manner. Surprisingly, the enzyme was able to catalyze the reaction using isocitrate as a substrate with much higher specific activity (8,579 units/mg). On the other hand, HICDH did not recognize 3-isopropylmalate as a substrate. A similar analysis was also performed with crude extract containing ICDH or IPMDH. ICDH catalyzed the reaction with both isocitrate (14,835 units/mg) and homoisocitrate (764 units/mg), whereas, as expected, IPMDH recognized 3-isopropylmalate (2,418 units/mg) as a substrate but did not utilize homoisocitrate and isocitrate as substrates at all. Therefore, we analyzed only HICDH and ICDH in detail in the following experiments.
Expression and Purification of HICDH--
16 mg of HICDH was
prepared through three purification steps from 1 liter of culture with
over 95% purity on SDS-PAGE (data not shown). We carried out
sedimentation equilibrium analysis to evaluate the quaternary structure
of HICDH. By using a partial specific volume of 0.745 that was
calculated from the amino acid composition, data were fitted best with
the dimer-tetramer equilibrium: equilibrium constants for monomer-dimer
and monomer-tetramer association of 1.19 × 1047
M1 and 3.80 × 1095
M
3, respectively. According to the
equilibrium constants, we estimate that HICDH is present as a mixture
of homodimer and homotetramer with a ratio of about 25 and 75%,
respectively, when the enzyme concentration is in the range of
0.25-1.0 mg/ml. We therefore treated HICDH as a homotetramer in the
kinetic analysis described below. The heat stability of HICDH and ICDH
was analyzed by two different assay methods. When melting temperatures
(Tm) were examined by incubating the purified enzyme
at various temperatures for 20 min, Tm of 93.6 and
77.2 °C were obtained for HICDH and ICDH, respectively. Next, we
determined the half-life (t1/2) of each enzyme at
90 °C. The half-life of HICDH was determined to be 16.7 h,
whereas that of ICDH was only 14 s. Thus, HICDH is much more
stable than ICDH.
Kinetic Properties of HICDH Activity--
To characterize HICDH in
detail, kinetic analysis was carried out with purified HICDH. As shown
above, because HICDH recognized both homoisocitrate and isocitrate as
the substrates, we used both compounds for the analysis. The result
shows that the kcat value (171 s1)
of HICDH is almost the same for the reaction with either homoisocitrate or isocitrate (Table II). However, the
Km value of HICDH for homoisocitrate was much larger
than that for isocitrate. Consequently, the catalytic efficiency
(kcat/Km) of the reaction for homoisocitrate is about 20 times smaller than that for isocitrate. When
a similar analysis was carried out for ICDH, ICDH also recognized homoisocitrate as a substrate, although the catalytic efficiency for
homoisocitrate was about 20 times lower than that for isocitrate. It
was of interest that the
kcat/Km value of HICDH for homoisocitrate was 2 times smaller than the corresponding value of ICDH
for homoisocitrate. Although the reaction with homoisocitrate was less
efficient than the reaction with isocitrate for both HICDH and ICDH,
the reason for the reduced efficiency differs between the two enzymes.
HICDH has a larger Km value for homoisocitrate,
whereas ICDH has a lower kcat value for the reaction with homoisocitrate.
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Site-directed Mutagenesis to Alter the Substrate Specificity of
HICDH--
As shown above, Thermus HICDH recognizes both
homoisocitrate and isocitrate as substrates, in contrast to HICDHs from
yeast, S. pombe and S. cerevisiae, which utilize
homoisocitrate but not isocitrate as the substrate
(3).2 Structural studies for
IPMDH and ICDH revealed that only a few amino acid residues in a loop
between -strand 3 and
-helix 4 determine the substrate
specificity of the enzymes of the same enzyme family (36-38). Since
HICDH shows significant amino acid sequence identity to both enzymes,
we hypothesized that a limited number of residues also determine the
substrate specificity even in HICDH. In order to elucidate the residues
determining the difference in the substrate specificity between yeast
and Thermus HICDHs, we constructed six altered HICDHs from
T. thermophilus HB27 in each of which 1-7 amino acid
replacements were introduced in the loop region. An altered enzyme,
HICDH/7Sc, containing the same sequence as that of S. cerevisiae HICDH in the loop displayed activity for homoisocitrate
but not for isocitrate (Fig. 5). On the
other hand, two other altered enzymes, HICDH/4Sc and HICDH/5Sc, containing YSSP sequence like HICDH/7Sc and the yeast enzymes, recognized isocitrate as well as homoisocitrate as substrates, although
the activity was significantly decreased in the altered enzymes. These
results suggested that Arg85 and/or Tyr86 were
required for recognizing isocitrate. Enzyme assays for HICDH/R85V, HICDH/2Sc, and HICDH/3Sc, all of which possessed Val at position 85, revealed that the altered enzymes completely lost activity for
isocitrate but acquired activity with 3-isopropylmalate as a
substrate.
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DISCUSSION |
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A gene coding for a new member of -decarboxylating
dehydrogenase, HICDH, has been cloned from an extreme thermophile,
Thermus thermophilus HB27, and its involvement in lysine
biosynthesis has been demonstrated. Although HICDH is indispensable for
lysine AAA biosynthesis and its primary function in vivo
must be the conversion of homoisocitrate to 2-oxoadipate, kinetic
analysis showed that the enzyme can also utilize isocitrate, an
intermediate of the tricarboxylic acid cycle, 20 times more efficiently
compared with using homoisocitrate as a substrate (Table II, Fig. 3).
This may indicate that the kinetic constant of an enzyme determined in vitro does not always reflect its physiological role, or
it may be possible that HICDH will substitute for ICDH in some cases. On the other hand, the catalytic efficiency in terms of
kcat/Km of ICDH for the ICDH
reaction is in a range similar to that of HICDH for the same reaction.
Therefore, this may suggest that ICDH can function in lysine AAA
biosynthesis as well.
HICDH and ICDH share similarities in primary sequence and reaction mechanism and are therefore thought to have diverged from a common ancestral enzyme (3). A common ancestor is thought to have broad substrate specificity (1), so an HICDH from T. thermophilus HB27 that can catalyze reactions contained in both lysine AAA biosynthesis and the tricarboxylic acid cycle may still have features of the ancestral-type enzyme. It should be noted that we recently found that homocitrate synthase, which catalyzes synthesis of homocitrate from 2-oxoglutarate and acetyl-CoA, the first reaction in lysine biosynthesis of T. thermophilus HB27, was able to catalyze the synthesis of citrate from oxaloacetate and acetyl-CoA, the corresponding reaction by citrate synthase in the tricarboxylic acid cycle, although homocitrate synthase has no structural similarity to citrate synthase (39). Taken together with our previous results on LysJ and LysK (17, 18), both of which are able to catalyze the reactions in lysine and arginine biosynthesis, dual functions and/or broad specificity may be common features of lysine biosynthetic enzymes of T. thermophilus HB27.
Amino acid residues responsible for binding the malate moiety of the
substrates are completely conserved in -decarboxylating dehydrogenases, including HICDH (Fig. 2). Therefore, the corresponding residues are presumably involved in substrate binding even in HICDH.
However, amino acid residues in the
-strand 3-
-helix 4 loop that
recognize the
-moiety (Fig. 1) are not conserved between ICDH and
IPMDH. According to the three-dimensional structure of IPMDH of
T. ferrooxidans, three residues (Glu88,
Leu91, and Leu92 in T. ferrooxidans
IPMDH numbering (38)) are specific determinants for recognition of
3-isopropylmalate (Fig. 6).
Leu91 and Leu92 make hydrophobic contacts with
the
-isopropyl group of 3-isopropylmalate, and Glu88
interacts with the nicotinamide ring of NAD+ for
stabilizing the Michaelis complex. Glu88 also serves to
prevent IPMDH from binding the
-carboxylate group of isocitrate via
charge repulsion. On the other hand, in ICDH from E. coli,
two residues (Ser113 and Asn115 in the
numbering of E. coli ICDH) are indicated by x-ray structural analysis to be responsible for isocitrate recognition (36). Ser113 forms a hydrogen bond to the
-carboxylate group
of isocitrate, and Asn115 interacts both with the
-carboxylate group of isocitrate and with NADP+. Due to
the polar environment of the
-moiety-binding site of the ICDH, the
hydrophobic isopropyl moiety of 3-isopropylmalate is excluded from the
site. In HICDH, regions probably recognizing the
-moiety are also
occupied by amino acid residues different from those in ICDH and IPMDH
(Figs. 1 and 2). Chen and Jeong (3) aligned amino acid sequences of
several enzymes in this protein family and suggested Tyr106
and Ile110 (numbering according to HICDH from S. cerevisiae) as the residues that were necessary for recognition of
the
-moiety of homoisocitrate. They speculated that the phenolic
hydroxyl group of Tyr106 formed a hydrogen bond to
-carboxylate of homoisocitrate analogous to Ser113 in
E. coli ICDH. In HICDH from T. thermophilus HB27,
however, the corresponding position is occupied by phenylalanine. This indicates that the mechanism for recognition of homoisocitrate has to
be reconsidered in HICDH.
|
Thermus HICDH recognizes both homoisocitrate and
isocitrate as substrates (Table II) in contrast to HICDHs from
S. pombe and S. cerevisiae, where the yeast
enzymes catalyze the reaction with only homoisocitrate and neither
isocitrate nor 3-isopropylmalate (3).2 To elucidate the
difference in the mechanism for substrate recognition between
Thermus and yeast HICDHs, we introduced amino acid
replacements into the -strand 3-
-helix 4 loop region of
Thermus HICDH. An altered enzyme, HICDH/7Sc, containing the
same sequence as that of S. cerevisiae HICDH in the loop
displayed activity for homoisocitrate but not for isocitrate,
indicating that the loop actually does contain the determinants for
substrate specificity in HICDH. Among the altered HICDHs, all of the
enzymes with Val at position 85 completely lost the ability to utilize
isocitrate as a substrate. These results indicate that
Arg85 is required for recognition of isocitrate as a
substrate in Thermus HICDH. However, this result looks
confusing, because the corresponding position (116 in E. coli ICDH) is also occupied by Val in ICDH. Val116 in
E. coli ICDH does not directly interact with substrate nor NADP+, but replacement of the residue by Leu or Ser
converted the ICDH to preference toward 3-isopropylmalate (9). This
suggests that substrate recognition of the loop residues differs
between these enzymes.
The phylogenetic tree of HICDH, ICDH, and IPMDH from various sources indicates that HICDH from T. thermophilus HB27 belongs to one group along with the HICDHs from two yeasts and DR1674 from D. radiodurans that is suggested to synthesize lysine through AAA (Fig. 3). According to the hicdh knockout experiment, the gene was shown to be involved in lysine biosynthesis. Nevertheless, unlike the case for yeast HICDHs, Thermus HICDH has higher activity for isocitrate. Our present study indicates that only a few amino acid replacements are enough to convert Thermus HICDH to an enzyme that is specific to homoisocitrate. Since HICDH/7Sc displayed extremely high thermostability comparable with that of the wild-type enzyme, which may ensure that the microorganism grows at extremely elevated temperatures, we may assume that it would be possible for the organism to acquire mutations that could generate a homoisocitrate-specific enzyme. We at present do not know what causes the enzyme to have dual functions or quite higher activity for isocitrate. Physiology and metabolism will have to be investigated in detail for T. thermophilus HB27 to understand this.
We previously reported that an anaerobic hyperthermophilic archaeaon,
Pyrococcus horikoshii, has a gene cluster that is very similar to that of T. thermophilus HB27, with each component
of the archaeon tightly linked to the corresponding counterpart in T. thermophilus HB27 in phylogenetic analysis, suggesting
that the archaeaon probably synthesizes lysine through AAA as in the case of T. thermophilus HB27 (16). However, PH1722, an HICDH homolog, in the gene cluster of P. horikoshii does not
belong to this group in the phylogenetic tree but rather to the IPMDH group (Fig. 3). Similar gene clusters were also found in two related thermoacidophilic archaea, Sulfolobus tokodaii strain
7 and Sulfolobus sulfataricus, whose genome sequences
have recently been determined (40, 41). Recently, expression of the
gene cluster was shown to be regulated by LysM, an Lrp-like
transcriptional regulator, depending on the presence or absence of
lysine (42), although the functions of the cluster have not yet been
analyzed. Based on these observations, it is speculated that the two
Sulfolobus species also synthesize lysine through AAA.
However, it is of interest that these microorganisms carry no
hicdh-homologous genes other than icd and
leuB in their genomes. IPMDH (LeuB) was isolated from
S. tokodaii strain 7 and analyzed in detail (2). The
Sulfolobus IPMDH cannot utilize homoisocitrate or isocitrate
as substrates (2).2 This may suggest that the HICDH
reaction of lysine AAA biosynthesis in Sulfolobus could be
catalyzed by a putative ICDH. In order to prove this hypothesis,
substrate specificity of the putative ICDH has to be analyzed. In any
case, PH1722 in Pyrococcus and the putative ICDHs in the
Sulfolobus species could be keys to elucidate evolution of
-decarboxylating dehydrogenase. It should be also noted that the
HICDH group is monophyletic, with a deep divergent point between IPMDH
and ICDH groups. This suggests that HICDH has not evolved from IPMDH or
ICDH but rather directly from an ancestral enzyme of
-decarboxylating dehydrogenase.
Amino acid sequence identity between HICDH and ICDH is
45%, and the basic mechanisms for reactions and substrate recognition to bind a malate moiety seem similar between the two enzymes. However,
although these enzymes also showed similar catalytic efficiency
(kcat/Km) for the reaction
with homoisocitrate or isocitrate, profiles in the kinetic parameters
were different; HICDH has larger kcat and
Km values, and ICDH has smaller kcat and Km values. In
addition, HICDH is a much more heat-stable enzyme than ICDH.
Determination of structures of both enzymes may lead us to understand
not only the detailed catalytic mechanism but also molecular evolution
along with the heat stability of these enzymes.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Hiromi Nishida (Institute of Molecular and Cellular Biosciences, The University of Tokyo) for his help in phylogenetic analysis and Dr. Tomoyuki Fujii (Department of Applied Biological Chemistry, The University of Tokyo) for his help in quaternary structure analysis of HICDH using analytical ultracentrifuge. We also thank Dr. Elinor T. Adman (University of Washington School of Medicine) for providing useful comments for completing the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and from the Noda Institute for Scientific Research.
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB075751, AB085838, and AB085839.
Present address: Harima Institute/Spring-8, Institute of Physical
and Chemical Research (RIKEN), Sayo-gun, Hyogo 679-5148, Japan.
§ To whom correspondence should be addressed: Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Tel.: 81-3-5841-3072; Fax: 81-3-5841-8030; E-mail: umanis@mail.ecc.u-tokyo.ac.jp.
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M205133200
2 J. Miyazaki, N. Kobashi, M. Nishiyama, and H. Yamane, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
IPMDH, 3-isopropylmalate dehydrogenase;
ICDH, isocitrate dehydrogenase;
AAA, -aminoadipate;
HICDH, homoisocitrate dehydrogenase.
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