Kinetics and product analysis of the reaction catalysed by recombinant homoaconitase from Thermus thermophilus

HACN (homoaconitase) is a member of a family of [4Fe-4S] cluster-dependent enzymes that catalyse hydration/dehydration reactions. The best characterized example of this family is the ubiquitous ACN (aconitase), which catalyses the dehydration of citrate to cis-aconitate, and the subsequent hydration of cis-aconitate to isocitrate. HACN is an enzyme from the alpha-aminoadipate pathway of lysine biosynthesis, and has been identified in higher fungi and several archaea and one thermophilic species of bacteria, Thermus thermophilus. HACN catalyses the hydration of cis-homoaconitate to (2R,3S)-homoisocitrate, but the HACN-catalysed dehydration of (R)-homocitrate to cis-homoaconitate has not been observed in vitro. We have synthesized the substrates and putative substrates for this enzyme, and in the present study report the first steady-state kinetic data for recombinant HACN from T. thermophilus using a (2R,3S)-homoisocitrate dehydrogenase-coupled assay. We have also examined the products of the reaction using HPLC. We do not observe HACN-catalysed 'homocitrate dehydratase' activity; however, we have observed that ACN can catalyse the dehydration of (R)-homocitrate to cis-homoaconitate, but HACN is required for subsequent conversion of cis-homoaconitate into homoisocitrate. This suggests that the in vivo process for conversion of homocitrate into homoisocitrate requires two enzymes, in simile with the propionate utilization pathway from Escherichia coli. Surprisingly, HACN does not show any activity when cis-aconitate is substituted for the substrate, even though other enzymes from the alpha-aminoadipate pathway can accept analogous tricarboxylic acid-cycle substrates. The enzyme shows no apparent feedback inhibition by L-lysine.     

The fungal α‐aminoadipate pathway for lysine biosynthesis requires two enzymes of the aconitase family for the isomerization of homocitrate to homoisocitrate

Fungi produce α‐aminoadipate, a precursor for penicillin and lysine via the α‐aminoadipate pathway. Despite the biotechnological importance of this pathway, the essential isomerization of homocitrate via homoaconitate to homoisocitrate has hardly been studied. Therefore, we analysed the role of homoaconitases and aconitases in this isomerization. Although we confirmed an essential contribution of homoaconitases from Saccharomyces cerevisiae and Aspergillus fumigatus, these enzymes only catalysed the interconversion between homoaconitate and homoisocitrate. In contrast, aconitases from fungi and the thermophilic bacterium Thermus thermophilus converted homocitrate to homoaconitate. Additionally, a single aconitase appears essential for energy metabolism, glutamate and lysine biosynthesis in respirating filamentous fungi, but not in the fermenting yeast S. cerevisiae that possesses two contributing aconitases. While yeast Aco1p is essential for the citric acid cycle and, thus, for glutamate synthesis, Aco2p specifically and exclusively contributes to lysine biosynthesis. In contrast, Aco2p homologues present in filamentous fungi were transcribed, but enzymatically inactive, revealed no altered phenotype when deleted and did not complement yeast aconitase mutants. From these results we conclude that the essential requirement of filamentous fungi for respiration versus the preference of yeasts for fermentation may have directed the evolution of aconitases contributing to energy metabolism and lysine biosynthesis.     

Enzymology and evolution of the pyruvate pathway to 2-oxobutyrate in Methanocaldococcus jannaschii

The archaeon Methanocaldococcus jannaschii uses three different 2-oxoacid elongation pathways, which extend the chain length of precursors in leucine, isoleucine, and coenzyme B biosyntheses. In each of these pathways an aconitase-type hydrolyase catalyzes an hydroxyacid isomerization reaction. The genome sequence of M. jannaschii encodes two homologs of each large and small subunit that forms the hydrolyase, but the genes are not cotranscribed. The genes are more similar to each other than to previously characterized isopropylmalate isomerase or homoaconitase enzyme genes. To identify the functions of these homologs, the four combinations of subunits were heterologously expressed in Escherichia coli, purified, and reconstituted to generate the iron-sulfur center of the holoenzyme. Only the combination of MJ0499 and MJ1277 proteins catalyzed isopropylmalate and citramalate isomerization reactions. This pair also catalyzed hydration half-reactions using citraconate and maleate. Another broad-specificity enzyme, isopropylmalate dehydrogenase (MJ0720), catalyzed the oxidative decarboxylation of beta-isopropylmalate, beta-methylmalate, and d-malate. Combined with these results, phylogenetic analysis suggests that the pyruvate pathway to 2-oxobutyrate (an alternative to threonine dehydratase in isoleucine biosynthesis) evolved several times in bacteria and archaea. The enzymes in the isopropylmalate pathway of leucine biosynthesis facilitated the evolution of 2-oxobutyrate biosynthesis through the introduction of a citramalate synthase, either by gene recruitment or gene duplication and functional divergence.     

The Methanococcus jannaschii dCTP deaminase is a bifunctional deaminase and diphosphatase

Most bacteria produce the dUMP precursor for thymine nucleotide biosynthesis using two enzymes: a dCTP deaminase catalyzes the formation of dUTP and a dUTP diphosphatase catalyzes pyrophosphate release. Although these two hydrolytic enzymes appear to catalyze very different reactions, they are encoded by homologous genes. The hyperthermophilic archaeon Methanococcus jannaschii has two members of this gene family. One gene, at locus MJ1102, encodes a dUTP diphosphatase, which can scavenge deoxyuridine nucleotides that inhibit archaeal DNA polymerases. The second gene, at locus MJ0430, encodes a novel dCTP deaminase that releases dUMP, ammonia, and pyrophosphate. Therefore this enzyme can singly catalyze both steps in dUMP biosynthesis, precluding the formation of free, mutagenic dUTP. Besides differing from the previously characterized Salmonella typhimurium dCTP deaminase in its reaction products, this archaeal enzyme has a higher affinity for dCTP and its steady-state turnover is faster than the bacterial enzyme. Kinetic studies suggest: 1) the archaeal enzyme specifically recognizes dCTP; 2) dCTP deamination and dUTP diphosphatase activities occur independently at the same active site, and 3) both activities depend on Mg(2+). The bifunctional activity of this M. jannaschii enzyme illustrates the evolution of a suprafamily of related enzymes that catalyze mechanistically distinct reactions.     

Identification of enzymes homologous to isocitrate dehydrogenase that are involved in coenzyme B and leucine biosynthesis in methanoarchaea

Two putative Methanococcus jannaschii isocitrate dehydrogenase genes, MJ1596 and MJ0720, were cloned and overexpressed in Escherichia coli, and their gene products were tested for the ability to catalyze the NAD- and NADP-dependent oxidative decarboxylation of DL-threo-3-isopropylmalic acid, threo-isocitrate, erythro-isocitrate, and homologs of threo-isocitrate. Neither enzyme was found to use any of the isomers of isocitrate as a substrate. The protein product of the MJ1596 gene, designated AksF, catalyzed the NAD-dependent decarboxylation of intermediates in the biosynthesis of 7-mercaptoheptanoic acid, a moiety of methanoarchaeal coenzyme B (7-mercaptoheptanylthreonine phosphate). These intermediates included (-)-threo-isohomocitrate [(-)-threo-1-hydroxy-1,2, 4-butanetricarboxylic acid], (-)-threo-iso(homo)(2)citrate [(-)-threo-1-hydroxy-1,2,5-pentanetricarboxylic acid], and (-)-threo-iso(homo)(3)citrate [(-)-threo-1-hydroxy-1,2, 6-hexanetricarboxylic acid]. The protein product of MJ0720 was found to be alpha-isopropylmalate dehydrogenase (LeuB) and was found to catalyze the NAD-dependent decarboxylation of one isomer of DL-threo-isopropylmalate to 2-ketoisocaproate; thus, it is involved in the biosynthesis of leucine. The AksF enzyme proved to be thermostable, losing only 10% of its enzymatic activity after heating at 100 degrees C for 10 min, whereas the LeuB enzyme lost 50% of its enzymatic activity after heating at 80 degrees C for 10 min.     

Activation of Pyk2/RAFTK induces tyrosine phosphorylation of alpha-synuclein via Src-family kinases

The hyperthermophilic archaeon Methanococcus jannaschii uses several non-canonical enzymes to catalyze conserved reactions in glycolysis and gluconeogenesis. A highly diverged gene from that organism has been proposed to function as a phosphoglycerate mutase. Like the canonical cofactor-independent phosphoglycerate mutase and other members of the binuclear metalloenzyme superfamily, this M. jannaschii protein has conserved nucleophilic serine and metal-binding residues. Yet the substrate-binding residues are not conserved. We show that the genes at M. jannaschii loci MJ0010 and MJ1612 encode thermostable enzymes with phosphoglycerate mutase activity. Phylogenetic analyses suggest that this gene family arose before the divergence of the archaeal lineage.     

Methanocaldococcus jannaschii uses a modified mevalonate pathway for biosynthesis of isopentenyl diphosphate

Archaea have been shown to produce isoprenoids from mevalonate; however, genome analysis has failed to identify several genes in the mevalonate pathway on the basis of sequence similarity. A predicted archaeal kinase, coded for by the MJ0044 gene, was associated with other mevalonate pathway genes in the archaea and was predicted to be the "missing" phosphomevalonate kinase. The MJ0044-derived protein was tested for phosphomevalonate kinase activity and was found not to catalyze this reaction. The MJ0044 gene product was found to phosphorylate isopentenyl phosphate, generating isopentenyl diphosphate. Unlike other known kinases associated with isoprene biosynthesis, Methanocaldococcus jannaschii isopentenyl phosphate kinase is predicted to be a member of the aspartokinase superfamily.     

The structure of (3R)-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Pseudomonas aeruginosa

Type II fatty acid biosynthesis systems are essential for membrane formation in bacteria, making the constituent proteins of this pathway attractive targets for antibacterial drug discovery. The third step in the elongation cycle of the type II fatty acid biosynthesis is catalyzed by beta-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase. There are two isoforms. FabZ, which catalyzes the dehydration of (3R)-hydroxyacyl-ACP to trans-2-acyl-ACP, is a universally expressed component of the bacterial type II system. FabA, the second isoform, as has more limited distribution in nature and, in addition to dehydration, also carries out the isomerization of trans-2- to cis-3-decenoyl-ACP as an essential step in unsaturated fatty acid biosynthesis. We report the structure of FabZ from the important human pathogen Pseudomonas aeruginosa at 2.5 A of resolution. PaFabZ is a hexamer (trimer of dimers) with the His/Glu catalytic dyad located within a deep, narrow tunnel formed at the dimer interface. Site-directed mutagenesis experiments showed that the obvious differences in the active site residues that distinguish the FabA and FabZ subfamilies of dehydratases do not account for the unique ability of FabA to catalyze isomerization. Because the catalytic machinery of the two enzymes is practically indistinguishable, the structural differences observed in the shape of the substrate binding channels of FabA and FabZ lead us to hypothesize that the different shapes of the tunnels control the conformation and positioning of the bound substrate, allowing FabA, but not FabZ, to catalyze the isomerization reaction.     

Structural and functional investigation of a putative archaeal selenocysteine synthase

Bacterial selenocysteine synthase converts seryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec) for selenoprotein biosynthesis. The identity of this enzyme in archaea and eukaryotes is unknown. On the basis of sequence similarity, a conserved open reading frame has been annotated as a selenocysteine synthase gene in archaeal genomes. We have determined the crystal structure of the corresponding protein from Methanococcus jannaschii, MJ0158. The protein was found to be dimeric with a distinctive domain arrangement and an exposed active site, built from residues of the large domain of one protomer alone. The shape of the dimer is reminiscent of a substructure of the decameric Escherichia coli selenocysteine synthase seen in electron microscopic projections. However, biochemical analyses demonstrated that MJ0158 lacked affinity for E. coli seryl-tRNA(Sec) or M. jannaschii seryl-tRNA(Sec), and neither substrate was directly converted to selenocysteinyl-tRNA(Sec) by MJ0158 when supplied with selenophosphate. We then tested a hypothetical M. jannaschii O-phosphoseryl-tRNA(Sec) kinase and demonstrated that the enzyme converts seryl-tRNA(Sec) to O-phosphoseryl-tRNA(Sec) that could constitute an activated intermediate for selenocysteinyl-tRNA(Sec) production. MJ0158 also failed to convert O-phosphoseryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). In contrast, both archaeal and bacterial seryl-tRNA synthetases were able to charge both archaeal and bacterial tRNA(Sec) with serine, and E. coli selenocysteine synthase converted both types of seryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). These findings demonstrate that a number of factors from the selenoprotein biosynthesis machineries are cross-reactive between the bacterial and the archaeal systems but that MJ0158 either does not encode a selenocysteine synthase or requires additional factors for activity.     

L-Aspartate semialdehyde and a 6-deoxy-5-ketohexose 1-phosphate are the precursors to the aromatic amino acids in Methanocaldococcus jannaschii

No orthologs are present in the genomes of the archaea encoding genes for the first two steps in the biosynthesis of the aromatic amino acids leading to 3-dehydroquinate (DHQ). The absence of these genes prompted me to examine the nature of the reactions involved in the archaeal pathway leading to DHQ in Methanocaldococcus jannaschii. Here I report that 6-deoxy-5-ketofructose 1-phosphate and l-aspartate semialdehyde are precursors to DHQ. The sugar, which is derived from glucose 6-P, supplies a "hydroxyacetone" fragment, which, via a transaldolase reaction, undergoes an aldol condensation with the l-aspartate semialdehyde to form 2-amino-3,7-dideoxy-D-threo-hept-6-ulosonic acid. Despite the fact that both hydroxyacetone and hydroxyacetone-P were measured in the cell extracts and confirmed to arise from glucose 6-P, neither compound was found to serve as a precursor to DHQ. This amino sugar then undergoes a NAD dependent oxidative deamination to produce 3,7-dideoxy-d-threo-hept-2,6-diulosonic acid which cyclizes to 3-dehydroquinate. The protein product of the M. jannaschii MJ0400 gene catalyzes the transaldolase reaction and the protein product of the MJ1249 gene catalyzes the oxidative deamination and the cyclization reactions. The DHQ is readily converted into dehydroshikimate and shikimate in M. jannaschii cell extracts, consistent with the remaining steps and genes in the pathway being the same as in the established shikimate pathway.     

Identification and characterization of an archaeon-specific riboflavin kinase

The riboflavin kinase in Methanocaldococcus jannaschii has been identified as the product of the MJ0056 gene. Recombinant expression of the MJ0056 gene in Escherichia coli led to a large increase in the amount of flavin mononucleotide (FMN) in the E. coli cell extract. The unexpected features of the purified recombinant enzyme were its use of CTP as the phosphoryl donor and the absence of a requirement for added metal ion to catalyze the formation of FMN. Identification of this riboflavin kinase fills another gap in the archaeal flavin biosynthetic pathway. Some divalent metals were found to be potent inhibitors of the reaction. The enzyme represents a unique CTP-dependent family of kinases.     

Identification of an archaeal 2-hydroxy acid dehydrogenase catalyzing reactions involved in coenzyme biosynthesis in methanoarchaea

Two putative malate dehydrogenase genes, MJ1425 and MJ0490, from Methanococcus jannaschii and one from Methanothermus fervidus were cloned and overexpressed in Escherichia coli, and their gene products were tested for the ability to catalyze pyridine nucleotide-dependent oxidation and reduction reactions of the following alpha-hydroxy-alpha-keto acid pairs: (S)-sulfolactic acid and sulfopyruvic acid; (S)-alpha-hydroxyglutaric acid and alpha-ketoglutaric acid; (S)-lactic acid and pyruvic acid; and 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid and 1-oxo-1,3,4, 6-hexanetetracarboxylic acid. Each of these reactions is involved in the formation of coenzyme M, methanopterin, coenzyme F(420), and methanofuran, respectively. Both the MJ1425-encoded enzyme and the MJ0490-encoded enzyme were found to function to different degrees as malate dehydrogenases, reducing oxalacetate to (S)-malate using either NADH or NADPH as a reductant. Both enzymes were found to use either NADH or NADPH to reduce sulfopyruvate to (S)-sulfolactate, but the V(max)/K(m) value for the reduction of sulfopyruvate by NADH using the MJ1425-encoded enzyme was 20 times greater than any other combination of enzymes and pyridine nucleotides. Both the M. fervidus and the MJ1425-encoded enzyme catalyzed the NAD(+)-dependent oxidation of (S)-sulfolactate to sulfopyruvate. The MJ1425-encoded enzyme also catalyzed the NADH-dependent reduction of alpha-ketoglutaric acid to (S)-hydroxyglutaric acid, a component of methanopterin. Neither of the enzymes reduced pyruvate to (S)-lactate, a component of coenzyme F(420). Only the MJ1425-encoded enzyme was found to reduce 1-oxo-1,3,4,6-hexanetetracarboxylic acid, and this reduction occurred only to a small extent and produced an isomer of 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid that is not involved in the biosynthesis of methanofuran c. We conclude that the MJ1425-encoded enzyme is likely to be involved in the biosynthesis of both coenzyme M and methanopterin.     

Identification and characterization of the 2-phospho-L-lactate guanylyltransferase involved in coenzyme F420 biosynthesis

Coenzyme F 420 is a hydride carrier cofactor functioning in methanogenesis. One step in the biosynthesis of coenzyme F 420 involves the coupling of 2-phospho- l-lactate (LP) to 7,8-didemethyl-8-hydroxy-5-deazaflavin, the F 420 chromophore. This condensation requires an initial activation of 2-phospho- l-lactate through a pyrophosphate linkage to GMP. Bioinformatic analysis identified an uncharacterized archaeal protein in the Methanocaldococcus jannaschii genome, MJ0887, which could be involved in this transformation. The predicted MJ0887-derived protein has domain similarity with other known nucleotidyl transferases. The MJ0887 gene was cloned and overexpressed, and the purified protein was found to catalyze the formation of lactyl-2-diphospho-5'-guanosine from LP and GTP. Kinetic constants were determined for the MJ0887-derived protein with both LP and GTP substrates and are as follows: V max = 3 micromol min (-1) mg (-1), GTP K M (app) = 56 microM, and k cat/ K M (app) = 2 x 10 (4) M (-1) s (-1) and LP K M (app) = 36 microM, and k cat/ K M (app) = 4 x 10 (4) M (-1) s (-1). The MJ0887 gene product has been designated CofC to indicate its involvement in the third step of coenzyme F 420 biosynthesis.     

In vitro conversion of propionate to pyruvate by Salmonella enterica enzymes: 2-methylcitrate dehydratase (PrpD) and aconitase Enzymes catalyze the conversion of 2-methylcitrate to 2-methylisocitrate

Salmonella enterica serovar Typhimurium LT2 catabolizes propionate through the 2-methylcitric acid cycle, but the identity of the enzymes catalyzing the conversion of 2-methylcitrate into 2-methylisocitrate is unclear. This work shows that the prpD gene of the prpBCDE operon of this bacterium encodes a protein with 2-methylcitrate dehydratase enzyme activity. Homogeneous PrpD enzyme did not contain an iron-sulfur center, displayed no requirements for metal cations or reducing agents for activity, and did not catalyze the hydration of 2-methyl-cis-aconitate to 2-methylisocitrate. It was concluded that the gene encoding the 2-methyl-cis-aconitate hydratase enzyme is encoded outside the prpBCDE operon. Computer analysis of bacterial genome databases identified the presence of orthologues of the acnA gene (encodes aconitase A) in a number of putative prp operons. Homogeneous AcnA protein of S. enterica had strong aconitase activity and catalyzed the hydration of the 2-methyl-cis-aconitate to yield 2-methylisocitrate. The purification of this enzyme allows the complete reconstitution of the 2-methylcitric acid cycle in vitro using homogeneous preparations of the PrpE, PrpC, PrpD, AcnA, and PrpB enzymes. However, inactivation of the acnA gene did not block growth of S. enterica on propionate as carbon and energy source. The existence of a redundant aconitase activity (encoded by acnB) was postulated to be responsible for the lack of a phenotype in acnA mutant strains. Consistent with this hypothesis, homogeneous AcnB protein of S. enterica also had strong aconitase activity and catalyzed the conversion of 2-methyl-cis-aconitate into 2-methylisocitrate. To address the involvement of AcnB in propionate catabolism, an acnA and acnB double mutant was constructed, and this mutant strain cannot grow on propionate even when supplemented with glutamate. The phenotype of this double mutant indicates that the aconitase enzymes are required for the 2-methylcitric acid cycle during propionate catabolism.     

Modified pathway to synthesize ribulose 1,5-bisphosphate in methanogenic archaea

Several sequencing projects unexpectedly uncovered the presence of genes that encode ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) in anaerobic archaea. RubisCO is the key enzyme of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway, a scheme that does not appear to contribute greatly, if at all, to net CO2 assimilation in these organisms. Recombinant forms of the archaeal enzymes do, however, catalyze a bona fide RuBP-dependent CO2 fixation reaction, and it was recently shown that Methanocaldococcus (Methanococcus) jannaschii and other anaerobic archaea synthesize catalytically active RubisCO in vivo. To complete the CBB pathway, there is a need for an enzyme, i.e., phosphoribulokinase (PRK), to catalyze the formation of RuBP, the substrate for the RubisCO reaction. Homology searches, as well as direct enzymatic assays with M. jannaschii, failed to reveal the presence of PRK. The apparent lack of PRK raised the possibility that either there is an alternative pathway to generate RuBP or RubisCO might use an alternative substrate in vivo. In the present study, direct enzymatic assays performed with alternative substrates and extracts of M. jannsachii provided evidence for a previously uncharacterized pathway for RuBP synthesis from 5-phospho-D-ribose-1-pyrophosphate (PRPP) in M. jannaschii and other methanogenic archaea. Proteins and genes involved in the catalytic conversion of PRPP to RuBP were identified in M. jannaschii (Mj0601) and Methanosarcina acetivorans (Ma2851), and recombinant Ma2851 was active in extracts of Escherichia coli. Thus, in this work we identified a novel means to synthesize the CO2 acceptor and substrate for RubisCO in the absence of a detectable kinase, such as PRK. We suggest that the conversion of PRPP to RuBP might be an evolutional link between purine recycling pathways and the CBB scheme.     

Cysteinyl-tRNA(Cys) formation in Methanocaldococcus jannaschii: the mechanism is still unknown

Most organisms form Cys-tRNA(Cys), an essential component for protein synthesis, through the action of cysteinyl-tRNA synthetase (CysRS). However, the genomes of Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, and Methanopyrus kandleri do not contain a recognizable cysS gene encoding CysRS. It was reported that M. jannaschii prolyl-tRNA synthetase (C. Stathopoulos, T. Li, R. Longman, U. C. Vothknecht, H. D. Becker, M. Ibba, and D. S?ll, Science 287:479-482, 2000; R. S. Lipman, K. R. Sowers, and Y. M. Hou, Biochemistry 39:7792-7798, 2000) or the M. jannaschii MJ1477 protein (C. Fabrega, M. A. Farrow, B. Mukhopadhyay, V. de Crécy-Lagard, A. R. Ortiz, and P. Schimmel, Nature 411:110-114, 2001) provides the "missing" CysRS activity for in vivo Cys-tRNA(Cys) formation. These conclusions were supported by complementation of temperature-sensitive Escherichia coli cysS(Ts) strain UQ818 with archaeal proS genes (encoding prolyl-tRNA synthetase) or with the Deinococcus radiodurans DR0705 gene, the ortholog of the MJ1477 gene. Here we show that E. coli UQ818 harbors a mutation (V27E) in CysRS; the largest differences compared to the wild-type enzyme are a fourfold increase in the K(m) for cysteine and a ninefold reduction in the k(cat) for ATP. While transformants of E. coli UQ818 with archaeal and bacterial cysS genes grew at a nonpermissive temperature, growth was also supported by elevated intracellular cysteine levels, e.g., by transformation with an E. coli cysE allele (encoding serine acetyltransferase) or by the addition of cysteine to the culture medium. An E. coli cysS deletion strain permitted a stringent complementation test; growth could be supported only by archaeal or bacterial cysS genes and not by archaeal proS genes or the D. radiodurans DR0705 gene. Construction of a D. radiodurans DR0705 deletion strain showed this gene to be dispensable. However, attempts to delete D. radiodurans cysS failed, suggesting that this is an essential Deinococcus gene. These results imply that it is not established that proS or MJ1477 gene products catalyze Cys-tRNA(Cys) synthesis in M. jannaschii. Thus, the mechanism of Cys-tRNA(Cys) formation in M. jannaschii still remains to be discovered.     

Characterization of homoisocitrate dehydrogenase involved in lysine biosynthesis of an extremely thermophilic bacterium,Thermus thermophilus HB27, and evolutionary implication of beta-decarboxylating dehydrogenase

Although the presence of an enzyme that catalyzes beta-decarboxylating dehydrogenation of homoisocitrate to synthesize 2-oxoadipate has been postulated in the lysine biosynthesis pathway through alpha-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 Arg(85) 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 Arg(85) is a key residue for both substrate specificity and evolution of beta-decarboxylating dehydrogenases.     

Crystal structure of the radical SAM enzyme catalyzing tricyclic modified base formation in tRNA

Wyosine and its derivatives, such as wybutosine, found in eukaryotic and archaeal tRNAs, are tricyclic hypermodified nucleosides. In eukaryotes, wybutosine exists exclusively in position 37, 3'-adjacent to the anticodon, of tRNA(Phe), where it ensures correct translation by stabilizing the codon-anticodon base-pairing during the ribosomal decoding process. Recent studies revealed that the wyosine biosynthetic pathway consists of multistep enzymatic reactions starting from a guanosine residue. Among these steps, TYW1 catalyzes the second step to form the tricyclic ring structure, by cyclizing N(1)-methylguanosine. In this study, we solved the crystal structure of TYW1 from Methanocaldococcus jannaschii at 2.4 A resolution. TYW1 assumes an incomplete TIM barrel with (alpha/beta)(6) topology, which closely resembles the reported structures of radical SAM enzymes. Hence, TYW1 was considered to catalyze the cyclization reaction by utilizing the radical intermediate. Comparison with other radical SAM enzymes allowed us to build a model structure complexed with S-adenosylmethionine and two [4Fe-4S] clusters. Mutational analyses in yeast supported the validity of this complex model structure, which provides a structural insight into the radical reaction involving two [4Fe-4S] clusters to create a complex tricyclic base.