Methanogen homoaconitase catalyzes both hydrolyase reactions in coenzyme B biosynthesis

Homoaconitase enzymes catalyze hydrolyase reactions in the alpha-aminoadipate pathway for lysine biosynthesis or the 2-oxosuberate pathway for methanogenic coenzyme B biosynthesis. Despite the homology of this iron-sulfur protein to aconitase, previously studied homoaconitases catalyze only the hydration of cis-homoaconitate to form homoisocitrate rather than the complete isomerization of homocitrate to homoisocitrate. The MJ1003 and MJ1271 proteins from the methanogen Methanocaldococcus jannaschii formed the first homoaconitase shown to catalyze both the dehydration of (R)-homocitrate to form cis-homoaconitate, and its hydration is shown to produce homoisocitrate. This heterotetrameric enzyme also used the analogous longer chain substrates cis-(homo)(2)aconitate, cis-(homo)(3)aconitate, and cis-(homo)(4)aconitate, all with similar specificities. A combination of the homoaconitase with the M. jannaschii homoisocitrate dehydrogenase catalyzed all of the isomerization and oxidative decarboxylation reactions required to form 2-oxoadipate, 2-oxopimelate, and 2-oxosuberate, completing three iterations of the 2-oxoacid elongation pathway. Methanogenic archaeal homoaconitases and fungal homoaconitases evolved in parallel in the aconitase superfamily. The archaeal homoaconitases share a common ancestor with isopropylmalate isomerases, and both enzymes catalyzed the hydration of the minimal substrate maleate to form d-malate. The variation in substrate specificity among these enzymes correlated with the amino acid sequences of a flexible loop in the small subunits.     

Isoleucine biosynthesis in Leptospira interrogans serotype lai strain 56601 proceeds via a threonine-independent pathway

Three leuA-like protein-coding sequences were identified in Leptospira interrogans. One of these, the cimA gene, was shown to encode citramalate synthase (EC 4.1.3.-). The other two encoded alpha-isopropylmalate synthase (EC 4.1.3.12). Expressed in Escherichia coli, the citramalate synthase was purified and characterized. Although its activity was relatively low, it was strictly specific for pyruvate as the keto acid substrate. Unlike the citramalate synthase of the thermophile Methanococcus jannaschii, the L. interrogans enzyme is temperature sensitive but exhibits a much lower K(m) (0.04 mM) for pyruvate. The reaction product was characterized as (R)-citramalate, and the proposed beta-methyl-d-malate pathway was further confirmed by demonstrating that citraconate was the substrate for the following reaction. This alternative pathway for isoleucine biosynthesis from pyruvate was analyzed both in vitro by assays of leptospiral isopropylmalate isomerase (EC 4.2.1.33) and beta-isopropylmalate dehydrogenase (EC 1.1.1.85) in E. coli extracts bearing the corresponding clones and in vivo by complementation of E. coli ilvA, leuC/D, and leuB mutants. Thus, the existence of a leucine-like pathway for isoleucine biosynthesis in L. interrogans under physiological conditions was unequivocally proven. Significant variations in either the enzymatic activities or mRNA levels of the cimA and leuA genes were detected in L. interrogans grown on minimal medium supplemented with different levels of the corresponding amino acids or in cells grown on serum-containing rich medium. The similarity of this metabolic pathway in leptospires and archaea is consistent with the evolutionarily primitive status of the eubacterial spirochetes.     

A comprehensive survey on isoleucine biosynthesis pathways in seven epidemic Leptospira interrogans reference strains of China

Previous studies have indicated that different species of Leptospira synthesize isoleucine via either pyruvate and/or threonine pathways. Seven epidemic Leptospira interrogans reference strains from China belonging to different serovars, together with three saprophytic strains of Leptospira biflexa and Leptospira meyeri, were analysed. The isoleucine biosynthesis properties were studied firstly by measuring the key enzymes of the two pathways, citramalate synthase (CimA, CE4.1.3.-) and threonine deaminase (IlvA, CE4.2.1.16), from cell extracts of the bacteria. Meanwhile, alpha-isopropylmalate synthase (LeuA, CE4.2.1.12), the key enzyme of leucine biosynthesis, was also measured as a control. It was found that all L. interrogans strains synthesized isoleucine via the pyruvate pathway exclusively, but L. biflexa and L. meyeri used both pathways. Dot-Blot and PCR amplification of both cimA and ilvA genes in the corresponding strains provided additional evidence consistent with the data of enzymatic assays. Although it is evident that leptospires' isoleucine biosynthesis may preferentially adapt either to the pyruvate pathway exclusively for pathogens or to the combination of both pyruvate and threonine pathways for saprophytes, broader sampling with careful genomospecies identification is needed for a solid conclusion.     

Alternative pathways for biosynthesis of leucine and other amino acids in Bacteroides ruminicola and Bacteroides fragilis

Bacteroides ruminicola is one of several species of anaerobes that are able to reductively carboxylate isovalerate (or isovaleryl-coenzyme A) to synthesize alpha-ketoisocaproate and thus leucine. When isovalerate was not supplied to growing B. ruminicola cultures, carbon from [U-14C]glucose was used for the synthesis of leucine and other cellular amino acids. When unlabeled isovalerate was available, however, utilization of [U-14C]glucose or [2-14C]acetate for leucine synthesis was markedly and specifically reduced. Enzyme assays indicated that the key enzyme of the common isopropylmalate (IPM) pathway for leucine biosynthesis, IPM synthase, was present in B. ruminicola cell extracts. The specific activity of IPM synthase was reduced when leucine was added to the growth medium but was increased by the addition of isoleucine plus valine, whereas the addition of isovalerate had little or no effect. The activity of B. ruminicola IPM synthase was strongly inhibited by leucine, the end product of the pathway. It seems unlikely that the moderate inhibition of the enzyme by isovalerate adequately explains the regulation of carbon flow by isovalerate in growing cultures. Bacteroides fragilis apparently also uses either the isovalerate carboxylation or the IPM pathway for leucine biosynthesis. Furthermore, both of these organisms synthesize isoleucine and phenylalanine, using carbon from 2-methylbutyrate and phenylacetate, respectively, in preference to synthesis of these amino acids de novo from glucose. Thus, it appears that these organisms have the ability to regulate alternative pathways for the biosynthesis of certain amino acids and that pathways involving reductive carboxylations are likely to be favored in their natural habitats.     

Alternative pathways for biosynthesis of leucine and other amino acids in Bacteroides ruminicola and Bacteroides fragilis.

Bacteroides ruminicola is one of several species of anaerobes that are able to reductively carboxylate isovalerate (or isovaleryl-coenzyme A) to synthesize alpha-ketoisocaproate and thus leucine. When isovalerate was not supplied to growing B. ruminicola cultures, carbon from [U-14C]glucose was used for the synthesis of leucine and other cellular amino acids. When unlabeled isovalerate was available, however, utilization of [U-14C]glucose or [2-14C]acetate for leucine synthesis was markedly and specifically reduced. Enzyme assays indicated that the key enzyme of the common isopropylmalate (IPM) pathway for leucine biosynthesis, IPM synthase, was present in B. ruminicola cell extracts. The specific activity of IPM synthase was reduced when leucine was added to the growth medium but was increased by the addition of isoleucine plus valine, whereas the addition of isovalerate had little or no effect. The activity of B. ruminicola IPM synthase was strongly inhibited by leucine, the end product of the pathway. It seems unlikely that the moderate inhibition of the enzyme by isovalerate adequately explains the regulation of carbon flow by isovalerate in growing cultures. Bacteroides fragilis apparently also uses either the isovalerate carboxylation or the IPM pathway for leucine biosynthesis. Furthermore, both of these organisms synthesize isoleucine and phenylalanine, using carbon from 2-methylbutyrate and phenylacetate, respectively, in preference to synthesis of these amino acids de novo from glucose. Thus, it appears that these organisms have the ability to regulate alternative pathways for the biosynthesis of certain amino acids and that pathways involving reductive carboxylations are likely to be favored in their natural habitats.     

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.     

Elucidation of an alternate isoleucine biosynthesis pathway in Geobacter sulfurreducens

The central metabolic model for Geobacter sulfurreducens included a single pathway for the biosynthesis of isoleucine that was analogous to that of Escherichia coli, in which the isoleucine precursor 2-oxobutanoate is generated from threonine. 13C labeling studies performed in G. sulfurreducens indicated that this pathway accounted for a minor fraction of isoleucine biosynthesis and that the majority of isoleucine was instead derived from acetyl-coenzyme A and pyruvate, possibly via the citramalate pathway. Genes encoding citramalate synthase (GSU1798), which catalyzes the first dedicated step in the citramalate pathway, and threonine ammonia-lyase (GSU0486), which catalyzes the conversion of threonine to 2-oxobutanoate, were identified and knocked out. Mutants lacking both of these enzymes were auxotrophs for isoleucine, whereas single mutants were capable of growth in the absence of isoleucine. Biochemical characterization of the single mutants revealed deficiencies in citramalate synthase and threonine ammonia-lyase activity. Thus, in G. sulfurreducens, 2-oxobutanoate can be synthesized either from citramalate or threonine, with the former being the main pathway for isoleucine biosynthesis. The citramalate synthase of G. sulfurreducens constitutes the first characterized member of a phylogenetically distinct clade of citramalate synthases, which contains representatives from a wide variety of microorganisms.     

Biosynthesis of phosphoserine in the Methanococcales

Methanococcus maripaludis and Methanocaldococcus jannaschii produce cysteine for protein synthesis using a tRNA-dependent pathway. These methanogens charge tRNA(Cys) with l-phosphoserine, which is also an intermediate in the predicted pathways for serine and cystathionine biosynthesis. To establish the mode of phosphoserine production in Methanococcales, cell extracts of M. maripaludis were shown to have phosphoglycerate dehydrogenase and phosphoserine aminotransferase activities. The heterologously expressed and purified phosphoglycerate dehydrogenase from M. maripaludis had enzymological properties similar to those of its bacterial homologs but was poorly inhibited by serine. While bacterial enzymes are inhibited by micromolar concentrations of serine bound to an allosteric site, the low sensitivity of the archaeal protein to serine is consistent with phosphoserine's position as a branch point in several pathways. A broad-specificity class V aspartate aminotransferase from M. jannaschii converted the phosphohydroxypyruvate product to phosphoserine. This enzyme catalyzed the transamination of aspartate, glutamate, phosphoserine, alanine, and cysteate. The M. maripaludis homolog complemented a serC mutation in the Escherichia coli phosphoserine aminotransferase. All methanogenic archaea apparently share this pathway, providing sufficient phosphoserine for the tRNA-dependent cysteine biosynthetic pathway.     

Amino Acid Biosynthesis in the Spirochete Leptospira: Evidence for a Novel Pathway of Isoleucine Biosynthesis

Radioactive carbon dioxide was incubated with growing cells of Leptospira interrogans serotypes semaranga and tarassovi, and the specific activities and distribution of the label within the cellular amino acids were determined. The origins of the carbon skeletons of all the acid-stable amino acids except isoleucine were found to be consistent with known biosynthetic pathways for these amino acids. Experiments using radioactive carbon dioxide and other tracers indicated that most of the isoleucine was synthesized by a pathway not involving threonine. The origin of the carbon skeleton of isoleucine consisted of two residues of pyruvate (carbons 2 and 3) and acetate of acetyl-coenzyme A by this pathway. Isotope competition studies indicated that the pathway was regulated by isoleucine. The results are discussed in relation to two proposed pathways of isoleucine biosynthesis involving citramalate as an intermediate.     

Mechanism of biosynthesis of 2-oxo-3-methylvalerate in Chlorobium vibrioforme.

The biosynthesis of 2-oxo-3-methylvalerate in Chlorobium vibrioforme was investigated by 13C nuclear magnetic resonance spectroscopy of the oxoacid formed from 13C-labeled acetate by washed suspensions. The threonine pathway could be excluded, and the results are in accord with a mechanism for the formation of 2-oxobutyrate from acetyl coenzyme A and pyruvate via citramalate.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> encodes a bacterial-type heterodimeric isopropylmalate isomerase involved in both Leu biosynthesis and the Met chain elongation pathway of glucosinolate formation

The last steps of the Leu biosynthetic pathway and the Met chain elongation cycle for glucosinolate formation share identical reaction types suggesting a close evolutionary relationship of these pathways. Both pathways involve the condensation of acetyl-CoA and a 2-oxo acid, isomerization of the resulting 2-malate derivative to form a 3-malate derivative, the oxidation-decarboxylation of the 3-malate derivative to give an elongated 2-oxo acid, and transamination to generate the corresponding amino acid. We have now analyzed the genes encoding the isomerization reaction, the second step of this sequence, in Arabidopsis thaliana. One gene encodes the large subunit and three encode small subunits of this enzyme, referred to as isopropylmalate isomerase (IPMI) with respect to the Leu pathway. Metabolic profiling of large subunit mutants revealed accumulation of intermediates of both Leu biosynthesis and Met chain elongation, and an altered composition of aliphatic glucosinolates demonstrating the function of this gene in both pathways. In contrast, the small subunits appear to be specialized to either Leu biosynthesis or Met chain elongation. Green fluorescent protein tagging experiments confirms the import of one of the IPMI small subunits into the chloroplast, the localization of the Met chain elongation pathway in these organelles. These results suggest the presence of different heterodimeric IPMIs in Arabidopsis chloroplasts with distinct substrate specificities for Leu or glucosinolate metabolism determined by the nature of the different small subunit. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

The Product of the LEU-3 Cistron as a Regulatory Element for the Production of the Leucine Biosynthetic Enzymes of Neurospora

A class of intracistronic (or closely linked) partial reversions of leu-3 mutations has been found to be conditionally constitutive with respect to the synthesis of isopropylmalate isomerase (specified by the leu-2 cistron) and beta-isopropylmalate dehydrogenase (specified by the leu-1 cistron), two of the enzymes of leucine biosynthesis in Neurospora. The intermediate level of enzyme production by these leu-3(cc) mutants is independent of the obligatory inducer effector, alpha-isopropylmalate, but dependent upon the presence of the branched-chain amino acids, isoleucine, valine and leucine. The properties of leu-3+, leu-3 and leu-3(cc) in heterokaryons indicate that the transnuclear regulatory activity of the leu-3 product varies specifically as a function of available effector molecules. The information presented suggests that the leu-3 cistron is responsible not only for the production of a "positive" regulatory substance necessary for the expression of the leu-1 and leu-2 cistrons, but that it probably serves also a coordinating role in the expression of many of the genes involved in branched-chain amino acid metabolism.     

Leucine synthesis in Corynebacterium glutamicum: enzyme activities, structure of leuA, and effect of leuA inactivation on lysine synthesis.

Enzymes and genes of the isopropylmalate pathway leading to leucine in Corynebacterium glutamicum were studied, and assays were performed to unravel their connection to lysine oversynthesis. The first enzyme of the pathway is inhibited by leucine (Ki = 0.4 mM), and all three enzyme activities of the isopropylmalate pathway are reduced upon addition of this amino acid to the growth medium. Three different DNA fragments were cloned, each resulting in an oversynthesis of one of the three enzymes. The leuA complementing fragment encoding the isopropylmalate synthase was sequenced. The leuA gene is 1,848 bp in size, encoding a polypeptide with an M(r) of 68,187. Upstream of leuA there is extensive hyphenated dyad symmetry and a putative leader peptide, which are features characteristic of attenuation control. In addition to leuA, the sequenced fragment contains an open reading frame with high coding probability whose disruption did not result in a detectable phenotype. Furthermore, the sequence revealed that this open reading frame separates leuA from lysC, which encodes the aspartate kinase initiating the synthesis of all amino acids of the aspartate family. The leuA gene was inactivated in three lysine-secreting strains by insertional mutagenesis. Fermentations were performed, and a roughly 50% higher lysine yield was obtained when appropriate leucine concentrations limiting for growth of the constructed strains were used.     

Identification of coenzyme M biosynthetic 2-phosphosulfolactate phosphatase. A member of a new class of Mg(2+)-dependent acid phosphatases.

Coenzyme M (CoM; 2-mercaptoethanesulfonic acid) is the terminal methyl carrier in methanogenesis. Methanogenic archaea begin the production of this essential cofactor by sulfonating phosphoenolpyruvate to form 2-phospho-3-sulfolactate. After dephosphorylation, this precursor is oxidized, decarboxylated and then reductively thiolated to form CoM. A thermostable phosphosulfolactate phosphohydrolase (EC 3.1.3.-) catalyzing the second step in CoM biosynthesis, was identified in the hyperthermophilic euryarchaeon Methanococcus jannaschii. The predicted ORF MJ1140 in the genome of M. jannaschii encodes ComB, a Mg2+-dependent acid phosphatase that is specific for 2-hydroxycarboxylic acid phosphate esters. Recombinantly expressed purified ComB efficiently hydrolyzes rac-2-phosphosulfolactate, (S)-2-phospholactate, phosphoglycolate and both enantiomers of 2-phosphomalate. In contrast to previously studied phosphoglycolate phosphatases, ComB has a low pH optimum for activity, a narrow substrate specificity and an amino acid sequence dissimilar to any biochemically characterized protein. Like other phosphatases that function via covalent phosphoenzyme intermediates, ComB can catalyze a transphosphorylation reaction. Homologs of comB are identified in all available cyanobacterial genome sequences and in genomes from phylogenetically diverse bacteria and archaea; most of these organisms lack homologs of other CoM biosynthetic genes. The broad and disparate distribution of comB homologs suggests that the gene has been recruited frequently into new metabolic pathways.     

A redox‐active isopropylmalate dehydrogenase functions in the biosynthesis of glucosinolates and leucine in Arabidopsis

We report a detailed functional characterization of an Arabidopsis isopropylmalate dehydrogenase (AtIPMDH1) that is involved in both glucosinolate biosynthesis and leucine biosynthesis. AtIPMDH1 shares high homology with enzymes from bacteria and yeast that are known to function in leucine biosynthesis. In plants, AtIPMDH1 is co‐expressed with nearly all the genes known to be involved in aliphatic glucosinolate biosynthesis. Mutation of AtIPMDH1 leads to a significant reduction in the levels of free leucine and of glucosinolates with side chains of four or more carbons. Complementation of the mutant phenotype by ectopic expression of AtIPMDH1, together with the enzyme’s substrate specificity, implicates AtIPMDH1 in both glucosinolate and leucine biosynthesis. This functional assignment is substantiated by subcellular localization of the protein in the chloroplast stroma, and the gene expression patterns in various tissues. Interestingly, AtIPMDH1 activity is regulated by a thiol‐based redox modification. This work characterized an enzyme in plants that catalyzes the oxidative decarboxylation step in both leucine biosynthesis (primary metabolism) and methionine chain elongation of glucosinolates (specialized metabolism). It provides evidence for the hypothesis that the two pathways share a common origin, and suggests a role for redox regulation of glucosinolate and leucine synthesis in plants.     

Wild-type isopropylmalate isomerase in Salmonella typhimurium is composed of two different subunits.

The isopropylmalate isomerase in Salmonella typhimurium is the second enzyme specific for leucine biosynthesis. It is a complex enzyme composed of two subunits which are coded for by two genes of the leucine operon, leuC and leuD. The two polypeptides have been shown to copurify through successive ammonium sulfate fractionations and have been identified on sodium dodecyl sulfate-polyacrylamide gels as having molecular weights of 51,000 (leuC gene product) and 23,500 (leuD gene product). They have also been shown to be fairly stable, since in vitro complementation of cell-free extracts of leuC and leuD mutant strains was demonstrated, with only a 40% loss of activity 16 h after preparation of the extracts. The native isopropylmalate isomerase was shown to have a Km for its substrate alpha-isopropylmalate of 3 x 10(-4)M.     

MAM3 catalyzes the formation of all aliphatic glucosinolate chain lengths in Arabidopsis

Chain elongated, methionine (Met)-derived glucosinolates are a major class of secondary metabolites in Arabidopsis (Arabidopsis thaliana). The key enzymatic step in determining the length of the chain is the condensation of acetyl-coenzyme A with a series of omega-methylthio-2-oxoalkanoic acids, catalyzed by methylthioalkylmalate (MAM) synthases. The existence of two MAM synthases has been previously reported in the Arabidopsis ecotype Columbia: MAM1 and MAM3 (formerly known as MAM-L). Here, we describe the biochemical properties of the MAM3 enzyme, which is able to catalyze all six condensation reactions of Met chain elongation that occur in Arabidopsis. Underlining its broad substrate specificity, MAM3 also accepts a range of non-Met-derived 2-oxoacids, e.g. converting pyruvate to citramalate and 2-oxoisovalerate to isopropylmalate, a step in leucine biosynthesis. To investigate its role in vivo, we identified plant lines with mutations in MAM3 that resulted in a complete lack or greatly reduced levels of long-chain glucosinolates. This phenotype could be complemented by reintroduction of a MAM3 expression construct. Analysis of MAM3 mutants demonstrated that MAM3 catalyzes the formation of all glucosinolate chain lengths in vivo as well as in vitro, making this enzyme the major generator of glucosinolate chain length diversity in the plant. The localization of MAM3 in the chloroplast suggests that this organelle is the site of Met chain elongation.     

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.