Methanogen Homoaconitase Catalyzes Both Hydrolyase Reactions in Coenzyme B Biosynthesis

Homoaconitase enzymes catalyze hydrolyase reactions in the α-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)2aconitate, cis-(homo)3aconitate, and cis-(homo)4aconitate, 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. Homoaconitase enzymes catalyze hydrolyase reactions in the α-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)2aconitate, cis-(homo)3aconitate, and cis-(homo)4aconitate, 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. In the final reaction of methanogenesis, the nickel metalloenzyme methyl-coenzyme M (CoM) 2The abbreviations used are: CoM, coenzyme M; CoB, coenzyme B; HCS, homocitrate synthase; HACN, homoaconitase; HICDH, homoisocitrate dehydrogenase; IPMI, isopropylmalate isomerase; CHES, 2-(cyclohexylamino)ethanesulfonic acid; TAPS, tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid; HPLC, high pressure liquid chromatography; LC-MS, liquid chromatography-mass spectrometry. reductase catalyzes the attack of the coenzyme B (CoB) thiol on the methyl thioether of methyl-CoM, releasing methane and forming a heterodisulfide compound (CoM-S-S-CoB) (1Horng Y.-C. Becker D.F. Ragsdale S.W. Biochemistry. 2001; 40: 12875-12885Crossref PubMed Scopus (51) Google Scholar). To reach the deeply buried active site of methyl-CoM reductase, CoB contains a long 7-mercaptoheptanoyl chain with an amide bond to phosphothreonine (2Noll K.M. Rinehart Jr., K.L. Tanner R.S. Wolfe R.S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4238-4242Crossref PubMed Scopus (78) Google Scholar, 3Ermler U. Grabarse W. Shima S. Goubeaud M. Thauer R.K. Science. 1997; 278: 1457-1462Crossref PubMed Scopus (438) Google Scholar). Methanogens make the 7-mercaptoheptanoate moiety of CoB from a 2-oxosuberate intermediate, which these cells produce using a series of 2-oxoacid elongation reactions (4White R.H. Biochemistry. 1989; 28: 860-865Crossref Scopus (15) Google Scholar). 2-Oxosuberate biosynthesis begins with 2-oxoglutarate from central metabolism (Fig. 1A). The homocitrate synthase enzyme (HCS) catalyzes the aldol-like addition of an acetyl group from acetyl-coenzyme A to 2-oxoglutarate to produce (R)-homocitrate. The homoaconitase enzyme (HACN) is proposed to catalyze the anti-elimination of water, forming the cis-homoaconitate intermediate, and its hydration is proposed to produce (2R,3S)-homoisocitrate. Finally, the NAD+-dependent homoisocitrate dehydrogenase enzyme (HICDH) oxidizes and decarboxylates the β-hydroxytricarboxylic acid to produce 2-oxoadipate. Analogous reactions are found in the citric acid (Krebs) cycle, the isopropylmalate pathway to leucine, the pyruvate pathway to 2-oxobutyrate, and the α-aminoadipate pathway (5Jensen R.A. Annu. Rev. Microbiol. 1976; 30: 409-425Crossref PubMed Scopus (826) Google Scholar). In the latter case, some bacteria and fungi use HCS, HACN, and HICDH enzymes to produce 2-oxoadipate for lysine biosynthesis (6Xu H. Andi B. Qian J. West A.H. Cook P.F. Cell Biochem. Biophys. 2006; 46: 43-64Crossref PubMed Scopus (127) Google Scholar). However, methanogens repeat this series of reactions two more times, using 2-oxoadipate to produce 2-oxopimelate and using 2-oxopimelate to produce 2-oxosuberate. The enzymes in the CoB pathway evolved from leucine biosynthetic proteins that catalyze analogous reactions in the isopropylmalate pathway to 2-oxoisocaproate (Fig. 1B). HCS was identified in the methanogen Methanocaldococcus jannaschii as a paralog of isopropylmalate synthase (7Howell D.M. Harich K. Xu H. White R.H. Biochemistry. 1998; 37: 10108-10117Crossref PubMed Scopus (39) Google Scholar). Remarkably, this HCS was reported to convert 2-oxoglutarate and acetyl-CoA to trans-homoaconitate, which was hydrated to form (S)-homocitrate. But in analogous reactions, HCS produces (R)-homo2citrate from 2-oxoadipate and (R)-homo3citrate from 2-oxopimelate. Although different acetyl-CoA condensing enzymes are known to have different stereospecificities, each enzyme usually forms only one product (8Bentley R. Bentley R. Molecular Asymmetry in Biology. Academic Press, New York1970: 90-163Google Scholar). The yeast HCS produces only (R)-homocitrate (9Thomas U. Kalyanpur M.G. Stevens C.M. Biochemistry. 1966; 5: 2513-2516Crossref PubMed Scopus (24) Google Scholar). (R)-Homocitrate is also a key component of the nitrogenase iron-molybdenum cofactor found in many bacteria and some methanogens (10Curatti L. Hernandez J.A. Igarashi R.Y. Soboh B. Zhao D. Rubio L.M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 17626-17631Crossref PubMed Scopus (78) Google Scholar). Subsequently the M. jannaschii HICDH was identified as a paralog of isopropylmalate dehydrogenase (11Howell D.M. Graupner M. Xu H. White R.H. J. Bacteriol. 2000; 182: 5013-5016Crossref PubMed Scopus (28) Google Scholar). This enzyme catalyzes the NAD+-dependent oxidation of threo-homoisocitrate, threo-(homo)2isocitrate, and threo-(homo)3isocitrate with similar specificity constants. Experiments using cell-free extract from Methanosarcina thermophila indicated that (–)-threo-homoisocitrate or (2R,3S)-homoisocitrate is the relevant diastereomer substrate for methanogen HICDH, similar to bacterial and yeast enzymes (7Howell D.M. Harich K. Xu H. White R.H. Biochemistry. 1998; 37: 10108-10117Crossref PubMed Scopus (39) Google Scholar, 12Yamamoto T. Miyazaki K. Eguchi T. Bioorg. Med. Chem. 2007; 15: 1346-1355Crossref PubMed Scopus (8) Google Scholar). The HACN enzyme in CoB biosynthesis has not yet been identified. It is predicted to differ from the previously characterized HACN enzymes that function in α-aminoadipate pathways for lysine biosynthesis in Saccharomyces cerevisiae (13Strassman M. Ceci L.N. J. Biol. Chem. 1966; 241: 5401-5407Abstract Full Text PDF PubMed Google Scholar), Aspergillus nidulans (14Weidner G. Steffan B. Brakhage A.A. Mol. Gen. Genet. 1997; 255: 237-247Crossref PubMed Scopus (40) Google Scholar), and the bacterium Thermus thermophilus (15Jia Y. Tomita T. Yamauchi K. Nishiyama M. Palmer D.R.J. Biochem. J. 2006; 396: 479-485Crossref PubMed Scopus (17) Google Scholar). Along with the homologous 2-methyl-cis-aconitate hydratase and dimethylmalate dehydratase proteins, those HACN proteins catalyze only half of an aconitase-like reaction (16Grimek T.L. Escalante-Semerena J.C. J. Bacteriol. 2004; 186: 454-462Crossref PubMed Scopus (23) Google Scholar, 17Kollmann-Koch A. Eggerer H. Hoppe-Seyler's Z. Physiol. Chem. 1984; 365: 847-857Crossref PubMed Scopus (10) Google Scholar). The HACN enzymes catalyze the hydration of cis-homoaconitate to form homoisocitrate (18Bhattacharjee J.K. Mortlock R.P. The Evolution of Metabolic Function. CRC Press, Boca Raton, FL1992: 47-80Google Scholar). No HACN has been shown to catalyze the dehydration of homocitrate to form cis-homoaconitate, but an aconitase enzyme was proposed to catalyze the dehydration of homocitrate in aerobic fungi and T. thermophilus. Methanogens have no aconitase, so their HACN must catalyze both half-reactions. Additionally, the methanogen HACN must use tricarboxylate substrates of varying chain length, whereas the HACN proteins from α-aminoadipate pathways need only recognize cis-homoaconitate. Genomes from methanogens that produce CoB contain two copies of genes annotated as encoding large and small subunits of [4Fe-4S]-dependent isopropylmalate isomerases. The large subunit paralogs share more than 50% amino acid sequence identity with each other and more than 40% identity with the T. thermophilus HACN subunits. The methanogen small subunits are likewise more similar to each other than to bacterial or eukaryotic sequences. These genes are scattered around the chromosomes of most methanogens, with little contextual information about gene function. Therefore bioinformatic analysis cannot distinguish the functions of these proteins. We previously identified one combination of M. jannaschii proteins (MJ0499 and MJ1277) as the isopropylmalate/citramalate isomerase that is involved in leucine and isoleucine biosyntheses (19Drevland R.M. Waheed A. Graham D.E. J. Bacteriol. 2007; 189: 4391-4400Crossref PubMed Scopus (52) Google Scholar). In this work, the MJ1003 and MJ1271 proteins from M. jannaschii formed an active homoaconitase enzyme. HACN activity evolved in parallel in the archaeal and fungal lineages from isopropylmalate isomerase or aconitase ancestors, respectively. Direct assays showed that this reconstituted ironsulfur enzyme catalyzed the hydration of cis-homoaconitate, cis-homo2aconitate, cis-homo3aconitate, and even cis-homo4aconitate with comparable specificity. cis-Aconitate and citraconate were not substrates, although the minimal substrate maleate was hydrated to produce d-malate. In a coupled reaction with HICDH, the homoaconitate analogs were converted to their corresponding 2-oxoacids, demonstrating that the homoisocitrate products are substrates for HICDH. In contrast to previously studied HACN proteins, the methanogen enzyme catalyzed both the dehydration of (R)-homocitrate to form cis-homoaconitate and the subsequent hydration reaction that forms homoisocitrate. HICDH oxidizes and decarboxylates homoisocitrate to make 2-oxoadipate. (S)-Homocitrate and trans-homoaconitate were inhibitors rather than substrates for HACN, so HACN cannot catalyze all of the predicted dehydratase reactions in the original pathway for CoB biosynthesis. Chemicals and Reagents—(R)-Homocitrate and (S)-homocitrate were synthesized through the diastereoselective alkylation of malic acid. (2R,3S)-Homoisocitrate was synthesized from dimethyl d-malate (20Ma G. Palmer D.R.J. Tetrahedron Lett. 2000; 41: 9209-9212Crossref Scopus (21) Google Scholar). p-Toluenesulfonic acid was recrystallized from ethyl acetate. Synthesis of 2-Oxoacids and Homoaconitic Acids—Massoudi et al. (21Massoudi H.H. Cantacuzene D. Wakselman C. de la Tour C.B. Synthesis. 1983; 12: 1010-1012Crossref Scopus (11) Google Scholar) used the unstable t-butyl ester of 2-oxoglutarate in a Wittig-Horner reaction to preferentially produce cis-homoaconitate. We have used the stable methyl ester of 2-oxoglutarate 4a to produce homoaconitate ester (Fig. (7Howell D.M. Harich K. Xu H. White R.H. Biochemistry. 1998; 37: 10108-10117Crossref PubMed Scopus (39) Google Scholar). ester was from the cis-homoaconitate ester The ester was with and from the to produce acid. The of the homoaconitate was The of the for each ester were of these were the of and and were the of these are in the was with in of was to the The reaction was with of and the was to with The was and The was to a in was in of for to produce acid Proc. 5: Scopus Google Scholar). The was and acid was from was the for using of was the for using of 2-oxoadipate was the of 2-oxoadipate in of with of and acid D.M. Y. J. Chem. PubMed Scopus Google Scholar). The was and the product was through a of was in of in of was with for in of was and the was for The reaction was with of and with The was with and to produce in as a The were on with and of the were as to produce acid and acid as of substrates were to with was from as for a acid was from a and Molecular the protein with was a from Xu and H. White (11Howell D.M. Graupner M. Xu H. White R.H. J. Bacteriol. 2000; 182: 5013-5016Crossref PubMed Scopus (28) Google Scholar). showed that this gene two and with the sequence reported the White L. J.A. L.M. R.A. R. A. J.C. Science. Scopus Google Scholar). The in a amino acid whereas the was Therefore the gene from of M. jannaschii using using and was the and of to and in the site of to of the from The was also identified in the sequences of these gene sequence was were in the the for and Molecular of the sequences. for the M. jannaschii dehydratase subunits were previously (19Drevland R.M. Waheed A. Graham D.E. J. Bacteriol. 2007; 189: 4391-4400Crossref PubMed Scopus (52) Google Scholar) and are in the and MJ1003 and MJ1271 proteins were in an previously (19Drevland R.M. Waheed A. Graham D.E. J. Bacteriol. 2007; 189: 4391-4400Crossref PubMed Scopus (52) Google Scholar). The cells were with the an of was the addition of and the was to a and for The MJ1003 and MJ1271 proteins were of the and (19Drevland R.M. Waheed A. Graham D.E. J. Bacteriol. 2007; 189: 4391-4400Crossref PubMed Scopus (52) Google Scholar). The protein was using a with a The HICDH protein was in an The cells were in and with HICDH protein and were as for the proteins. of the a MJ1003 and MJ1271 proteins were with and as previously (19Drevland R.M. Waheed A. Graham D.E. J. Bacteriol. 2007; 189: 4391-4400Crossref PubMed Scopus (52) Google Scholar). The was activity was activity was in a with cis-homoaconitate as A of the of was reconstituted using and was using a with an of was in an with an of the protein were and were for and analysis was using the with a from G. J.A. Biochemistry. 41: PubMed Scopus Google Scholar). The was using the with the addition of H. Biochem. 1983; PubMed Scopus Google Scholar). The protein was the using as a The for each were to the with each of were in with and substrate The and substrate was for and for The reactions were the addition of and the in was were from the of the reaction for for for and and for of the were in the reaction of hydrolyase activity catalyzed the of of substrate to product were of to the (19Drevland R.M. Waheed A. Graham D.E. J. Bacteriol. 2007; 189: 4391-4400Crossref PubMed Scopus (52) Google Scholar). The inhibitors were in assays HACN, and of substrate these reactions were the addition of cis-homoaconitate, and hydrolyase activity was The of maleate hydration products was using dehydrogenase and dehydrogenase enzymes (19Drevland R.M. Waheed A. Graham D.E. J. Bacteriol. 2007; 189: 4391-4400Crossref PubMed Scopus (52) Google Scholar). of hydration of in the was a coupled reaction with HICDH the of to (15Jia Y. Tomita T. Yamauchi K. Nishiyama M. Palmer D.R.J. Biochem. J. 2006; 396: 479-485Crossref PubMed Scopus (17) Google Scholar). and HICDH were as which was to a final of The reaction were to and the reactions were with of The were using the of the reaction of activity catalyzed the of The were as of reactions cis-homoaconitate analogs and HACN were for A of the reaction product was using a with a was with an with and in a of for were and products from with and cis-homo4aconitate were using and in The were and A was used to cis-homoaconitate analogs whereas products of reactions homoaconitate and were for The reaction was to with and with ethyl and with The were The was in and using a The was to a to and with a from to with acid in a of was in the the analysis of coupled reaction and HICDH were to the reaction To form the of 2-oxoacid the was to with and of was The were for and to with and with The was as The was to a and with a from to in acid a of was in the of the MJ1003 and MJ1271 proteins were previously in and the was to and The subunits to form a similar to the homologous isopropylmalate isomerase (19Drevland R.M. Waheed A. Graham D.E. J. Bacteriol. 2007; 189: 4391-4400Crossref PubMed Scopus (52) Google Scholar). The was with and to an iron-sulfur The and of a the the not catalyze the hydration of citraconate or the dehydration of To this homoaconitase cis-homoaconitate was synthesized using the Wittig-Horner reaction (Fig. The reconstituted catalyzed the hydration of cis-homoaconitate with a activity of in a activity was and with activity No was required in this although activity was with in the of was only of the activity with In the MJ1003 and small proteins that also formed a no activity with analysis of the reaction products showed a in substrate and the of one corresponding to homocitrate (Fig. No homoisocitrate was analysis of the reaction identified corresponding to the of homocitrate or homoisocitrate and corresponding to the of a hydrolyase that the iron-sulfur in the is for 2-Oxosuberate biosynthesis three of 2-oxoacid Therefore the methanogen HACN was predicted to catalyze the isomerization of homocitrate as as and the catalyzed the hydration of cis-homoaconitate, cis-homo2aconitate, cis-homo3aconitate, and even the cis-homo4aconitate substrates with similar (Fig. no hydration activity was in a was used as a substrate the was than the of of was dehydratase activity in reactions or for HACN were from the of the hydration reactions for all substrates The specificity for the substrates are although the for longer chain The is similar for all three relevant substrates but is for Therefore the methanogen HACN not substrates with in their for the of hydrolyase activity was the of in homoaconitate activity was the of in homoaconitate in a Despite its for long substrates, HACN catalyzed the hydration of a minimal of homoaconitate with a the HACN reactions maleate d-malate or and no analysis (19Drevland R.M. Waheed A. Graham D.E. J. Bacteriol. 2007; 189: 4391-4400Crossref PubMed Scopus (52) Google Scholar). Therefore the of this hydrolyase reaction is the as in the reactions, a similar of to the active The for maleate is than its for substrates However, the for maleate is high with for the tricarboxylate of HACN with M. jannaschii HICDH enzyme was previously shown to catalyze the oxidative decarboxylation of and (11Howell D.M. Graupner M. Xu H. White R.H. J. Bacteriol. 2000; 182: 5013-5016Crossref PubMed Scopus (28) Google Scholar). To the of the HACN hydration reaction of cis-homoaconitate to coupled reactions were with HACN and HICDH the of This coupled reaction activity with and a of The 2-oxoadipate product of these enzymes was identified as the (Fig. The for cis-homoaconitate in this coupled reaction is similar to that in the However, the of the of product HICDH. were for and substrates, 2-oxopimelate and 2-oxosuberate, (Fig. B and The cis-homo4aconitate substrate was also hydrated to produce that HICDH to form (Fig. Although cis-homo4aconitate is not both the HACN and HICDH enzymes recognize tricarboxylate substrates with two to in their for the coupled of HACN hydrolyase activity with not activity with not activity with in a and of analysis showed that the product of homoaconitate hydration was that the methanogen HACN catalyzed both in the isomerization of However, the of homocitrate in this pathway was The M. jannaschii HCS was previously reported to produce (S)-homocitrate from 2-oxoglutarate and but from 2-oxoadipate (7Howell D.M. Harich K. Xu H. White R.H. Biochemistry. 1998; 37: 10108-10117Crossref PubMed Scopus (39) Google Scholar). To which homocitrate is a substrate for HACN, each with HACN and HICDH in coupled (S)-Homocitrate was not a substrate for these enzymes (Fig. However, (R)-homocitrate was and with a activity of to a of for In assays the of the HACN protein catalyzed the dehydration of (R)-homocitrate with a of The of dehydration is than the of cis-homoaconitate as for aconitase J. Biol. Chem. Full Text PDF PubMed Google Scholar). of using of cis-homoaconitate and (R)-homocitrate the HACN hydrolyase activity no activity was in the of (S)-homocitrate. HACN on whereas (S)-homocitrate the In reactions cis-homoaconitate, hydrolyase activity was 50% the and No was with citraconate to some and many use a homoaconitase enzyme in the α-aminoadipate pathway for lysine biosynthesis. with the homologous aconitase the HACN enzyme is to catalyze the isomerization of homocitrate to homoisocitrate. the previously studied catalyzed only the hydration of cis-homoaconitate, forming homoisocitrate. The M. jannaschii enzyme is the first HACN shown to catalyze both hydrolyase analysis showed that this enzyme preferentially homoaconitate to Direct assays that (R)-homocitrate was to form assays with HICDH showed that homoaconitate was also converted to homoisocitrate. The was used to that (R)-homocitrate converted to 2-oxoadipate, that the methanogen HACN catalyzes the complete isomerization of (R)-homocitrate to (2R,3S)-homoisocitrate. The of HACN for and cis-homoaconitate analogs that cannot catalyze all of the dehydratase reactions in CoB biosynthesis proposed previously (7Howell D.M. Harich K. Xu H. White R.H. Biochemistry. 1998; 37: 10108-10117Crossref PubMed Scopus (39) Google Scholar). enzymes are required in that or 2-oxoacid elongation through a pathway using only (R)-homocitrate analogs as shown in D.E. White R.H. PubMed Scopus Google Scholar). The of M. jannaschii cis-homoaconitate hydration is similar to the for the T. thermophilus HACN, which has a similar but a (15Jia Y. Tomita T. Yamauchi K. Nishiyama M. Palmer D.R.J. Biochem. J. 2006; 396: 479-485Crossref PubMed Scopus (17) Google Scholar). the M. jannaschii HACN cis-homoaconitate analogs with two to in the all with similar specificity constants. This substrate specificity is to the hydration of which has a The M. jannaschii HACN also catalyzes the hydration of the minimal substrate maleate that has no This reaction produces that the for the maleate and homoaconitate substrates are the maleate the longer chain analogs of cis-homoaconitate have been as substrates for the yeast or T. thermophilus HACN, so is this substrate specificity is to the methanogen with the of homoaconitate hydration catalyzed the M. jannaschii HACN, the of (R)-homocitrate dehydration is The was in the analysis of the M. jannaschii IPMI, which catalyzed the dehydration of and with of more than of than the specificity for citraconate hydration (19Drevland R.M. Waheed A. Graham D.E. J. Bacteriol. 2007; 189: 4391-4400Crossref PubMed Scopus (52) Google Scholar). the for this reaction and distinguish the different of substrate to all of the information about the reaction of and HACN proteins has been from aconitase H. Chem. Rev. PubMed Scopus Google Scholar). of aconitase with substrate analogs that the protein long substrate H. H. Biochemistry. PubMed Scopus Google Scholar). The of three and form with the of The is with in both and HACN proteins. The is in the small subunit proteins of and Therefore these two cannot substrate specificity. to the small subunit proteins of and contains a flexible loop with The sequence of this loop is and to the of substrate archaeal subunits have a sequence of whereas archaeal HACN subunits have a sequence of The in the and of this are in proteins, which the of their In contrast the HACN subunits have and that form an or bond with the have only one even these use both the isopropylmalate pathway and the α-aminoadipate pathway. Therefore a isomerase functions in both that the protein have a of The sequence for the flexible loop is which contains a of from both HACN and To substrate specificity of HACN to a substrate also the amino in the HACN to the substrate specificity of the enzyme. The aconitase that the and H. Chem. Rev. PubMed Scopus Google Scholar). This the of the yeast and bacterial HACN proteins to catalyze the dehydration of homocitrate The yeast HACN have a flexible loop sequence of the or form a bond with the of cis-homoaconitate. The bacterial have a loop sequence of that or amide with the substrate more than in these proteins. However, in the or of the loop also so required to this is and for the HACN specificity. The among 2-oxoacid elongation reactions of the cycle, leucine and the α-aminoadipate pathway has been for with the that the enzymes share common (5Jensen R.A. Annu. Rev. Microbiol. 1976; 30: 409-425Crossref PubMed Scopus (826) Google Scholar). The homocitrate to the superfamily. It is to isopropylmalate synthase and the but not to the common H. G. Thauer R.K. J. Bacteriol. 2007; 189: PubMed Scopus (52) Google Scholar), demonstrating HACN, and all to the of We have shown that M. jannaschii HACN and have substrate specificities. The and isopropylmalate to protein superfamily. We have also shown that M. jannaschii HICDH and isopropylmalate dehydrogenase recognize dehydratase products with Therefore the of an with substrate specificity the of each 2-oxoacid elongation pathway. This is with the parallel of HACN activity the dehydratase enzyme (Fig. and the We and for with and and for with

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