phosphothreonine and 7-mercaptoheptanoic-acid

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.

Topics: 3-Isopropylmalate Dehydrogenase; Alcohol Oxidoreductases; Cloning, Molecular; Enzyme Stability; Gene Expression; Heating; Heptanoic Acids; Isocitrate Dehydrogenase; Leucine; Methanococcus; Phosphothreonine; Sulfhydryl Compounds

The biochemical mechanism for the formation of the amide bond in N-(7-mercaptoheptanoyl)-L-threonine phosphate (HS-HTP) has been studied by measuring the incorporation of L-[3-(3)H]threonine into N-(7-mercaptoheptanoyl)-L-threonine (HS-HT) by cell extracts (CE) of Methanosarcina thermophila incubated with different precursors. Synthesis of HS-HT was observed from L-[3-(3)H]threonine and 7-mercaptoheptanoic acid (HS-H) when the incubations were conducted with either crude CE or Sephadex column-purified CE. In the presence of CE, the synthesis of HS-HT was found to be inhibited 66% by preincubation of the extract with ATPase, indicating that ATP was involved in the biosynthesis. In spite of this indication of ATP involvement in the coupling reaction, incubation of the crude CE with L-[3-(3)H]threonine, HS-H, and ATP was found to inhibit the formation of HS-HT. In contrast, the synthesis of HS-HT in the presence of Sephadex column-purified CE was found to be stimulated by the addition of ATP. Incubation of the crude CE with the CoA thioester of 7-mercaptoheptanoic acid (HS-HCoA) or the mixed disulfide formed between coenzyme M and 7-mercaptoheptanoic acid did not stimulate the biosynthesis. The biosynthesis of HS-HT was found to be strongly inhibited by an ethanol extract of the crude CE. This inhibition was found to be attributed to the HS-HTP present in the extract. Stimulation of HS-HT biosynthesis 300-fold was observed when the Sephadex column-purified CE was incubated with L-[3-(3)H]threonine and 7-mercaptoheptanoyl phosphate (HS-H-P). Data indicate that HS-HT is produced by the phosphorylation of HS-H to HS-H-P with ATP, which then reacts with L-threonine to produce HS-HT.

Topics: Adenosine Triphosphate; Amides; Coenzymes; Heptanoic Acids; Mesna; Methanosarcina; Phosphorylation; Phosphothreonine; Sulfhydryl Compounds; Threonine

The biosynthetic steps involved in the conversion of alpha-ketosuberate to 7-mercaptoheptanoic acid were studied in cell-free extracts of methanogenic bacteria. The pathway was established by measuring the incorporation of stable isotopically labeled precursors into the S-methyl ether methyl ester derivative of the enzymatically generated 7-mercaptoheptanoic acid by using gas chromatography-mass spectrometry (GC-MS). Quantitation of the 7-mercaptoheptanoic acid produced in the incubations with the substrates was accomplished by using an internal standard of 6-mercaptohexanoic acid. [4,4,6,6-2H4]-2-Oxosuberic acid, [7-2H]-7-oxoheptanoic acid, [2-2H]-2(RS)-(5-carboxypentyl)thiazolidine-4(R)-carboxylic acid, and S-(6-carboxyhexyl)cysteine were each shown to be converted to 7-mercaptoheptanoic acid. Incubation of cell extracts with a mixture of 2(RS)-(5-carboxypentyl)thiazolidine-4(R)-carboxylic acid and [2-2H]-2-(RS)-(5-carboxypentyl)-[34S]thiazolidine-4(R)-carboxylic acid showed that both 34S and 2H are incorporated into the 7-mercaptoheptanoic acid but only after separation of the cysteine from the [7-2H]-7-oxyheptanoic acid portion of the molecule. Furthermore, the sulfur from the cysteine was incorporated into the thiol only after its elimination from the cysteine and subsequent mixing with an unlabeled sulfur source which had a molecular weight of sufficient size that it was excluded from Sephadex G-25. Hydrogen sulfide was found to supply the sulfur for the production of the 7-mercaptoheptanoic acid in a reaction that was shown to obtain its reducing equivalents from hydrogen via an F420-dependent hydrogenase.

Topics: Cysteine; Dicarboxylic Acids; Euryarchaeota; Heptanoic Acids; Keto Acids; Models, Biological; Phosphothreonine; Sulfhydryl Compounds; Thiazoles; Threonine