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Out of 27 strains of methionine auxotrophs of Brevibacterium flavum, 14 strains did not grow on homoserine but grew on O-acetylhomoserine, and all were found to lack homoserine O-acetyltransferase [EC 2.3.1.31] alone. Another 3 strains did not grow on O-acetylhomoserine but grew on homocysteine, and the two strains tested were found to lack O-acetylhomoserine sulfhydrylase (AHS) alone, without any changes in the activities of cystathionine gamma-synthase [EC4.2.99.9] and beta-cystathionase [EC 4.4.1.8]. Prototrophic revertants of the AHS-lacking mutants showed concomitant reversion of AHS activity. None of the methionine auxotrophs grew on cystathionine. From these results it was concluded that the methionine biosynthetic pathway of this bacterium involves formation of O-acetylhomoserine from homoserine by the action of homoserine O-acetyltransferase, and direct formation of homocysteine from O-acetylhomoserine by the AHS reaction. AHS synthesis was strongly repressed by methionine. AHS was purified to 70% purity. The purified preparation was activated by pyridoxal phosphate after treatment with hydroxylamine. The enzyme showed a molecular weight of 360,000, an optimum pH of 8.7 for activity, and specifically reacted with O-acetyl-L-homoserine and showed with O-acetyl-L-serine one hundredth as much activity as that with O-acetylo -homoserine, but did not show activity with O-succinyl-L-homoserine, homoserine, or serine. The Km values for O-acetylhomoserine and H2S were 2.0 mM and 0.08 mM, respectively. The enzyme was inhibited 50, 23. and 29% by 10 mM L-methionine, l-homoserine, and O-acetyl-L-serine, respectively, but it was not inhibited by cystathionine or S-adenosyl-L-methionine.

Fumarase (EC 4.2.1.2) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC 14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 30) corresponded to the sequence (residues 6 to 35) of the deduced product of the fumarase gene of C. glutamicum (GenBank accession no. BAB98403). The molecular mass of the native enzyme was 200 kDa. The protein was a homotetramer, with a 50-kDa subunit molecular mass. The homotetrameric and stable properties indicated that the enzyme belongs to a family of Class II fumarase. Equilibrium constants (K(eq)) for the enzyme reaction were determined at pH 6.0, 7.0, and 8.0, resulting in K(eq)=6.4, 6.1, and 4.6 respectively in phosphate buffer and in 16, 19, and 17 in non-phosphate buffers. Among the amino acids and nucleotides tested, ATP inhibited the enzyme competitively, or in mixed-type, depending on the buffer. Substrate analogs, meso-tartrate, D-tartrate, and pyromellitate, inhibited the enzyme competitively, and D-malate in mixed-type.

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Homoserine O-acetyltransferase [EC 2.3.1.31] partially purified from Brevibacterium flavum was found to be specifically inhibited by the metabolic end products methionine and S-adenosylmethionine only when the enzymatic reaction was performed in the presence of cysteine or dithiothreitol, or after the preincubation of the enzyme with either of the sulfhydryl compounds. p-Hydroxymercuribenzoate desensitized the enzyme to inhibition. Concentrations of methionine and S-adenosylmethionine giving 50% inhibition were 4.8 and 0.26 mM, respectively, and 0.5 mM S-adenosylmethionine showed almost complete inhibition. No synergistic action by the two inhibitors was found. Optimum pHs were 7.5 and 8.5 for the inhibition by methionine and S-adenosylmethionine, respectively. The inhibitions by the former and the latter were of mixed type and non-competitive respectively, with respect to both substrates, homoserine and acetyl-CoA. Plots of the reaction rate against concentration of the inhibitors were sigmoidal, indicating the presence of co-operativity. N-Formylmethionine, alpha-methylmethionine, trifluoromethionine, selenomethionine, ethionine or S-adenosylhomocysteine inhibited the enzyme to almost the same extent as methionine or S-adenosylmethionine. The enzyme irreversibly lost sensitivity to inhibition during extraction or storage. Sensitivity was retained by the addition of cysteine, dithiothreitol, homoserine (substate), or glycerol.

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