Methionine Biosynthesis in Brevibacterium flavum: Properties and Essential Role of O-Acetylhomoserine Sulfhydrylase

Abstract

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.

Partial purification and some properties of homoserine O-acetyltransferase of a methionine auxotroph of Saccharomyces cerevisiae

“…Cystathionine is then cleaved through the .-cystathionase reaction to produce L-homocysteine. Butin some microorganisms, O-acetylhomoserine can also be sulfhydrylated with H2S through catalysis by O-acetylhomnoserine sulfhydrylase, giving rise to Lhomocysteine directly (7,17,19,20,30,31).The enzyme homoserine O-acetyltransferase (EC 2.3. 1.31), which catalyzes transfer of the acetyl group from acetyl coenzyme A (acetyl-CoA) to the 0 atom of homoserine (see equation 1 below), also catalyzes the acetyl exchange reaction between O-acetylhomoserine and [14C]homoserine (see equation 2) (9, 23).…”

Occurrence of Transsulfuration in Synthesis of l -Homocysteine in an Extremely Thermophilic Bacterium, Thermus thermophilus HB8

“…One is an OAH sulfhydrylase lacking CTT ␥-synthase activity of B. flavum that has no CTT ␥-synthase (23), and the other is a CTT ␥-synthase of Bacillus sphaericus (12), which also has an activity of OAH sulfhydrylase that is not functional to directly synthesize homocysteine in the cell. The increase in the synthesis of OAH sulfhydrylase with abundant cysteine (or glutathionine) in the medium is, therefore, considered to be a result of substrate induction of CTT ␥-synthase.…”

“…We have recently found activities of CTT ␥-synthase, CTT ␤-lyase, and OAH sulfhydrylase in a cell extract of an extremely thermophilic bacterium, Thermus thermophilus HB8, in addition to a very high OAS sulfhydrylase activity (35). On the other hand, Kosuge et al (17,18) have reported that T. thermophilus HB27 synthesizes homocysteine through direct sulfhydrylation of OAH and that transsulfuration is not func-tional, as reported for Saccharomyces cerevisiae (6) and Brevibacterium flavum (23), by demonstrating that mutant strains deficient in a gene homologous to S. cerevisiae MET17 require homocysteine but not CTT to grow. In order to determine which pathway is physiologically active for the synthesis of homocysteine in T. thermophilus HB8, transsulfuration or direct sulfhydrylation of OAH, we examined the activities of enzymes involved in the pathway leading to methionine in extracts of cells fed with various sulfur sources, and we estimated the amounts of the enzymes synthesized.…”

“…Thus, many organisms obtain homocysteine by incorporating sulfur that is first assimilated into cysteine, via CTT. However, a few microorganisms synthesize homocysteine directly through a replacement reaction of OAH with sulfide that is catalyzed by OAH sulfhydrylase (EC 4.2.99.10) (6,23,34).Little is known about sulfur metabolism and the related enzymes of bacteria living under extreme conditions such as alkaline pH or high temperature. Some archaea have been reported to synthesize cysteine from methionine through reversed transsulfuration from homocysteine to cysteine (36), but an OAS sulfhydrylase (EC 4.2.99.8) has been purified from an archaeon and characterized in detail, suggesting that the enzyme is functional as a cysteine synthase (1).…”

“…Thus, many organisms obtain homocysteine by incorporating sulfur that is first assimilated into cysteine, via CTT. However, a few microorganisms synthesize homocysteine directly through a replacement reaction of OAH with sulfide that is catalyzed by OAH sulfhydrylase (EC 4.2.99.10) (6,23,34).…”