Feedback Inhibition by Methionine and S-Adenosylmethionine, and Desensitization of Homoserine O-Acetyltransferase in Brevibacterium flavum

Shiio, Isamu; Ozaki, Hachiro

doi:10.1093/oxfordjournals.jbchem.a133342

Cited by 14 publications

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“…The lack of feedback inhibition of homoserine O-acetyltransferase activity is highly significant since the concentrations of inhibitors in the reaction were at least 10 times higher than the K m values for the substrates, which were 0.46 Ϯ 0.07 and 0.2 Ϯ 0.06 mM for homoserine and acetyl coenzyme A, respectively. This result for the MetX Leptospira enzyme is in contrast to the feedback inhibition by methionine and S-adenosylmethionine found for the similar enzymes from Bacillus subtilis, Bacillus polymyxa, and Brevibacterium flavum (3,21,23).…”

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Homoserine O-acetyltransferase, involved in the Leptospira meyeri methionine biosynthetic pathway, is not feedback inhibited

et al. 1997

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The Leptospira meyeri serovar semaranga metX gene was identified by complementation of an Escherichia coli metA mutant, i.e., devoid of homoserine O-succinyltransferase. However, the MetX protein exhibited a homoserine O-acetyltransferase activity in agreement with its similarity to homoserine O-acetyltransferases. Reverse transcription-PCR analysis demonstrated that metX is the second gene of an operon.The first step of the biosynthetic pathway leading to methionine is esterification of homoserine (6). However, the precursor differs according to the organism. In members of the family Enterobacteriaceae, O-succinyl-homoserine is formed by acylation of homoserine in the presence of O-succinyl-coenzyme A, catalyzed by homoserine O-succinyltransferase, the metA gene product (7,18). In gram-positive bacteria of the genus Bacillus and in fungi, esterification of homoserine occurs by an acetylation (3, 9, 13) catalyzed by a homoserine O-acetyltransferase.The genus Leptospira consists of two nomenspecies, Leptospira interrogans sensu lato (pathogenic) and Leptospira biflexa sensu lato (nonpathogenic), which belong to the order Spirochaetales (4). Previous studies indicated that Leptospira spp. synthesize most of their amino acids by the same biosynthetic pathways as those used by Escherichia coli (5).Our interest in methionine biosynthesis in Leptospira arose from the fact that radiotracer studies of biosynthetic pathways did not include any data for the methionine pathway (5). In this paper, we describe isolation and transcriptional organization of the metX gene, as well as the enzymatic characterization and regulation of its product, homoserine O-acetyltransferase.(This work represents a portion of a thesis submitted by P. Bourhy to the University of Paris VII, Paris, France, for the Ph.D.)Cloning of a DNA fragment from Leptospira meyeri which can complement an E. coli metA mutant. We had previously constructed a recombinant cosmid library and cloned the Leptospira metY gene, which complemented E. coli metB mutants (1). The size of the cloned DNA fragment (25 kbp) in the pb10 recombinant cosmid suggested that other methionine biosynthetic genes might be found on this fragment. In order to test this hypothesis, E. coli RC709 (metF63 pro-22; R. Clowes) and AB1932 (metA28 argH1 thi-1 lacY1 lacZ4 galK2 xyl-4 [or -5] tsx-6 F Ϫ ; E. A. Adelberg) were used for complementation experiments in supplemented M9 minimal medium (20) at 30°C. Both strains were electroporated with pb10. pb10 weakly complemented metA but not metF mutants. Further subcloning experiments allowed us to locate the Leptospira DNA able to complement E. coli metA and metB mutants more precisely within a 6.8-kbp fragment. Plasmid pb12 carrying a 6.8-kbp PstI fragment from pb10 in the pBR322 vector (22) still complemented E. coli metB and metA mutants (Fig.

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Homoserine O-acetyltransferase, involved in the Leptospira meyeri methionine biosynthetic pathway, is not feedback inhibited

et al. 1997

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“…(2) This enzyme has not yet been well characterized except for a few bacteria (16,22,26,27). Shiio and colleagues (16,22) have recently reported on feedback control by the end product methionine in the case of the enzyme from Brevibacterium flavum.…”

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confidence: 99%

“…Shiio and colleagues (16,22) have recently reported on feedback control by the end product methionine in the case of the enzyme from Brevibacterium flavum. Wyman et al have described purification of the enzyme to near homogeneity (26) and also its regulation in whole cells (27) of Bacillus polymyxa.…”

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confidence: 99%

“…1.31), which catalyzes transfer of the acetyl group from acetyl coenzyme A (acetyl-CoA) to (2) This enzyme has not yet been well characterized except for a few bacteria (16,22,26,27). Shiio and colleagues (16,22) have recently reported on feedback control by the end product methionine in the case of the enzyme from Brevibacterium flavum. Wyman et al have described purification of the enzyme to near homogeneity (26) and also its regulation in whole cells (27) of Bacillus polymyxa.…”

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

Yamagata¹

1987

J Bacteriol

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A wild-type strain and six methionine auxotrophs of Saccharomyces cerevisiae were cultured in a synthetic medium supplemented with 0.1 mM L-cysteine or L-methionine and analyzed for the synthesis of homoserine O-acetyltransferase (EC 2.3.1.31). Among them, four muitant strains exhibited enzyme activity in cell extracts, Methionine added to the synthetic medium at concentrations higher than 0.1 mM repressed enzyme synthesis in two of these strains. The enzyme was partially purified (3,500-fold) from an extract of a mutant strkin through ammonium sulfate fractionation and chromatography on columns of DEAE-cellulose, PhenylSepharose C1-4B, and Sephadex G-150. The enzyme exhibited optimal p1I at 7.5 for activity and at 7.8 for stability. The reaction product was ascertained to be O-acetyl-L-homoserine by confirming that it produced L-homocysteine in an O-acetyl-L-homoserine sulfhydrylase reaction. The Km for L-homoserine was 1.0 mM, and for acetyl coenzyme A it was 0.027 mM. The molecular weight of the enzyme was esimated to be approximately 104,000 by Sephadex G-150 column chromatography anid 101,000 by sucrose density gradient centrifugation.The isoelectric point was at pH 4.0. Of the hydroxy amino acids examined, the enzyme showed reactivity only to L-homoserine. Succinyl coenzyme A was not an acyl donor. In the absence of L-homoserine, acetyl coenzyme A was deacylated by the enzyme, with a Km of 0.012 mM. S-Adenosylmethionine and S-adenosylhomocysteine slightly inhibited the enzyme, but nmethionine had no effect.It is well established that in microorganisms other than a few enteric bacteria O-acetyl-L-homoserine is an essential member of the biosynthetic pathway from L-homoserine to L-methionine (9, 23). In many cases, O-acetylhomoserine synthesizes cystathionine with L-cysteine through the cystathionine y-synthase reaction. 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).(2) This enzyme has not yet been well characterized except for a few bacteria (16,22,26,27). Shiio and colleagues (16,22) have recently reported on feedback control by the end product methionine in the case of the enzyme from Brevibacterium flavum. Wyman et al. have described purification of the enzyme to near homogeneity (26) and also its regulation in whole cells (27) of Bacillus polymyxa. This protein is, however, subject to rapid and irreversible inactivation after extraction (26).Regulatory properties of the enzyme of Saccharomyces cerevisiae have been reported (4, 6), based on observation of the acety...

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“…Dehydratase I might possibly be converted into dehydratase I1 by desensitization during extraction from E. gracilis cells like in homoserine 0-acetyltransferase (Shiio & Ozaki, 1981), but this is not the case. We found that unusual accumulation of isoleucine during the growth inhibition by threonine was due to an insufficiently controlled formation of 2-oxobutyrate from exogenous threonine (Oda et al, 1982b).…”

Section: Discussionmentioning

confidence: 99%

Occurrence and Some Properties of Two Threonine Dehydratases in Euglena gracilis

Oda¹,

Nakano²,

Kitaoka³

1983

Microbiology

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Two threonine dehydratases, I and 11, were found to occur in Euglena gracilis and each enzyme was purified partially. Like the biosynthetic threonine dehydratases of other organisms, dehydratase I was feedback-inhibited by isoleucine and not affected by adenylates. Dehydratase I1 was not influenced by branched-chain amino acids and adenylates. Occurrence of dehydratase 11, with a much higher activity than dehydratase I, suggests that dehydration of threonine is not the key point in the regulation of isoleucine biosynthesis in E. gracih.

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Feedback Inhibition by Methionine and S-Adenosylmethionine, and Desensitization of Homoserine O-Acetyltransferase in Brevibacterium flavum

Cited by 14 publications

References 0 publications

Homoserine O-acetyltransferase, involved in the Leptospira meyeri methionine biosynthetic pathway, is not feedback inhibited

Homoserine O-acetyltransferase, involved in the Leptospira meyeri methionine biosynthetic pathway, is not feedback inhibited

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

Occurrence and Some Properties of Two Threonine Dehydratases in Euglena gracilis

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