Cobalamin and the synthesis of methionine by ultrasonic extracts of <i>Escherichia coli</i>

Guest, J. R.; Helleiner, C. W.; Cross, Michael; Woods, D. D.

doi:10.1042/bj0760396

Cited by 66 publications

(37 citation statements)

References 14 publications

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“…The reaction was stopped (and proteins precipitated) ikfethionine synthesis by cell-free extracts. This assay was based on the experiments of Guest et al (1960) and was done by the procedure described by Rowbury & Woods (1 961). When required DL-homocysteine ( 5 m~) was replaced as substrate by L-cystathionine ( 5 mM) or DL-allocystathionine (10 mM).…”

Section: Extract Of Heated Escherichia Coli (Strain ~~1 5 )mentioning

confidence: 99%

“…DLAllocystathionine was obtained from the California Corporation for Biochemical Research (Los Angeles, U.S.A.). Other special chemicals were as described by Guest et al (1960).…”

Section: Extract Of Heated Escherichia Coli (Strain ~~1 5 )mentioning

confidence: 99%

“…dry wt./ml.) and subjected to ultrasonic vibration and further treatment as described by Guest et al (1960); in some experiments the dialysed extract was also treated with Dowex-1 resin by the method of these authors. The protein content of the final preparation was determined spectrophotometrically according to Layne …”

Section: Introductionmentioning

confidence: 99%

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Repression by Methionine of Cystathionase Formation in Escherichia coli

Rowbury

Woods

1964

Journal of General Microbiology

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SUMMARYCystathionase catalyses the formation of homocysteine from cystathionine; its formation in cultures of Escherichia coli is repressed by the presence of methionine in the growth medium, and to a similar extent to that shown with homocysteine methylase (the enzyme complex which catalyses the conversion homocysteine -+ methionine). Cystathionase, again like homocysteine methylase, is formed rapidly without concomitant growth when repressed organisms are transferred to a medium free from methionine ; such enzyme formation is prevented by chloramphenicol, suggesting that de novo synthesis of protein is required. The co-repression by methionine of the two enzyme systems fortifies the evidence that cystathionase is a component of the normal pathway of methionine synthesis by E . coli and consequently that its substrate, cystathionine, is a normal intermediate. Cystathionase preparations also formed pyruvate from cysteine; this activity paralleled that of cystathionase itself when the methionine status of the medium was changed, and it is concluded that the same protein is responsible for both activities. INTRODUCTIONIn previous work in this laboratory Wijesundera & Woods (1962) showed that L-cystathionine was converted to homocysteine, pyruvate and ammonia by a cystathionase enzyme present in several strains of Escherichia coli. Since homocysteine is undoubtedly the immediate precursor of methionine in this organism (see review by Guest & Woods, 1962), these observations supported the view, originating from the properties of methionine auxotrophs of 23. coli and other micro-organisms, that cystathionine is an earlier intermediate, though there remained some evidence to the contrary (see Discussion). The final reaction by which homocysteine is converted to methionine is a complicated one requiring folic acid derivatives and, in certain circumstances, also cobalamin as cofactors (Guest & Woods, 1962). However, the formation of the enzyme complex (homocysteine methylase), and indeed individual components of it, is repressed by the presence of methionine in the culture medium; the enzymes are, however, rapidly resynthesized when methionine is removed (Rowbury & Woods, 1961 ; Foster, Rowbury & Woods, 1963). The main objective of the present work was to examine the effect of growth with methionine on the cystathionase content of E. coli with conditions under which it could be strictly compared with the known effect on the homoc ys t eine met hy lase complex. TO Xhcrob. xxxvDownloaded from www.microbiologyresearch.org by

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Section: Extract Of Heated Escherichia Coli (Strain ~~1 5 )mentioning

confidence: 99%

“…DLAllocystathionine was obtained from the California Corporation for Biochemical Research (Los Angeles, U.S.A.). Other special chemicals were as described by Guest et al (1960).…”

Section: Extract Of Heated Escherichia Coli (Strain ~~1 5 )mentioning

confidence: 99%

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Repression by Methionine of Cystathionase Formation in Escherichia coli

Rowbury

Woods

1964

Journal of General Microbiology

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“…This role of homocysteine has been amply confirmed with suspensions of intact organisms and with cell-free enzyme preparations (Gibson & Woods, 1960; Guest, Helleiner, Cross & Woods, 1960). I n the case of cystathionine closer investigation has given apparently contradictory results.…”

Section: Introductionmentioning

confidence: 81%

O-Succinylhomoserine as an Intermediate in the Synthesis of Cystathionine by Escherichia coli

Rowbury

Woods

1964

Journal of General Microbiology

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SUMMARYFurther evidence that cystathionine is a precursor of methionine synthesis in Escherichia coli was obtained by showing its synthesis by cellfree extracts of an auxotroph which required homocysteine or methionine for growth. Succinate, as well as homoserine and cysteine, was an obligatory substrate and maximal synthesis was dependent on the further addition of glucose, adenosine triphosphate and coenzyme A. The reaction was further studied by using mixtures of extracts of auxotrophs of which each alone was unable to synthesize cystathionine. At least two enzymes were required: the first, present in one strain, formed a heat-stable intermediate from homoserine and succinate; while the second, present in the other strain, converted this intermediate to cystathionine in the presence of cysteine. The intermediate was formed enzymically from homoserine and succinyl-coenzyme A alone and was ninhydrin-positive ; this suggested that it might be O-succinylhomoserine. This compound was prepared by reaction of succinyl chloride or succinic anhydride with homoserine in the presence of perchloric acid; its chromatographic and chemical properties were closely similar to those of the intermediate. The synthetic product, like that formed enzymically, gave cystathionine when incubated with cysteine and a cell-free extract of the relevant E . coli auxotroph. It is concluded that O-succinylhomoserine is an intermediate in the formation of cystathionine, and consequently of methionine by E. coli.

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“…The isolation of the B12 coenzyme had revitalized the vitamin B12 field and the report from D.D. Woods laboratory (Guest et a!., 1960) that this vitamin was involved in methionine biosynthesis was especially intriguing. Could another coenzyme form of vitamin B I 2 be functioning in this reaction?…”

Section: The Early Decisionsmentioning

confidence: 99%

A revisit to bacterial protein synthesis: The search for the role of GTP

Weissbach

1998

Protein Science

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Cobalamin and the synthesis of methionine by ultrasonic extracts of Escherichia coli

Cited by 66 publications

References 14 publications

Repression by Methionine of Cystathionase Formation in Escherichia coli

Repression by Methionine of Cystathionase Formation in Escherichia coli

O-Succinylhomoserine as an Intermediate in the Synthesis of Cystathionine by Escherichia coli

A revisit to bacterial protein synthesis: The search for the role of GTP

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