The Gene-Enzyme Relationships of Proline Biosynthesis in Escherichia coli

Hayzer, David J.; Leisinger, Th.

doi:10.1099/00221287-118-2-287

Cited by 104 publications

(98 citation statements)

References 7 publications

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“…Amino acid analysis confirmed NMR findings : glutamic acid was not substantially accumulated in response to upshock in contrast to Gramnegative bacteria (Measures, 1975). The small increase seen at 2 h (Table 1) may be due to increased activity in the biosynthetic pathway leading to proline (Hayzer & Leisinger, 1980). No evidence was obtained of significant accumulation of any of the other neutral amino acids found in other Gram-positive bacteria.…”

Section: Discussionsupporting

confidence: 66%

“…Measures (1975) showed that the glutamate biosynthetic enzymes (GDH & GOGAT) of many bacteria were K+-stimulated and Giaever et al (1988) showed that the trehalose phosphate synthase of E. coli is both osmotically induced and K+-activated. The genes and enzymes of proline biosynthesis have been characterized in a number of micro-organisms including E. coli (Hayzer & Leisinger, 1980), Pseudomonas aeruginosa (Krishna & Leisinger, 1979) and Saccharomyces cerevisiae (Tomenchok & Brandriss, 1987). Proline biosynthesis in all of these organisms occurs via a series of three enzymic reactions and regulation of synthesis is exerted primarily through feedback inhibition of y-glutamyl kinase, the first enzyme of the pathway, by proline.…”

Section: Discussionmentioning

confidence: 99%

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The effects of osmotic upshock on the intracellular solute pools of Bacillus subtilis

Whatmore

Chudek²,

Reed

1990

Journal of General Microbiology

261

327

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The effects of hypersaline treatment (osmotic upshock) on solute accumulation have been studied in the Grampositive bacterium BaciZZus subtiZis. Natural abundance 3C NMR spectroscopy studies revealed only proline as a major organic osmoticum in cells grown in defined medium (no exogenous organic solutes) and this finding was confirmed by amino acid analysis. Intracellular concentrations of both K+ and proline rose markedly after osmotic upshock. K+ influx from the medium was rapid (< 1 h) but proline synthesis was a slower process (5-9 h). Proline synthesis appeared to be dependent on the prior accumulation of K+ and it is possible that K+ serves in some manner as the signal for increased proline synthesis. In cells upshocked in medium enriched in glycine betaine the endogenous synthesis of proline was repressed and glycine betaine served as the sole organic osmoticum. K+ was also accumulated under these conditions.

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Section: Discussionsupporting

confidence: 66%

Section: Discussionmentioning

confidence: 99%

The effects of osmotic upshock on the intracellular solute pools of Bacillus subtilis

Whatmore

Chudek²,

Reed

1990

Journal of General Microbiology

261

327

View full text Add to dashboard Cite

show abstract

“…All the yields and degrees of purification were calculated from the activity measurable after the first DEAE-cellulose step when pyrroline-5-carboxylic acid reductase, which interferes with the assay of glutamate-semialdehyde dehydrogenase, was absent. From earlier experiments [9], one can calculate that the first two steps resulted in an approximate 25% loss of activity, the actual final yield and purification was, therefore, 31 ' %, and 1200-fold, respectively. The final enzyme preparation contained less than 1 % of a contaminating protein as determined by disc-gel electrophoresis with 5 "/, and 7.5 polyacrylamide at pH 4.3 and 8.9.…”

Section: Pur$cution Of Glulamate-semialdehyde Dehydrogenasementioning

confidence: 99%

“…The final enzyme preparation contained less than 1 % of a contaminating protein as determined by disc-gel electrophoresis with 5 "/, and 7.5 polyacrylamide at pH 4.3 and 8.9. There was no detectable y-glutamyl kinase activity as assayed by the method of Hayzer and Leisinger [9]. …”

Section: Pur$cution Of Glulamate-semialdehyde Dehydrogenasementioning

confidence: 99%

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Proline Biosynthesis in Escherichia coli. Purification and Characterisation of Glutamate-Semialdehyde Dehydrogenase

Hayzer

Leisinger²

1982

Eur J Biochem

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Glutamate-semialdehyde dehydrogenase, catalysing the reduction in vivo of y-glutamyl phosphate to glutamate 5-semialdehyde in the pathway of proline biosynthesis in Escherichia coli, has been purified to homogeneity. High initial levels of the enzyme were achieved by using a multicopy ColEl-proA,B hybrid plasmid. The protein has a molecular weight of 1.89 x lo5 and consists of four identical subunits of molecular weight 4.7 x lo4 each. The pH optimum is 7.0 and the protein is stable for at least 10 min between pH 6.0-9.0 and for long periods at pH 7.0. It is rapidly inactivated at temperatures greatcr than 50 "C. The enzyme is very sensitive to inhibition byp-chloromercuribenzoate, copper and nickel ions.The synthesis of L-proline by Escherichia coli involves the reduction of L-glutamic acid to glutamate hemialdehyde, mediated by two consecutive enzymic steps. A y-glutamyl kinase (ATP : L-glutamate 5-phosphotransferase, EC 2.7.2.1 1) activates the y-carboxyl group of glutamate before its reduction to an aldehyde by NADPH-dependent glutamatesemialdehyde dehydrogenase [~-glutamate-5-semialdehyde : NADP+ oxidoreductase (phosphorylating), EC 1.2.1.41, or y-glutamyl-phosphate reductase]. Glutamate 5-semialdehyde undergoes spontaneous Schiff-base formation and cyclisation to L-1-pyrroline-5-carboxylic acid which is subsequently reduced to proline by an NADPH-dependent pyrroline-5-carboxylic acid reductase [L-proline : NAD(P) + 5-0x1-doreductase, EC 1.5.1.21. All three biosynthetic enzymes from Pseudomonas aeruginosa PA01 have been partially purified and characterized [1,2] whereas the first two of the E. coli biosynthetic enzymes have been subjected to investigation whilst in a very crude state [3,4]. Pyrroline-5-carboxylic acid reductase of E. coli, a highly active and readily measurable enzyme, has been partially purified and characterized [ 5 ] . The availability of pure samples of the first two enzymes of the pathway would enable a study to be made of the problems of the true nature of the activated intermediate in the pathway, generally assumed to by y-glutamyl phosphate, and whether there is aggregation between the kinase and glutamatesemialdehyde dehydrogenase. The function of such an enzyme complex would be to protect the glutamyl phosphate from a hostile nucleophilic and aqueous environment. To date these studies have been limited by the use of crude cell extracts but have provided suggestive evidence of the existence of an aggregate [6-81. This paper describes a method for the purification of the second enzyme in the proline biosynthetic pathway and its physical properties. MATERIALS AND METHODS Bacrerial StrainThe construction of Eschevichia coli strain CSH52/ pLC44-11 [ihi araA(lac pro)strA recA($J8Odlaci)(pLC44-1 I)] has been described by Hayzer and Leisinger [9].Large-Scale Fermentation of E. coli CSH52/pLC44-1 I A preculture of 300 ml of medium M63 [lo] plus glucose (0.5% w/v) in a 1-1 flask was grown overnight at 37°C with vigorous aeration. The entire amount was used to inocu1;lte 7 1 of the same med...

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Pyrroline‐5‐carboxylate synthase and proline biosynthesis: From osmotolerance to rare metabolic disease

et al. 2010

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Pyrroline-5-carboxylate synthase (P5CS) is a bifunctional enzyme that exhibits glutamate kinase (GK) and c-glutamyl phosphate reductase (GPR) activities. The enzyme is highly relevant in humans because it belongs to a combined route for the interconversion of glutamate, ornithine and proline. The deficiency of P5CS activity in humans is associated with a rare, inherited metabolic disease. It is well established that some bacteria and plants accumulate proline in response to osmotic stress. The alignment of P5CSs from different species and analysis of the solved structures of GK and GPR reveal high sequence and structural conservation. The information acquired from different mutant enzymes with increased osmotolerant properties, together with the position of the insertion found in the longer human isoform, permit the delimitation of the regulatory site of GK and P5CS and the proposal of a model of P5CS architecture. Additionally, the GK moiety of the human enzyme has been modeled and the known clinical mutations and polymorphisms have been mapped.

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The Gene-Enzyme Relationships of Proline Biosynthesis in Escherichia coli

Cited by 104 publications

References 7 publications

The effects of osmotic upshock on the intracellular solute pools of Bacillus subtilis

The effects of osmotic upshock on the intracellular solute pools of Bacillus subtilis

Proline Biosynthesis in Escherichia coli. Purification and Characterisation of Glutamate-Semialdehyde Dehydrogenase

Pyrroline‐5‐carboxylate synthase and proline biosynthesis: From osmotolerance to rare metabolic disease

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