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

Abstract

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...

Analysis of theEscherichia coli proBAlocus by DNA and protein sequencing

“…The induced protein of molecular weight 41.5k comigrated with purified GPR, and the induced protein of molecular weight of 37.0k comigrated with purified GK. The molecular weight of GPR is somewhat lower than that previously reported (18). Densitometric analysis of the Coomassie stained SDS-polyacrylamide gels indicated that GK and GPR together represented approximately 20-25% of the total soluble protein in the cell, with GPR accumulating in a 2 to 3-fold molar excess over GK (See Discussion).…”

Cloning of genes for proline and leucine biosynthesis from Brucella abortus by functional complementation in Escherichia coli

“…The gene is about 1300 bp for proA, which is what would be expected for a protein of the size seen. The protein is similar in size to the subunit molecular mass of 42000 Da seen for the E. coli enzyme (Hayzer & Leisinger, 1982) or 44000 Da for the Serratia enzyme (Omori et al, 1991). For leuB, the gene is 1400 bp, which could code for a protein of 51 000 Da, more than the 46000 Da observed.…”

Proline biosynthesis from L-ornithine in Clostridium sticklandii: purification of Δ1-pyrroline-5-carboxylate reductase, and sequence and expression of the encoding gene, proC

“…The activities of y-glutamyl kinase and glutamate-y-semialdehyde dehydrogenase were assayed as described before (Hayzer ek Leisinger, 1982;Smith et al, 1984;Limauro et al, 1996). The hydroxamate assay was used to detect y-glutamyl kinase.…”