Interaction between primary and secondary metabolism in Streptomyces coelicolor A3(2): role of pyrroline-5-carboxylate dehydrogenase.
The activity of the proline catabolic enzyme pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12) was induced up to three-hundred-fold by the addition of three hundred proline to the growth medium of the Gram-positive bacterium Streptomyces coelicolor A3(2). Rifampicin, an inhibitor of RNA polymerase activity, abolished induction, implying that regulation was at the level of activation of gene transcription. The enzyme was purified and SDS-PAGE of the highly purified enzyme preparation revealed a single subunit with M(r) 68,000. A single band of protein, which also stained for enzyme activity, was observed after native gel electrophoresis. The M(r) of the enzyme was estimated to be approximately 265,000 by native gel electrophoresis and approximately 305,000 by gel filtration, which indicated that the enzyme had a tetrameric quaternary structure. The apparent Km for pyrroline-5-carboxylate was 109 +/- 7.3 microM, whilst that for NAD+ was 43.3 +/- 2.5 microM. Product inhibition by NADH (apparent Ki 0.6mM) was observed. The observed Vmax was 22.0 +/- 1 mol min-1 (mg protein)-1. Neither 1 nor 5 mM proline had any effect on enzyme activity, whilst glutamate was a very weak inhibitor.
Smith
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DD
Department of Biological Sciences, University of Warwick, Conventry, UK.
Wood
N J
NJ
Hodgson
D A
DA
D050842:1-Pyrroline-5-Carboxylate Dehydrogenase; D018533:Biomass; D002701:Chloramphenicol / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism* / Q000502:physiology*; D011392:Proline / Q000378:metabolism / Q000494:pharmacology*; D012293:Rifampin / Q000494:pharmacology; D013302:Streptomyces / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D013997:Time Factors
D009243:NAD; D002701:Chloramphenicol; D011392:Proline; D050842:1-Pyrroline-5-Carboxylate Dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D012293:Rifampin