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 d ehydrogenase (EC 1.5.1.12) was induced up to threehundredfold by the a ddition of three hundred proline to the growth medium of the Gram-posi tive bacterium Streptomyces coelicolor A3(2). Rifampicin, an inhibitor of RNA polymerase activity, abolished induction, implying that regula tion was at the level of activation of gene transcription. The enzyme was purified and SDS-PAGE of the highly purified enzyme preparation re vealed a single subunit with M(r) 68 000. A single band of protein, wh ich also stained for enzyme activity, was observed after native gel el ectrophoresis. The M(r) of the enzyme was estimated to be approximatel y 265 000 by native gel electrophoresis and approximately 305 000 by g el filtration, which indicated that the enzyme had a tetrameric quater nary structure. The apparent K-m for pyrroline-5-carboxylate was 109 /- 7.3 mu M, whilst that for NAD(+) was 43.3 +/- 2.5 mu M. Product inh ibition by NADH (apparent K-i 0.6 mM) was observed. The observed V-max was 22.0 +/- 1 mol min(-1) (mg protein)(-1). Neither 1 nor 5 mM proli ne had any effect on enzyme activity, whilst glutamate was a very weak inhibitor.