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

Clostridium sticklandii utilizes combinations of amino acids for growth by Stickland reactions. Proline is an efficient electron acceptor in these reactions and is reduced to 5-aminovalerate. Proline can be partly synthesized from ornithine by the action of ornithine aminotransferase and Delta(1-)pyrroline-5-carboxylate (PCA) reductase. Both enzymes were present in crude extracts of C. sticklandii in sufficient activity of 0.93 nkat (mg protein)(-1) and 4.3 nkat (mg protein)(-1), respectively, whereas enzymes involved in proline biosynthesis from glutamate were not detected. PCA reductase was purified to homogeneity in a three-step procedure involving ammonium sulfate precipitation, affinity chromatography with Procion Red and gel filtration on Sephadex GF200. The homogeneous enzyme was most likely an octamer of 230 kDa with a subunit size of 25 kDa as obtained by SDS-PAGE and 28 9 kDa as calculated from the sequence. Apparent K-m values for PCA and NADH were 0 19 mM and 0 025 mM, respectively. The enzyme also catalysed in vitro the reverse reaction, the oxidation of proline, at alkaline ph values above 8 and higher substrate concentrations (apparent K-m values: 1.55 mM for proline and 10.5 mM for NAD at ph 10.0). Studies with growing cells of C. sticklandii and [N-15]proline revealed that proline is not oxidized in vivo because N-15 was solely detected by HPLC-MS in 5-aminovalerate as the product of proline reduction. The proC gene encoding PCA reductase of C. sticklandii was cloned, sequenced and heterologously expressed in Escherichia coli. The enzyme exhibited high homologies to PCA reductases from different sources. Thus, C. sticklandii is able to synthesize the electron acceptor proline from ornithine (a degradation product of arginine) by action of ornithine aminotransferase and PCA reductase.