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

Abstract

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 A1-pyrroline-5-carboxylate (PCA) reductase. Both enzymes were present in crude extracts of C. sticklandii in sufficient activity of 0.93 nkat (mg protein)-l and 4 3 nkat (mg protein)'l, respectively, whereas enzymes involved in proline biosynthesis from glutamate were not detected. PCA reductase was purified t o homogeneity in a three-step procedure involving ammonium sulfate precipitation, affinity chromatography with Procion Red and gel filtration on Sephadex GF2OO. 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 Km values for PCA and NADH were 019 mM and 0025 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 Km values: 1-55 mM for proline and 10-5 mM for NAD at pH 10-0). Studies with growing cells of C. sticklandii and [15N]proline revealed that proline is not oxidized in vivo because 15N 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.

Crystal Structures of Δ1-Pyrroline-5-carboxylate Reductase from Human Pathogens Neisseria meningitides and Streptococcus pyogenes

“…To date, a broad range of oligomeric states of P5CR has been reported, extending from 125 kDa (suggesting tetramer) for yeast P5CR 37 to 200-340 kDa (octamer-dodecamer) observed for P5CRs purified from Clostridium sticklandii, Raltus norvegicus, Homo sapiens, Escherichia coli, and Spinacia oleracea. 7,10,[38][39][40] As shown by the crystal structures, these enzymes can exist as a dimer or as multimers of dimers. In Sp-P5CR, five dimers are arranged into a decamer resembling an hourglass-shaped barrel ( Figure 4).…”

Clostridium sticklandii, a specialist in amino acid degradation:revisiting its metabolism through its genome sequence

“…C. sticklandii can utilize glycine and ornithine both as an electron donor and an electron acceptor in the Stickland reaction. Ornithine can be reduced to 5-aminovaleric acid through the formation of D-proline as an intermediate, or it can be oxidized to acetate, D-alanine and ammonia [15,29,30]. Glycine can be directly oxidized by NAD + into methylene-THF, CO 2 and ammonia via the glycine cleavage system (gcv-system) or reduced to acetyl phosphate by glycine reductase [31].…”

Amino Acid Catabolic Pathways of Lactic Acid Bacteria

“…Pro might also be utilized by pyrroline-5-carboxylate reductase (EC 1.5.1.2). Although the reaction catalyzed by this enzyme is reversible in some cases, it preferentially acts in the biosynthesis of Pro (Kenklies et al 1999). The inspection of available genomic sequences reveals that the homologs of pyrroline-5-carboxylate reductase are present in E. faecalis, streptococci, L. lactis, and Lb.…”