Identification of d-Proline Reductase fromClostridium sticklandiias a Selenoenzyme and Indications for a Catalytically Active Pyruvoyl Group Derived from a Cysteine Residue by Cleavage of a Proprotein

Abstract

Highly active D-proline reductase was obtained from Clostridium sticklandii by a modified purification scheme. The cytoplasmic enzyme had a molecular mass of about 870 kDa and was composed of three subunits with molecular masses of 23, 26, and 45 kDa. The 23-kDa subunit contained a carbonyl group at its N terminus, which could either be labeled with fluorescein thiosemicarbazide or removed by o-phenylenediamine; thus, Nterminal sequencing became feasible for this subunit. L-[14 C]proline was covalently bound to the 23-kDa subunit if proline racemase and NaBH 4 were added. Selenocysteine was detected in the 26-kDa subunit, which correlated with an observed selenium content of 10.6 g-atoms in D-proline reductase. No other non-proteinaceous cofactor was identified in the enzyme. A 4.8-kilobase pair (kb) EcoRI fragment was isolated and sequenced containing the two genes prdA and prdB. prdA coding for a 68-kDa protein was most likely translated as a proprotein that was posttranslationally cleaved at a threonine-cysteine site to give the 45-kDa subunit and most probably a pyruvoyl-containing 23-kDa subunit. The gene prdB encoded the 26-kDa subunit and contained an in frame UGA codon for selenocysteine insertion. prdA and prdB were transcribed together on a transcript of 4.5 kb; prdB was additionally transcribed as indicated by a 0.8-kb mRNA species.

Analysis of Proline Reduction in the Nosocomial Pathogen Clostridium difficile

“…3B). The organization of this operon is also identical to that found in C. sticklandii, with the exception of a duplication of prdE in C. difficile (21). Located downstream, and potentially within the same operon of the prd genes, is an open reading frame predicted to encode the proline racemase (prdF).…”

“…Studies by Andreesen and his colleagues have also demonstrated that glycine derivatives (betaine and sarcosine) can also act as Stickland acceptors in E. acidaminophilum (1,33), and the enzymes catalyzing the reduction of these amino acids are likely to contain the core subunits from the glycine reductase (1). Kabisch et al also uncovered a selenoprotein subunit in the D-proline reductase (21). It should be noted that in the initial description of C. sticklandii, Stadtman and McClung reported that the closest related strain (based on biochemical characteristics) was the poorly understood C. difficile (45).…”

Selenium in Biology: Facts and Medical Perspectives

“…Most prokaryotic selenoproteins, however, are unique and catalyze highly-varied processes that have not been discovered in eukarya. In the clostridial cluster XI (Kreimer and Andreesen, 1995;Wagner et al, 1999;Kabisch et al, 1999), selenoproteins are vital for energy production, particularly under stress, and appear to be important for additional metabolic performances and pathogenicity. A better understanding of such unique pathways in clinically relevant microorganisms might provide a rational basis for therapeutic intervention.…”

Anaerobic 4-hydroxyproline utilization: Discovery of a new glycyl radical enzyme in the human gut microbiome uncovers a widespread microbial metabolic activity

“…12 , 13 Notably, ∼65% of the Clostridiales species that possess the prd operon, which is essential for Pro reduction, also encode HypD. 13 , 14 In these species, the conserved proC gene (encoding P5CR) adjacent to hypD provides a strong indication that Hyp is metabolized to Pro and used as an electron acceptor. In Stickland fermentation, Pro is metabolized via a 2-step pathway to 5-aminovalerate, which often accumulates as an end product (Fig.…”

“…2A). 14 , 15 To test whether HypD-encoding Clostridiales consume Hyp via this pathway, we examined two strains encoding both hypD and the prd operon ( C. difficile  630Δerm and Terrisporobacter glycolicus DSM 1288). We confirmed that they generate 5-aminovalerate when grown in the presence of Hyp (Fig.…”