Identification and characterization of bifunctional proline racemase/hydroxyproline epimerase from archaea: discrimination of substrates and molecular evolution.

Seiya Watanabe,Hisashi Nishiwaki,Yasuo Watanabe,Yoshiaki Tanimoto,Paul Taylor

doi:10.1371/journal.pone.0120349

Abstract

Proline racemase (ProR) is a member of the pyridoxal 5’-phosphate-independent racemase family, and is involved in the Stickland reaction (fermentation) in certain clostridia as well as the mechanisms underlying the escape of parasites from host immunity in eukaryotic Trypanosoma. Hydroxyproline epimerase (HypE), which is in the same protein family as ProR, catalyzes the first step of the trans-4-hydroxy-L-proline metabolism of bacteria. Their substrate specificities were previously considered to be very strict, in spite of similarities in their structures and catalytic mechanisms, and no racemase/epimerase from the ProR superfamily has been found in archaea. We here characterized the ProR-like protein (OCC_00372) from the hyperthermophilic archaeon, Thermococcus litoralis (TlProR). This protein could reversibly catalyze not only the racemization of proline, but also the epimerization of 4-hydroxyproline and 3-hydroxyproline with similar kinetic constants. Among the four (putative) ligand binding sites, one amino acid substitution was detected between TlProR (tryptophan at the position of 241) and natural ProR (phenylalanine). The W241F mutant showed a significant preference for proline over hydroxyproline, suggesting that this (hydrophobic and bulky) tryptophan residue played an importance role in the recognition of hydroxyproline (more hydrophilic and bulky than proline), and substrate specificity for hydroxyproline was evolutionarily acquired separately between natural HypE and ProR. A phylogenetic analysis indicated that such unique broad substrate specificity was derived from an ancestral enzyme of this superfamily.

Highlights

L-Proline can serve as a complete source of carbon and energy or of nitrogen for organisms
L-Proline racemase (EC 5.1.1.4; ProR) first catalyzes the racemization of L-proline to D-proline, followed by reductive cleavage to yield 5-aminopentanoate by D-proline reductase (EC 1.21.4.1). This pathway is only operative in certain clostridia including Clostridium sticklandii [2] and Clostridium difficile [3], clinically significant nosocomial pathogens, and Trypanosoma species including Trypanosoma cruzi [4, 5] and Trypanosoma vivax [6], the causative agents of Chagas disease and animal trypanosomiasis, respectively
Since T3Lhyp is metabolized to L-proline via a Pyr2C intermediate [9,10,11], the gene cluster may be related to both the metabolism of L-proline and T3LHyp

Summary

Introduction

L-Proline can serve as a complete source of carbon and energy or of nitrogen for organisms. L-Proline racemase (EC 5.1.1.4; ProR) first catalyzes the racemization of L-proline to D-proline, followed by reductive cleavage to yield 5-aminopentanoate by D-proline reductase (EC 1.21.4.1). This pathway is only operative in certain clostridia including Clostridium sticklandii [2] and Clostridium difficile [3], clinically significant nosocomial pathogens, and Trypanosoma species including Trypanosoma cruzi [4, 5] and Trypanosoma vivax [6], the causative agents of Chagas disease and animal trypanosomiasis, respectively. In the former, the pathway is involved in the so-called “Stickland reaction (fermentation)”, whereas ProR in the latter has been implicated in the mechanisms underlying the escape of parasites from host immunity as a B-cell mitogen

Methods

Results

Conclusion