title: Identification and characterization of bifunctional proline racemase/hydroxyproline epimerase from archaea: discrimination of substrates and molecular evolution.
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.
authors.affiliation: Faculty of Agriculture, Ehime University, Matsuyama, Ehime, 790-8566, Japan.
authors.forename: Seiya
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Faculty of Agriculture, Ehime University, Matsuyama, Ehime, 790-8566, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Tanimoto
authors.affiliation: Faculty of Agriculture, Ehime University, Matsuyama, Ehime, 790-8566, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Nishiwaki
authors.affiliation: Faculty of Agriculture, Ehime University, Matsuyama, Ehime, 790-8566, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Watanabe
mesh_terms: D000591:Amino Acid Isomerases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D015870:Gene Expression; D006909:Hydroxyproline / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D011392:Proline / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity; D019712:Thermococcus / Q000145:classification / Q000201:enzymology* / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; D011994:Recombinant Proteins; C026215:3-hydroxyproline; D011392:Proline; D000591:Amino Acid Isomerases; C022451:proline racemase; C021420:hydroxyproline epimerase; D006909:Hydroxyproline