title: Mechanistic and Evolutionary Insights from the Reciprocal Promiscuity of Two Pyridoxal Phosphate-dependent Enzymes.
abstract: Enzymes that utilize the cofactor pyridoxal 5'-phosphate play essential roles in amino acid metabolism in all organisms. The cofactor is used by proteins that adopt at least five different folds, which raises questions about the evolutionary processes that might explain the observed distribution of functions among folds. In this study, we show that a representative of fold type III, the Escherichia coli alanine racemase (ALR), is a promiscuous cystathionine β-lyase (CBL). Furthermore, E. coli CBL (fold type I) is a promiscuous alanine racemase. A single round of error-prone PCR and selection yielded variant ALR(Y274F), which catalyzes cystathionine β-elimination with a near-native Michaelis constant (Km = 3.3 mm) but a poor turnover number (kcat ≈10 h(-1)). In contrast, directed evolution also yielded CBL(P113S), which catalyzes l-alanine racemization with a poor Km (58 mm) but a high kcat (22 s(-1)). The structures of both variants were solved in the presence and absence of the l-alanine analogue, (R)-1-aminoethylphosphonic acid. As expected, the ALR active site was enlarged by the Y274F substitution, allowing better access for cystathionine. More surprisingly, the favorable kinetic parameters of CBL(P113S) appear to result from optimizing the pKa of Tyr-111, which acts as the catalytic acid during l-alanine racemization. Our data emphasize the short mutational routes between the functions of pyridoxal 5'-phosphate-dependent enzymes, regardless of whether or not they share the same fold. Thus, they confound the prevailing model of enzyme evolution, which predicts that overlapping patterns of promiscuity result from sharing a common multifunctional ancestor.
authors.affiliation: From the Institute of Natural and Mathematical Sciences, Massey University, Auckland 0632.
authors.forename: Valerie W C
authors.initials: VW
authors.lastname: Soo
authors.affiliation: the School of Biological Sciences, University of Auckland, Auckland 1142, and.
authors.forename: Yuliana
authors.initials: Y
authors.lastname: Yosaatmadja
authors.affiliation: the School of Biological Sciences, University of Auckland, Auckland 1142, and.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Squire
authors.affiliation: the Department of Biochemistry, University of Otago, Dunedin 9054, New Zealand wayne.patrick@otago.ac.nz.
authors.forename: Wayne M
authors.identifier: 0000-0002-2718-8053
authors.initials: WM
authors.lastname: Patrick
mesh_terms: D000411:Alanine Racemase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D008190:Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D011732:Pyridoxal Phosphate; D008190:Lyases; C019965:cystathionine beta-lyase; D000411:Alanine Racemase
keywords: directed evolution; enzyme mechanism; protein evolution; protein structure; pyridoxal phosphate