Send to

Choose Destination
J Biol Chem. 2016 Sep 16;291(38):19873-87. doi: 10.1074/jbc.M116.739557. Epub 2016 Jul 29.

Mechanistic and Evolutionary Insights from the Reciprocal Promiscuity of Two Pyridoxal Phosphate-dependent Enzymes.

Author information

From the Institute of Natural and Mathematical Sciences, Massey University, Auckland 0632.
the School of Biological Sciences, University of Auckland, Auckland 1142, and.
the Department of Biochemistry, University of Otago, Dunedin 9054, New Zealand


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.


directed evolution; enzyme mechanism; protein evolution; protein structure; pyridoxal phosphate

[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center