4WR3

Y274F alanine racemase from E. coli

4WR3 の概要

• エントリーDOI: 10.2210/pdb4wr3/pdb
• 分子名称: Alanine racemase, biosynthetic, PYRIDOXAL-5'-PHOSPHATE, SULFATE ION, ... (5 entities in total)
• 機能のキーワード: isomerase
• 由来する生物種: Escherichia coli
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 158488.68

構造登録者

Squire, C.J.,Yosaatmadja, Y.,Patrick, W.M. (登録日: 2014-10-23, 公開日: 2015-11-04, 最終更新日: 2023-09-27)

主引用文献

Soo, V.W.,Yosaatmadja, Y.,Squire, C.J.,Patrick, W.M.
Mechanistic and Evolutionary Insights from the Reciprocal Promiscuity of Two Pyridoxal Phosphate-dependent Enzymes.
J.Biol.Chem., 291:19873-19887, 2016

PubMed 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.

PubMed: 27474741
DOI: 10.1074/jbc.M116.739557

実験手法

X-RAY DIFFRACTION (1.9 Å)