4itg

P113S mutant of E. coli Cystathionine beta-lyase MetC

spinqualitylabelsall models 4itg, resolution 1.74Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 4itg is a 2 chain structure with sequence from Escherichia coli K-12. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.74Å Ligands: EPE, LLP Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function METC_ECOLI Publication Abstract from PubMed 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 beta-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 beta-elimination with a near-native Michaelis constant (Km = 3.3 mm) but a poor turnover number (kcat approximately 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. Mechanistic and Evolutionary Insights from the Reciprocal Promiscuity of Two Pyridoxal Phosphate-dependent Enzymes.,Soo VW, Yosaatmadja Y, Squire CJ, Patrick WM J Biol Chem. 2016 Sep 16;291(38):19873-87. doi: 10.1074/jbc.M116.739557. Epub, 2016 Jul 29. PMID:27474741 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Cystathionine beta-lyase

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