期刊
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
卷 1854, 期 2, 页码 146-155出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbapap.2014.12.003
关键词
Pyridoxal phosphate; Dialkylglycine decarboxylase; Directed evolution; Genetic selection; Conformational change; Stereoelectronic effect
资金
- National Institutes of Health [GM54779]
Dialkylglycine decarboxylase (DGD) is an unusual pyridoxal phosphate dependent enzyme that catalyzes decarboxylation in the first and transamination in the second half-reaction of its ping-pong catalytic cycle. Directed evolution was employed to alter the substrate specificity of DGD from 2-aminoisobutyrate (AIB) to 1-aminocyclohexane-1-carboxylate (AC6C). Four rounds of directed evolution led to the identification of several mutants, with clones in the final rounds containing five persistent mutations. The best clones show similar to 2.5-fold decrease in K-M and similar to 2-fold increase in k(cat), giving a modest similar to 5-fold increase in catalytic efficiency for AC6C Additional rounds of directed evolution did not improve catalytic activity toward AC6C Only one (S306F) of the five persistent mutations is close to the active site. S306F was observed in all 33 clones except one, and the mutation is shown to stabilize the enzyme toward denaturation. The other four persistent mutations are near the surface of the enzyme. The S306F mutation and the distal mutations all have significant effects on the kinetic parameters for AIB and AC6C. Molecular dynamics simulations suggest that the mutations alter the conformational landscape of the enzyme, favoring a more open active site conformation that facilitates the reactivity of the larger substrate. We speculate that the small increases in k(cat)/K-M for AC6C are due to two constraints. The first is the mechanistic requirement for catalyzing oxidative decarboxylation via a concerted decarboxylation/proton transfer transition state. The second is that DGD must catalyze transamination at the same active site in the second half-reaction of the ping-pong catalytic cycle. (C) 2014 Elsevier B.V. All rights reserved.
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