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Crystal structure of PhaB from Ralstonia eutropha in complex with Acetoacetyl-CoA and NADP
Structural highlights
FunctionPHAB_CUPNH Catalyzes the chiral reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA. Is involved in the biosynthesis of polyhydroxybutyrate (PHB), which is accumulated as an intracellular energy reserve material when cells grow under conditions of nutrient limitation.[1] [2] [3] [4] Publication Abstract from PubMedNicotinamide adenine dinucleotide phosphate (NADPH)-dependent acetoacetyl-CoA reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with beta-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. Out of approximately twenty thousand mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants respectively exhibited a 2.4 and 3.5-fold higher kcat value compared to that of wild-type enzyme. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis between the wild-type and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn play an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity. Directed evolution and structural analysis of NADPH-dependent acetoacetyl-CoA reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics.,Matsumoto K, Tanaka Y, Watanabe T, Motohashi R, Ikeda K, Tobitani K, Yao M, Tanaka I, Taguchi S Appl Environ Microbiol. 2013 Aug 2. PMID:23913421[5] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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