3VZP: Crystal Structure of Phab From Ralstonia Eutropha

Citation:
Abstract
NADPH-dependent acetoacetyl-coenzyme A (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. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited kcat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played 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.
PDB ID: 3VZPDownload
MMDB ID: 112932
PDB Deposition Date: 2012/10/15
Updated in MMDB: 2013/12 
Experimental Method:
x-ray diffraction
Resolution: 1.79  Å
Source Organism:
Similar Structures:
Biological Unit for 3VZP: tetrameric; determined by author and by software (PISA)
Molecular Components in 3VZP
Label Count Molecule
Proteins (4 molecules)
4
Acetoacetyl-coa Reductase
Molecule annotation
Chemicals (16 molecules)
1
7
2
2
3
7
* Click molecule labels to explore molecular sequence information.

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