Directed evolution and structural characterization of a simvastatin synthase.
Enzymes from natural product biosynthetic pathways are attractive candidates for creating tailored biocatalysts to produce semisynthetic pharmaceutical compounds. LovD is an acyltransferase that converts the inactive monacolin J acid (MJA) into the cholesterol-lowering lovastatin. LovD can also synthesize the blockbuster drug simvastatin using MJA and a synthetic alpha-dimethylbutyryl thioester, albeit with suboptimal properties as a biocatalyst. Here we used directed evolution to improve the properties of LovD toward semisynthesis of simvastatin. Mutants with improved catalytic efficiency, solubility, and thermal stability were obtained, with the best mutant displaying an approximately 11-fold increase in an Escherichia coli-based biocatalytic platform. To understand the structural basis of LovD enzymology, seven X-ray crystal structures were determined, including the parent LovD, an improved mutant G5, and G5 cocrystallized with ligands. Comparisons between the structures reveal that beneficial mutations stabilize the structure of G5 in a more compact conformation that is favorable for catalysis.
Gao
Xue
X
Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Xie
Xinkai
X
Pashkov
Inna
I
Sawaya
Michael R
MR
Laidman
Janel
J
Zhang
Wenjun
W
Cacho
Ralph
R
Yeates
Todd O
TO
Tang
Yi
Y
D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution; D019143:Evolution, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D019821:Simvastatin / Q000737:chemistry / Q000378:metabolism*
D011994:Recombinant Proteins; D019821:Simvastatin; D000217:Acyltransferases