Rational design of a minimal and highly enriched CYP102A1 mutant library with improved regio-, stereo- and chemoselectivity.
A minimal CYP102A1 mutant library of only 24 variants plus wild type was constructed by combining five hydrophobic amino acids (alanine, valine, phenylalanine, leucine and isoleucine) in two positions. Both positions are located close to the centre of the haem group. The first, position 87, has been shown to mediate substrate specificity and regioselectivity in CYP102A1. The second hotspot, position 328, was predicted to interact with all substrates during oxidation and has previously been identified by systematic analysis of 31 crystal structures and 6300 sequences of cytochrome P450 monooxygenases. By systematically altering the size of the side chains, a broad range of binding site shapes was generated. All variants were functionally expressed in E. coli. The library was screened with four terpene substrates geranylacetone, nerylacetone, (4R)-limonene and (+)-valencene. Only three variants showed no activity towards all four terpenes, while eleven variants demonstrated either a strong shift or improved regio- or stereoselectivity during oxidation of at least one substrate as compared to CYP102A1 wild type.
Seifert
Alexander
A
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Vomund
Sandra
S
Grohmann
Katrin
K
Kriening
Sebastian
S
Urlacher
Vlada B
VB
Laschat
Sabine
S
Pleiss
Jürgen
J
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D053138:Cyclohexenes / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000077222:Limonene; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D019151:Peptide Library; D011487:Protein Conformation; D015202:Protein Engineering; D012717:Sesquiterpenes / Q000737:chemistry; D013379:Substrate Specificity; D013729:Terpenes / Q000737:chemistry
D001426:Bacterial Proteins; D053138:Cyclohexenes; D019151:Peptide Library; D012717:Sesquiterpenes; D013729:Terpenes; D003577:Cytochrome P-450 Enzyme System; C506706:valencene; C009884:geranylacetone; D000077222:Limonene; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases