Engineering enzymes for improved catalytic efficiency: a computational study of site mutagenesis in epothilone-B hydroxylase.
Epothilone F, 21-hydroxyl-epothilone B, is an intermediate in the synthesis of BMS-310705, an antitumor compound that has been evaluated in Phase I clinical trials. A bioconversion process utilizing the Gram-positive bacterium Amycolatopsis orientalis was used to prepare epothilone F from epothilone B. In order to improve the yield of epothilone F, a mutagenesis program was performed with the goal of engineering the epothilone-B hydroxylase (EBH) enzyme to improve the yield of epothilone F through oxidative biotransformation. The mutations in EBH increased the yield of epothilone F from 21% in the recombinant expression system to higher than 80% utilizing the best EBH mutants. The studies described here show how a homology model of EBH was used to obtain an understanding of the possible mechanism that led to improved yield of epothilone F in the mutated enzymes. A novel aspect of this study is that it provides some insight into how mutations distant from the binding site can affect enzyme activity.
Nayeem
Akbar
A
Computer Aided Drug Design, Division of Molecular Biosciences, Bristol-Myers Squibb Company, PO Box 5400, Princeton, NJ 08543-5400, USA. akbar.nayeem@bms.com
Chiang
Shu-Jen
SJ
Liu
Suo-Win
SW
Sun
Yuhua
Y
You
Li
L
Basch
Jonathan
J
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics*; D003198:Computer Simulation; D003577:Cytochrome P-450 Enzyme System; D034261:Epothilones / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment; D040681:Structural Homology, Protein
D001426:Bacterial Proteins; D034261:Epothilones; C426625:epothilone F; C434963:epothilone K; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C093788:epothilone B