STRUCTURAL INSIGHTS INTO THE SUBSTRATE SPECIFICITY OF BAEYER-VILLIGER MONOOXYGENASES

FERRONI, F.M.; SMITH, M.; OPPERMAN, D.J.

Viena

Congreso; BIOTRANS 2015; 2015

Institute of Applied Synthetic Chemistry, TU Wien

Baeyer-Villiger monooxygenases (BVMOs) are valuable oxidative biocatalysts that catalyze the oxidation of linear or cyclic ketones into the corresponding esters or lactones using NADH/NADPH as electron donor and molecular oxygen as oxidative reactant [1]. During the past decade there has been an increase in research efforts towards discovering new BVMOs and improving/altering the activity and specificity of already characterized BVMOs, mostly cyclohexanone monooxygenase (CHMO) and phenylacetone monooxygenase (PAMO) [2]. We recently reported on four closely related BVMOs from the fungus Aspergillus flavus [3]. Whole-cell biotransformation against a battery of substrates showed that of the four BVMOs, BVMOAFL838 showed the best conversion with a range of aliphatic ketones ranging in length from C8 to C12, while BVMOAFL210 was the only BVMO from this subgroup that converted cyclic and substituted cyclic ketones efficiently. To investigate these differences in substrate specificity, these two enzymes were recombinantly expressed in E. coli with a C-terminal 6His-tag and purified using immobilized metal affinity and gel-filtration chromatography. Both enzymes were crystallized and the three-dimensional structures determined using X-ray diffraction. Although the enzymes were crystallized in the presence of NADP and substrate, no electron density could be observed for either. Both enzymes showed the highest overall structural similarity to PAMO and also contained the characteristic bulge in the active site, which is absent in CHMO.Differences in the amino acids lining the active site and substrate access channel, as well as the loop structure, were mapped and compared. The structures also revealed the different possible positions of surface loop structures involved in co-factor binding.[1] Leisch H., Morley K. and P.C.K. Lau, Chem Rev 2011, 111, 4165-4222.[2] Reetz M.T., Daligault F., Brunner B., Hinrichs H. and A. Deege, Angew Chem Int Ed Engl 2004, 43, 4078-4081.[3] Ferroni F.M., Smit M.S. and D.J. Opperman, J Mol Catal. B: Enzym 2014, 107, 47-54.