Akihiro Imura

Crystal structures of cyclohexanone monooxygenase reveal complex domain movements and a sliding cofactor

Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O(2) as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP(+) in two distinct states, to resolutions of 2.3 and 2.2 A. The two conformations reveal domain shifts around multiple linkers and loop movements, involving conserved arginine 329 and tryptophan 492, which effect a translation of the nicotinamide resulting in a sliding cofactor. Consequently, the cofactor is ideally situated and subsequently repositioned during the catalytic cycle to first reduce the flavin and later stabilize formation of the Criegee intermediate. Concurrent movements of a loop adjacent to the active site demonstrate how this protein can effect large changes in the size and shape of the substrate binding pocket to accommodate a diverse range of substrates. Finally, the previously identified BVMO signature sequence is highlighted for its role in coordinating domain movements. Taken together, these structures provide mechanistic insights into CHMO-catalyzed Baeyer-Villiger oxidation.

Enantioselective synthesis of 20(S)-camptothecin using an enzyme-catalyzed resolution

The important key intermediate of a 20(S)-camptothecin synthesis was prepared enantioselectively using an enzyme-catalyzed resolution. A commercially available papain was found to exhibit the highest enantioselectivity with moderate activity, and the (S)-enantiomer of 99% ee was obtained as the remaining substrate.

Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis

There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of (R)‐2‐benzyloxy‐propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3–5°C increase in the apparent melting temperature (Tm) of the mutants over the native BcEST that has a Tm of 50°C was outperformed by TtEST, a naturally occurring homologue with a Tm of 65°C. Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations. This work expands the repertoire of characterized members of the α/β‐fold hydrolase superfamily. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally occurring thermostable biocatalyst that outperforms experimentally evolved homologues that carry out the same hydrolysis.

Enantioselective synthesis of a key intermediate of Levofloxacin using microbial resolution

Abstract The important key intermediate in the synthesis of Levofloxacin, (S)-7,8-difluoro-2,3-dihydro-3-methyl-4H-1,4-benzoxazine, was prepared enantioselectively by microbial resolution. When lyophilized microorganism selected from soil was treated with the corresponding amide, the (S)-amine was obtained with high enantiomeric purity (99% ee).

RESOLUTION OF CIS-2-FLUOROCYCLOPROPANECARBOXYLIC ACID BY A MICROBIAL ENANTIOSELECTIVE HYDROLYSIS

Abstract The important key intermediate of quinolone analogue synthesis, (1S,2S)-2-fluorocyclopropanecarboxylic acid, was prepared enantioselectively by a microbial resolution. One of the strains with the highest enzymatic specificity was selected from soil and when lyophilized cells were treated with corresponding ester, the remaining (1S,2S)-ester was obtained with high enantiomeric purity (98% e.e.).