No abstract

Baeyer-Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMO ); this used NADH 4200-fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79 %) compared to NADPH (58 %) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen-bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.

The regulatory region of two divergently oriented transcriptional units involved in the formation of the gas-evolving hydrogenase (isoenzyme 3) of Escherichia coli was investigated. DNA sequence analysis revealed the existence of a 210 bp non-coding region containing two sequences showing homology to -24/-12 NtrA-dependent promoters. These sequences were arranged in a divergent orientation entirely consistent with their being involved in transcribing the divergent operons. Through S1 protection experiments it could be shown that transcription of both promoters was NtrA-dependent and that it was regulated in an identical manner: oxygen repressed expression, as did anaerobic growth in the presence of nitrate; transcription was induced in cells grown anaerobically in the absence of exogenous electron acceptors and formate was found to be obligately required for this anaerobic induction. Lying at an approximately equal distance between both promoters was a short stretch of DNA which showed similarity to the sequence previously identified (Birkmann and Böck, 1989a) as being necessary for formate induction of the fdhF gene.

The regulation of synthesis of the hydrogenase which is a component of the formate hydrogen-lyase complex was studied by means of a strain of Escherichia coli possessing a transcriptional fusion of the hydrogenase gene (hyd) with the lacZ gene (hyd::lac fusion). Formation of active hydrogenase in the wild strain requires the presence of nickel in the medium; transcription of the hyd gene, however, is independent from the presence of Ni2+. Ni2+ addition to Ni2+-prestarved cells did not lead to any activation of presumptive hydrogenase apoprotein. Regulatory mutants were isolated in which nitrate repression of hyd::lac expression was relieved. Two main classes of regulatory mutants were identified: (i) Mutants with a defect in nitrate reductase; (ii) mutants with a cis-dominant regulatory mutation closely linked to the hyd::lac fusion. In the presence of formate which acts as an inducer, the hyd::lac fusion was also expressed under aerobic conditions. The results infer that nitrate repression of transcription of the hydrogenase structural gene is not effected by nitrate itself but requires the function of the electron transport chain leading to nitrate and that mutations in the promoter/operator region of the hyd cistron may confer insensitivity to redox control both by oxygen and nitrate.

Substrate inhibition can be caused by substrate binding to the enzyme–product complex and can be controlled rationally by targeting enzyme access tunnels.