Stereoselectivity, kinetics, and engineering of flavin/deazaflavin oxidoreductases for use in industrial biocatalysis

Abstract

Biocatalytic approaches are widely recognized as valuable tools for producing pharmaceuticals and fine chemicals with high chemical and optical purity. A range of industrial syntheses that require complex structures and/or a high degree of stereoselectivity, which are often technically challenging in chemosynthesis, find relevance in biocatalysis. Moreover, traditional chemical synthesis continues to face concerns about environmental costs and viability. Biocatalysts, such as enzymes, can address both concerns due to their high specificity and inherent renewability, as well as their generally mild operating conditions. Due to their unusual deazaflavin cofactor, F420, the flavin/deazaflavin oxidoreductase (FDOR) family have attracted attention as potential biocatalysts. A low-potential obligate hydride donor with chemistry more akin to nicotinamides than conventional flavins, F420 plays a crucial role in methanogenesis, antibiotic biosynthesis, and oxidative stress resistance. The taxonomic distribution of F420 is restricted to archaea and a few lineages of bacteria, but it is totally absent from eukaryotes. The FDOR enzymes are the largest family of F420-dependent enzymes in Mycobacteria, which donate hydrides into substrates such as biliverdin, aflatoxins, quinones and fatty acids using the reduced form of the cofactor. Several enzymes in the FDOR-A subgroup were screened for substrate range and activity, leading to the identification of two particularly promiscuous enzymes MSMEG_2027 and MSMEG_2850 that belong to the FDOR-A1 subgroup. They were further characterised by X-ray crystallography, and induced-fit docking (IFD) studies rationalised the opposite stereochemical outcome compared to the OYE family. A kinetic study and chiral GC/MS analysis revealed rather low turnover and moderate level of enantioselectivity in some cases. To improve the catalytic rate and stereoselectivity of MSMEG_2027 and MSMEG_2850, we applied protein engineering which was aided by computational predictions. There were several mutations that improved stereoselectivity and activity. The IFD and MM-GBSA (Molecular Mechanics Generalized Born Surface Area solvation) energy calculations facilitated predictions of binding poses and binding free energies, which in turn allowed us to rationalize mutational effects and provide further insight into FDOR hydrogenation mechanisms. The development of an efficient production system for F420 will be another challenge in the application of FDORs for biocatalysis. A modified biosynthetic pathway for F420 was identified in Mycobacteria that bypasses the yet-unknown lactate kinase that has been proposed to produce the 2-phospholactyl moiety in methanogenic archaea. This pathway has been successfully integrated into Escherichia coli, allowing for F420-dependent enzymatic activity in vivo. The limiting precursor in F420-producing E. coli was identified as phosphoenolpyruvate (PEP) by using a genomic-scale metabolic model. The overexpression of PEP synthase and growth on gluconeogenic carbon sources led to a ~40-fold increase in the space-time yield of F420 compared to the widely used recombinant Mycobacterium smegmatis system. In the FDOR superfamily, there are two major groups, the FDOR-As and the FDOR-Bs. There has been evidence that FDOR-As can function as enantioselective ene-reductases, but FDOR-Bs have not been investigated for their use in asymmetric alkene reduction. Eight FDOR-B enzymes from four different subgroups (B1, B3, B4, and B6) were examined for their biocatalytic activity, kinetic properties, and stereoselectivity. Stereochemical comparisons were made between FDOR-As, FDOR-Bs, and OYEs. Computational modelling and docking analysis rationalised the observed catalysis and proposed a mechanism for the catalytic behaviour.