Old yellow enzymes from extremophiles: finding and characterizing potential biocatalysts

English: The old yellow enzyme (OYE) family is a diverse group of flavoenzymes that catalyse the asymmetric reduction of activated C=C bonds of a wide variety of α/β-unsaturated carbonyl compounds. OYEs are attractive as biocatalysts due to the ability to perform transhydrogenation with high stereospecificity (Stuermer et al., 2007). The number of functionally and structurally characterised OYEs has grown over the past decade, as have the enzyme family’s substrate spectrum. Vital in the search for new industrial biocatalysts among the OYE family is the structural and functional characterisation of new OYE homologues (Oberdorfer et al., 2011; Toogood et al., 2010; Williams and Bruce, 2002). This study investigated the sequence-based evolutionary relationship among the vast number of OYE homologues in the Proteobacteria, Firmicutes and Archaea, with particular attention paid to two substrate-binding residues in the catalytic site and a single residue implicated in the modulation of the redox potential of enzymebound FMN. A strong correlation was identified between grouping of OYE homologues through advanced maximum likelihood evolutionary analysis, and the grouping of OYEs into subgroups through the identity of the above-mentioned three target residues. Two OYE homologues were selected for cloning, heterologous expression and subsequent characterisation. The first OYE (CmOYE) was selected from the mesophile Cupriavidus metallidurans CH3 and belongs to the previously identified “thermophilic-like” subclass of OYEs (Toogood et al., 2010), which includes mainly OYEs from thermophiles, but also OYEs YqjM from B. subtilis (Kitzing et al., 2003) and XenA (P. putida; Griese et al., 2006) from mesophiles. CmOYE was regarded as an ideal target, as it adds to the short list of mesophilic OYEs from the subclass. The second OYE (SsOYE) was selected form the hyperthermophile Sulfolobus solfataricus P2 and belongs to an as-yet uncharacterised subclass of OYEs. SsOYE was regarded as an ideal target due to its potentially high thermal stability (an ideal characteristic in biocatalysts) and unconventional OYE structure (bearing high similarity to the two-domain enoyl-CoA reductase from E. coli, as revealed with the aid of homology modelling form the translated nucleotide sequence). Both targeted OYEs were successfully cloned and heterologously expressed in E. coli as both unmodified and modified (with addition of N-terminal His6-tag). Due to the presence of rare codons in the gene sequence of SsOYE, the protein was expressed in E. coli in the presence of the plasmid pLySSRARE2. Purification of CmOYE and SsOYE through IMAC and size-exclusion chromatography provided homogenous protein solutions and revealed that CmOYE and SsOYE are present in solution as a monomers, with the monomeric nature of SsOYE possibly due to the presence of a second domain. Temperature and pH profiles of the two OYEs revealed an optimum temperature and pH for CmOYE that corresponds well to the optimum growth conditions of the source organism. The optimum catalytic temperature of SsOYE was identified to be significantly lower than that of the source organism, while the optimum catalytic pH was higher than the optimum growth pH of S. solfataricus P2. Steady-state kinetics performed for both CmOYE and SsOYE with 2-cyclohexenone as substrate revealed that catalytic efficiency and affinity differed vastly between the two OYE homologues. While the Km for CmOYE was found to be comparable with the close homologue from Thermus scotoductus SA-01 (CrS; Opperman et al., 2010), catalytic efficiency for both CmOYE and SsOYE was revealed to be significantly lower than that observed for the close homologues YqjM, XenA and CrS. SsOYE revealed a more limited substrate scope compared to CmOYE. Maleimides were identified as good substrates for both, corresponding to activities reported for YqjM, XenA and CrS. No conversions were observed for cyclic enones with methyl substitutions on the Cβ position. However, certain compounds previously reported to act well as substrates for YqjM and XenA were not accepted as substrates by either CmOYE or SsOYE, or resulted in significantly lower conversion as reported. Neither CmOYE nor SsOYE exhibited activity towards citral, an enal with a methyl group on Cβ that has been reported to act as substrate for YqjM and XenA but not for CrS. CmOYE exhibited marginal activity towards the Cα methyl-substituted 2-methylcyclopentanone and none towards 2-methylcyclohexenone, two compounds for which conversions have been reported for YqjM. Two isomers of carvone were successfully reduced by both CmOYE and SsOYE, an activity not exhibited by XenA. Ketoisophorone, a known substrate for YqjM, resulted in marginal conversion for both SsOYE and CmOYE, with SsOYE exhibiting almost double the conversion observed for CmOYE. CmOYE catalysed the conversion of carvones and 2-cyclohexenone in the absence of nicotinamide cofactor, but in conjunction with a light-driven cofactor regeneration approach. Utilisation of this cofactor-regeneration approach failed to produce any positive results in SsOYE. Further functional characterisation involved investigating the enzymes’ ability to catalyse the dehydrogenation of saturated ketones. This phenomenon has been reported for the OYE from the thermophile Geobacillus kaustophilus (Schittmayer et al., 2011) in the absence of nicotinamide cofactor and utilizing only molecular oxygen, but has been attributed to elevated reaction temperatures (70°C). Although not successful for SsOYE, CmOYE catalysed the conversion of cyclohexanone and (+)-dihydrocarvone to their corresponding unsaturated compounds at 25°C. Lastly, crystallisation of CmOYE was performed for the collection of X-ray crystallographic data for future structural characterisation. The enzyme was successfully crystallised and diffraction data collected at a resolution range of 57.99 - 1.93Å. The study demonstrated that predicting functional characteristics from sequence data remains problematic for members of the OYE family. Although the use of three catalytically important target residues as fingerprint is useful for elucidating the evolutionary relationship among the vast number of OYE homologues, these groupings do not necessarily result in the clustering of OYEs with similar functional characteristics. The need for more functional and structural data of OYE homologues (especially OYE homologues belonging to yet uncharacterised subgroups) remains if correlation between sequence similarity and functional similarity among OYE homologues is to be elucidated.