Old yellow enzymes from extremophiles: finding and characterizing potential biocatalysts

[摘要] English: The old yellow enzyme (OYE) family is a diverse group of flavoenzymes that catalyse theasymmetric reduction of activated C=C bonds of a wide variety of α/β-unsaturated carbonylcompounds. OYEs are attractive as biocatalysts due to the ability to perform transhydrogenationwith high stereospecificity (Stuermer et al., 2007). The number of functionallyand structurally characterised OYEs has grown over the past decade, as have the enzymefamily's substrate spectrum.Vital in the search for new industrial biocatalysts among the OYE family is the structural andfunctional characterisation of new OYE homologues (Oberdorfer et al., 2011; Toogood et al.,2010; Williams and Bruce, 2002). This study investigated the sequence-based evolutionaryrelationship among the vast number of OYE homologues in the Proteobacteria, Firmicutesand Archaea, with particular attention paid to two substrate-binding residues in the catalyticsite and a single residue implicated in the modulation of the redox potential of enzymeboundFMN. A strong correlation was identified between grouping of OYE homologuesthrough advanced maximum likelihood evolutionary analysis, and the grouping of OYEs intosubgroups through the identity of the above-mentioned three target residues.Two OYE homologues were selected for cloning, heterologous expression and subsequentcharacterisation. The first OYE (CmOYE) was selected from the mesophile Cupriavidusmetallidurans CH3 and belongs to the previously identified 'thermophilic-like subclass ofOYEs (Toogood et al., 2010), which includes mainly OYEs from thermophiles, but alsoOYEs 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 ofmesophilic OYEs from the subclass. The second OYE (SsOYE) was selected form thehyperthermophile Sulfolobus solfataricus P2 and belongs to an as-yet uncharacterisedsubclass of OYEs. SsOYE was regarded as an ideal target due to its potentially highthermal 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 withthe aid of homology modelling form the translated nucleotide sequence).Both targeted OYEs were successfully cloned and heterologously expressed in E. coli asboth unmodified and modified (with addition of N-terminal His6-tag). Due to the presence ofrare 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 IMACand size-exclusion chromatography provided homogenous protein solutions and revealedthat CmOYE and SsOYE are present in solution as a monomers, with the monomeric natureof SsOYE possibly due to the presence of a second domain. Temperature and pH profilesof the two OYEs revealed an optimum temperature and pH for CmOYE that correspondswell to the optimum growth conditions of the source organism. The optimum catalytictemperature of SsOYE was identified to be significantly lower than that of the sourceorganism, 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 assubstrate revealed that catalytic efficiency and affinity differed vastly between the two OYEhomologues. While the Km for CmOYE was found to be comparable with the closehomologue from Thermus scotoductus SA-01 (CrS; Opperman et al., 2010), catalyticefficiency for both CmOYE and SsOYE was revealed to be significantly lower than thatobserved for the close homologues YqjM, XenA and CrS.SsOYE revealed a more limited substrate scope compared to CmOYE. Maleimides wereidentified as good substrates for both, corresponding to activities reported for YqjM, XenAand CrS. No conversions were observed for cyclic enones with methyl substitutions on theCβ position. However, certain compounds previously reported to act well as substrates forYqjM and XenA were not accepted as substrates by either CmOYE or SsOYE, or resulted insignificantly lower conversion as reported. Neither CmOYE nor SsOYE exhibited activitytowards citral, an enal with a methyl group on Cβ that has been reported to act as substratefor YqjM and XenA but not for CrS. CmOYE exhibited marginal activity towards the Cαmethyl-substituted 2-methylcyclopentanone and none towards 2-methylcyclohexenone, twocompounds for which conversions have been reported for YqjM. Two isomers of carvonewere successfully reduced by both CmOYE and SsOYE, an activity not exhibited by XenA.Ketoisophorone, a known substrate for YqjM, resulted in marginal conversion for bothSsOYE and CmOYE, with SsOYE exhibiting almost double the conversion observed forCmOYE.CmOYE catalysed the conversion of carvones and 2-cyclohexenone in the absence ofnicotinamide cofactor, but in conjunction with a light-driven cofactor regeneration approach.Utilisation of this cofactor-regeneration approach failed to produce any positive results inSsOYE. Further functional characterisation involved investigating the enzymes' ability tocatalyse the dehydrogenation of saturated ketones. This phenomenon has been reportedfor the OYE from the thermophile Geobacillus kaustophilus (Schittmayer et al., 2011) in theabsence of nicotinamide cofactor and utilizing only molecular oxygen, but has beenattributed to elevated reaction temperatures (70°C). Although not successful for SsOYE,CmOYE catalysed the conversion of cyclohexanone and (+)-dihydrocarvone to theircorresponding unsaturated compounds at 25°C.Lastly, crystallisation of CmOYE was performed for the collection of X-ray crystallographicdata for future structural characterisation. The enzyme was successfully crystallised anddiffraction data collected at a resolution range of 57.99 - 1.93Å.The study demonstrated that predicting functional characteristics from sequence dataremains problematic for members of the OYE family. Although the use of three catalyticallyimportant target residues as fingerprint is useful for elucidating the evolutionary relationshipamong the vast number of OYE homologues, these groupings do not necessarily result inthe clustering of OYEs with similar functional characteristics. The need for more functionaland structural data of OYE homologues (especially OYE homologues belonging to yetuncharacterised subgroups) remains if correlation between sequence similarity andfunctional similarity among OYE homologues is to be elucidated.

[发布机构] University of the Free State