Old yellow enzymes from extremophiles: finding and characterizing potential biocatalysts

Abstract

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.

Afrikaans: Die ‘old yellow enzyme’ (OYE) familie is 'n diverse groep flavo-ensieme wat die asimmetriese reduksie van geaktiveerde C=C verbindings van 'n wye verskeidenheid α/β- onversadigde karbonielverbindings, kataliseer. OYEs is gesogte biokataliste as gevolg van die vermoë om trans-hidrogenasie met 'n hoë stereoselektiwiteit uit te voer (Stuermer et al. 2007). Die aantal funksioneel- en struktureel gekarakteriseerde OYEs het oor die afgelope dekade gegroei, en so ook die ensiem-familie se substraat spektrum. Die strukturele en funksionele karakterisering van nuwe OYE homoloë (Oberdorfer et al, 2011; Toogood et al, 2010; Williams en Bruce, 2002) is noodsaaklik in die soektog na nuwe industriële biokataliste onder die OYE familie is. Hierdie studie het die aminosuuropeenvolging-gebaseerde evolusionêre verband tussen die groot aantal OYE homoloë in die proteobakterieë, firmikiete en archaea, ondersoek. Besondere aandagis gegee aan twee substraat-bindende residu’s in die katalitiese setel, asook 'n enkele residuwat in die regulering van die ensiem-gebonde FMN se redokspotensiaal, geïmpliseer is. 'n Sterk korrelasie tussen die groepering van OYE homoloë deur middel van gevorderde maksimum waarskynlikheid evolusionêre analise, en die groepering van OYEs in die subgroepe deur die identiteit van die bogenoemde drie teiken-residu’s, is geïdentifiseer. Twee OYE homoloë is gekies vir klonering, heteroloë uitdrukking en die daaropvolgende karakterisering. Die eerste OYE (CmOYE) is gekies uit die mesofiel Cupriavidus metallidurans CH3 en behoort aan die voorheen geïdentifiseerde "termofiel-agtige" subklas van OYEs (Toogood et al, 2010), wat hoofsaaklik OYEs van termofiele, maar ook OYEs YqjM uit B. subtilis (Kitzing et al., 2003) en XenA (P. putida; Griese, et al., 2006) van mesofiele, insluit. CmOYE is beskou as 'n ideale teiken, omdat dit bydra tot die min voorbeelde van mesofiele OYEs in hierdie subklas. Die tweede OYE (SsOYE) van die hipertermofiel Sulfolobus solfataricus P2 behoort aan 'n tans ongekarakteriseerde subklas van OYEs. SsOYE is beskou as 'n ideale teiken as gevolg van die potensiële hoë termiese stabiliteit (ʼn ideale kenmerk in biokataliste), asook die onkonvensionele OYE struktuur (soortgelyk aan die twee-domein enoiel-KoA reduktase van E. coli, soos blootgelê met die hulp van homologie modellering vanaf die aminosuur opeenvolging). Beide OYEs is suksesvol gekloneer en heteroloog uitgedruk in E. coli as beide ongemodifiseerde en gemodifiseerde (met die byvoeging van die N-terminale His6-tag) proteïen. As gevolg van die teenwoordigheid van seldsame kodons in die basispaaropeenvolging van SsOYE, is die proteïen in E. coli uitgedruk in die teenwoordigheid van die plasmied pLySSRARE2. Homogene proteïen-oplossings is verkry deur suiwering van CmOYE en SsOYE deur IMAC en uitsluitingskromatografie. Die laasgenoemde suiweringstegniek het ook aangedui dat CmOYE as 'n monomeer bestaan. SsOYE as ook ʼn monomeer in oplossing teenwoordig is, moontlik as gevolg van die teenwoordigheid van die tweede domein. Temperatuur en pH profiele van die twee OYEs se aktiwiteit het aangedui dat CmOYE 'n optimale temperatuur en pH het wat ooreenstem met die optimum groeitoestande van die bron organisme. Die optimum katalitiese temperatuur van SsOYE blyk aansienlik laer as dié van die bron organisme te wees, terwyl die optimum katalitiese pH hoër is as die optimum groei pH van S. solfataricus P2. Gestadigte-toestand kinetika is uitgevoer vir beide CmOYE en SsOYE met 2-sikloheksenoon as substraat wat aangedui het dat die katalitiese doeltreffendheid en affiniteit aansienlik tussen die twee OYE homoloë verskil. Terwyl die Km vir CmOYE vergelykbaar met die van die OYE vanaf Thermus scotoductus SA-01 (CrS; Opperman et al, 2010) is, is die katalitiese doeltreffendheid vir beide CmOYE en SsOYE beduidend laer is as wat vir die homoloë YqjM, Xena en CRS waargeneem is SsOYE toon aktiwiteit teenoor ʼn meer beperkte omvang substrate as CmOYE. Maleimiedes is as goeie substrate vir beide geïdentifiseer, wat ooreenstem met die aktiwiteite gerapporteer vir YqjM, XenA en CrS. Geen omskakelings is waargeneem vir sikliese enone met metiel-vervangings op die Cβ posisie nie. Sekere verbindings wat voorheen as goeiesubstrate vir YqjM en XenA beskou is, is egter nie as substrate deur CmOYE óf SsOYE geïdentifiseer nie, of het gelei tot beduidend laer aktiwiteite as wat vir homoloë gerapporteer is. Nie CmOYE of SsOYE toon aktiwiteit teenoor sitral nie, 'n enal met 'n metielgroep op Cβ wat wel as substraat vir YqjM en XenA optree, maar nie vir CrS nie. CmOYE toon marginale aktiwiteit met die Cα metiel-gesubstitueerde 2-metielsiklopentanoon en geen aktiwiteit met 2-metielsikloheksanoon nie, twee substrate waarvoor aktiwiteit met YqjM gerapporteer is. Twee isomere van karvoon is suksesvol deur beide CmOYE en SsOYE omgeskakel, 'n aktiwiteit wat nie vir XenA gerapporteer is nie. Beide SsOYE en CmOYE het baie lae aktiwiteit met ketoisoforoon, ʼn bekende substraat vir YqjM, getoon. Vir SsOYE is byna dubbel die omskakeling as wat vir CmOYE waargeneem is, met ketoisoforoon getoon. CmOYE het die omskakeling van karvoon en 2-sikloheksenoon in die afwesigheid van ʼn nikotienamied kofaktor, maar in samewerking met 'n lig-gedrewe kofaktor regenereringsmetode, gekataliseer. Gebruik van hierdie kofaktor-regenereringsbenadering het geen positiewe resultate met SsOYE geproduseer nie. Verdere funksionele karakterisering is gedoen deur die ensieme se vermoë om die dehidrogenasie van versadigde ketone te kataliseer, te ondersoek. Hierdie verskynsel is vir die OYE van die termofiel Geobacillus kaustophilus (Schittmayer et al,. 2011) in die afwesigheid van nikotienamied ko-faktor en met die gebruik van slegs molekulêre suurstof, gerapporteer. Hierdie verskynsel is egtertoe geskryf aan verhoogde reaksie temperature (70°C). Hoewel geen sukses met SsOYE behaal is nie, het CmOYE die omskakeling van sikloheksanoon en (+)-dihidrokarvoon na die ooreenstemmende onversadigde verbindings by 25°C gekataliseer. Laastens is die kristallisasie van CmOYE vir die versameling van X-straal kristallografiese data vir toekomstige strukturele karakterisering, uitgevoer. Die ensiem was suksesvol gekristalliseer en diffraksie data is versamel by 'n resolusie van 57.99-1.93 Å. Dié studie het getoon dat die voorspelling van funksionele eienskappe vanaf aminosuuropeenvolging steeds problematies vir lede van die OYE familie is. Hoewel die gebruik van die drie geïdentifiseerde teiken-residue as vingerafdruk nuttig is vir aanduidings van die evolusionêre verhouding tussen die groot aantal OYE homoloë, kom hierdie groeperinge nie noodwendig ooreen met die groepering van OYEs met soortgelyke funksionele eienskappe nie. Die behoefte vir meer funksionele en strukturele data van OYE homoloë (veral OYE homoloë wat aan tans ongekarakteriseerde subgroepe behoort) bly groot as 'n korrelasie tussen aminosuuropeenvolging en funksionaliteit tussen OYE homoloë gevind wil word.