Over-expression, purification and characterization of Adh5p from Saccharomyces cerevisiae

Abstract

English: In the past few decades much research has been conducted on yeast alcohol dehydrogenases with emphasis on their role in S. cerevisiae. There are seven known Adh proteins that were identified in S. cerevisiae. Six of them are intensively studied on gene and translational level. After sequencing of S. cerevisiae chromosome II (Feldman et al. 1994), ADH5 was identified which is 76% and 77% identical to ADH1 and ADH2 respectively. Later studies on global localization on genes showed that ADH5 is localized in the cytoplasm (Hue et al. 2003). No further research has been performed on ADH5. In this study Adh5p was over-expressed, purified and characterized towards its primary substrate, ethanol, and preferred co-factor NAD+. Furthermore, indepth kinetic studies were performed using various alcohols, increasing in chain length and branching. Adh5p is capable of oxidizing alcohols from two carbons to ten carbons. However, the catalytic efficiency decreases with increasing chain length. Results showed that Adh5p functions in vitro the same as Adh1p and Adh2p, sharing a primary substrate (ethanol). Adh1p and Adh2p are capable of converting more substrate per unit enzyme per second Adh5p. The second part of this study was to determine if Adh5p could substitute the catalytic function of Adh1p in vivo. For this purpose, ADH5 expression needed to be similar to Adh1p levels in the cell. Thus an expression vector was used containing ADH5 gene flanked with the promoter and terminator regions of ADH1. S. cerevisiae TΔ123 was constructed with ADH4 and ADH5 still intact. S. cerevisiae TΔ123 was transformed with a pRS413 and pRS423 construct containing the ADH1 promoter and terminator fused to the ADH5 ORF. Growth was monitored in chemically defined media containing 7 g l-1 ethanol or 8 g l-1 glucose. Growth parameters were also compared to the S. cerevisiae W303-1A and the adh quadruple deletion strain (Q1) containing only ADH1. The S. cerevisiae TΔ123::ADH5_S and S. cerevisiae TΔ123::ADH5_M constructs were capable of limited growth on both glucose and ethanol as carbon source When comparing the biomass yield of S. cerevisiae TΔ123::ADH5_S and S. cerevisiae TΔ123::ADH5_M to the biomass yield for both S. cerevisiae W303- 1A and S. cerevisiae Q1 the constructs delivered a much lower biomass yield. To verify the expression of Adh5p, a Micro BCA™ protein assay kit supplied by Pierce (Smith et al. 1985) was used. Protein concentrations were determined at various time intervals. Cell homogenization was standardized, S. cerevisiae Q1 cells were diluted to fit the OD600 obtained for both the S. cerevisiae TΔ123::ADH5_S and S. cerevisiae TΔ123::ADH5_M strains. To conclude, Adh5p is capable of oxidizing various alcohols, but ethanol is its primary substrate. Furthermore Adh5p is not capable of replacing Adh1p in cellular metabolic function. The low turnover number illustrated by Adh5p and the lack thereof to reduce acetaldehyde is the most prominent cause of cellular death. Unlike Adh1p, Adh5p is not capable of reducing acetaldehyde to ethanol and thus not capable of NAD+ - NADH regeneration.

Afrikaans: Gedurende die afgelope paar jaar is daar baie navorsing gedoen op gis ADH, hoofsaaklik die van S. cerevisiae. Daar is tans sewe ADH’s geïdentifiseer in S. cerevisiae, ses van die sewe is reeds gekarakteriseer en baie navorsing is reeds gedoen op geen vlak. Dit was egter na Feldman en sy kollegas in 1994 bevestig het dat een van die ADH’s (ADH5), wat tot op hede nog geen navorsing op gedoen is nie, sekere verwantskappe deel met Adh1p en Adh2p, op basispaaropeenvolgende vlak, naamlik 76% en 77%. Geen navorsing was tot op hede op ADH5 gedoen nie. In hierdie studie was Adh5p uitgedruk en gekarakteriseer teenoor die proteïen se primêre substraat, naamlik etanol en ko-faktor naamlik NAD+. Diepgaande studies was uitgevoer op verskeie ander substrate, alkohole wat se koolstof lengte toeneem vanaf twee tot tien koolstof atome. Hoe langer die alkohol, hoe vinniger neem die effektiwiteit van die ensiem af. Resultate wys dat Adh5p dieselfde funksioneer as Adh1p en Adh2p. Die hoof- en kritieke verskil is dat Adh1p en Adh2p die vermoë besit om meer substraat per eenheid ensiem om te skakel per sekonde as Adh5p, en is dus baie meer effektief is as Adh5p. Die tweede helfte van die studie was om te bepaal of Adh5p die funksie van Adh1p kan oorneem in vivo. ADH5 uitdrukkingsvlakke moes dus verhoog word sodat dit vergelykbaar kan wees met ADH1 vlakke. ‘n Uitdrukkingsplasmied was gemaak wat die ADH5 geen bevat, gekoppel aan die promotor en termineerder gebiede van ADH1. Die konstruk was in die TΔ123 mutant in getransformeer. Die getransformeerde gisstam, S. cerevisiae TΔ123::ADH5_S en S. cerevisiae TΔ123::ADH5_M, was daaropvolgend gebruik om die groeikromme te monitor in chemies gedefinieerde medium. Die media het 7 g l- 1 etanol of 8 g l-1 glukose bevat. Groeiparameters van die delesiekonstrukte was vergelyk met S. cerevisiae W303-1A en S. cerevisiae Q1 stamme. Wanneer daar vergelykinge getref word tussen die biomassa-opbrengs van albei verwysingsstamme en die delesiekonstrukte kan daar afgelei word dat die biomassa-opbrengs van die konstrukte sowat tien maal laer is. Proteïen konsentrasies was op verskeie tydsintervalle geneem om sodoende die hoeveelheid proteïen in elke stam te kan vergelyk. Die konsentrasies van al die stamme waar Adh1p uitgedruk is, was vergelykbaar met die van die konstrukte. Om saam te vat, Adh5p is wel in staat daarvan om verskeie alkohole te oksideer, maar etanol is wel die primêre substraat. Adh5p is nie in staat om asetaldehied te reduseer nie en dus lei dit tot ‘n toename van die toksiese produk wat lei tot die afsterwing van die sel. Adh5p kan dus nie NAD+ - NADH herwin nie, maar speel tog ʼn belangrike rol in etanol metabolisme sodat gisselle onder optimum kondisies kan funksioneer.