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

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.