Subunit and cofactor binding of Saccharomyces cerevisiae sulfite reductase - towards developing wine yeast with lowered ability to produce hydrogen sulfide

Abstract

Wine yeast, in common with other industrial Saccharomyces cerevisiae yeast, can produce hydrogen sulfide during alcoholic fermentation. The aim of this work was to evaluate a genetic strategy for reducing hydrogen sulfide production by lowering the activity of NADPH-dependent sulfite reductase, a key enzyme in the biosynthesis of the sulfur-containing amino acids. The role of amino acids predicted to be necessary for cofactor binding was investigated by comparisons with other proteins in the ferredoxin family. Substitutions in the glycine-rich loop domain (glycines 891 and 893) and at serine 953 of the alpha subunit, Met10p, appear to strongly affect NADPH binding. However, mutations in two amino acids that are completely conserved within the NADPH binding domain of the ferredoxin reductase family, lysine 959 and cysteine 987, have minimal effect on enzyme activity. Serine 820 is indicated to be essential for efficient FAD binding. The two-hybrid system was used to confirm that Met10p interacts with Met5p, the predicted beta subunit of sulfite reductase. Binding of the subunits could only be demonstrated in the absence of methionine, indicating that an additional factor, possibly siroheme, is required to mediate this interaction. Strategies to decrease, but not eliminate, sulfite reductase activity in the cell using identified mutants were tested, and the findings are discussed with the a view to producing improved wine strains.