Purification and properties of two oxidoreductases catalyzing the enantioselective reduction of diacetyl and other diketones from baker's yeast

Abstract

The NADPH-linked diacetyl reductase system from the cytosolic fraction of Saccharomyces cerevisiae has been resolved into two oxidoreductases catalyzing irreversibly the enantioselective reduction of diacetyl (2,3-butanedione) to (9-and (R)-acetoin (3-hydroxy-2-butanone) [so-called (S)-and (R)-diacetyl reductases] (EC 1.1.1.5) which have been isolated to apparent electrophoretical purity.The clean-up procedures comprising streptomycin sulfate treatment, Sephadex G-25 filtration, DEAESepharose CL-6B column chromatography, affinity chromatography on Matrex Gel Red A and Superose 6 prep grade filtration led to 120-fold and 368-fold purifications, respectively.The relative molecular mass of the (R)-diacetyl reductase, estimated by means of HPLC filtration on Zorbax G F 250 and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was 36 000. The (R)-enzyme was most active at pH 6.4 and accepted in addition to diacetyl C5-, C6-2,3-diketones, 1,2-~yclohexanedione, 2-0x0 aldehydes and short-chain 2-and 3-0x0 esters as substrates. The enzyme was characterized by high enantioselectivity and regiospecificity. The K,,, values for diacetyl and 2,3-pentanedione were determined as 2.0 mM.The M , of the (3-diacetyl reductase was determined as 75000 by means of HPLC filtration on Zorbax G F 250. The enzyme decomposed into subunits of M , 48000 and 24000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The optimum pH was 6.9. The purified @)-enzyme reduced stereospecifically a broad spectrum of substrates, comprising 2,3-, 2,4-and 2,5-diketones, %-ox0 aldehydes, 1 ,2-cyclohexanedione and methyl ketones as well as 3-, 4-and 5-0x0 esters. The 2,3-and 2,4-diketones are transformed to the corresponding (S)-2-hydroxy ketones; 2,5-hexanedione, however, was reduced to (S,S)-2,5-hexanediol. The K,,, values for diacetyl and 2,3-pentanedione were estimated as 2.3 and 1.5 mM, respectively. Further characterization of the (S)-diacetyl reductase revealed that it is identical with the so-called'(S)-enzyme', involved in the enantioselective reduction of 3-, 4-and 5-0x0 esters in baker's yeast.From the pioneering work of Neuburg and Nord [l], it is known that preparative reductions of diacetyl by actively fermenting baker's yeast lead to levorotary 2,3-butanediol. Various phytochemical transformations of diacetyl homologues and diketo compounds have been applied to obtain optically active hydroxy ketones and diols, useful building blocks in organic chemistry [2,3]. Diacetyl is also a physiological metabolite mainly formed in yeast by the chemical oxidative decarboxylation of 2-acetolactate, an intermediate in the biosynthesis of valine and leucine [4-71. It is responsible for the well-known off-flavor called 'buttery note' in fermented beverages, especially beer 181.The importance of diacetyl in the brewing process has been a major reason for various investigations of the enzymes involved in its reduction to acetoin (so-called diacetyl reductases) and further to 2,3-butanediol (2,3-butanediol deCorrespondence to J. Heidlas,

Role of Saccharomyces cerevisiae Oxidoreductases Bdh1p and Ara1p in the Metabolism of Acetoin and 2,3-Butanediol

“…Lanes 1, 2, 5, 6, 9, and 10 show the expression levels of Bdh1p-6His in the cell extracts of strains WCG4-11/22a (lanes 1 and 2), FY834 (lanes 5 and 6), and FY834 bdh1⌬ (lanes 9 and 10). Lanes 3,4,7,8,11,and 12 show the expression of Bdh2p-6His in the cell extracts of strains WCG4-11/22a (lanes 3 and 4), FY834 (lanes 7 and 8), and FY834 bdh1⌬ (lanes 11 and 12). effect of the oxygen supply during yeast fermentation on the formation of 2,3-butanediol through acetoin was previously observed (15).…”

Enzymology of Vicinal Diketone Reduction in Brewer's Yeast

“…It was shown by Hardwick et al 7 that alcohol dehydrogenase (ADH 1; EC 1.1.1.1) will reduce diacetyl and pentanedione, albeit rather less efficiently than it deals with acetaldehyde, but it was also shown that yeast contains other enzymes capable of reducing the VDKs. Others have reported specific diacetyl reductases (EC 1.1.1.5) in yeast 8,14,16 . Attention has been drawn 10 to the challenges involved in unraveling the literature on the diversity of oxidoreductases in an organism such as yeast.…”

Characterization of partially purified alcohol dehydrogenase from Rhodococcus sp. strain GK1

“…The ADH from strain GK1 did not oxidize 2,3 butanediol or acetoin (Table 2), in common with two oxidoreductases from S. cerevisiae (Heidlas and Tressl 1990).…”

“…A wide variety of bacteria (Seitz et al 1963 ;Ui et al 1987 ;Hummel et al 1989 ;Gibson et al 1991 ;Blomquist et al 1993) and yeasts (Louis-Eugène et al 1988 ;Verduyn et al 1988 ;Legeay et al 1989 ;Heidlas and Tressl 1990 ;Hang and Woodams 1993 ;Olguin-Castillo et al 1994) display a diacetyl reductase. Chicken liver has also been described as a source of this enzyme (Gibson et al 1991).…”