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The genes involved in the 2,3-butanediol pathway coding for ct-acetolactate decarboxylase, a-acetolactate synthase (ae-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiela terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budAl gene, coding for ot-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrgena genes budB and budC, coding for at-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long cx-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic a-ALS enzymes encoded by the genes ilvB, ilvG, and ilvl. The K. terrigena a-ALS is also shown to complement an anabolic ce-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.

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The primary and secondary structure of glutaredoxin-3 (Grx3), a glutathione-disulfide oxidoreductase from Escherichia coli, has been determined. The amino acid sequence of Grx3 consists of 82 residues and contains a redox-active motif, Cys-Pro-Tyr-Cys, typical of the glutaredoxin family. Sequence comparison reveals a homology (33% identity) to that of glutaredoxin-1 (Grx1) from E. coli as well as to other members of the thioredoxin superfamily. In addition to the active site cysteine residues, Grx3 contains one additional cysteine (Cys 65 ) corresponding to one of the two non-active site (or structural) cysteine residues present in mammalian glutaredoxins. The sequence-specific 1 H and 15 N nuclear magnetic resonance assignments of reduced Grx3 have been obtained. From a combined analysis of chemical shifts, 3J HN␣ coupling constants, sequential and medium range NOEs, and amide proton exchange rates, the secondary structure of reduced Grx3 was determined and found to be very similar to that inferred from amino acid sequence comparison to homologous proteins. The consequences of the proposed structural similarity to Grx1 are that Grx3, while possessing a largely intact GSH binding cleft, would have a very different spatial distribution of charged residues, most notably surrounding the active site cysteine residues and occurring in the proposed hydrophobic protein-protein interaction area. These differences may contribute to the observed very low K cat of Grx3 as a reductant of insulin disulfides or as a hydrogen donor for ribonucleotide reductase. Thus, despite an identical active site disulfide motif and a similar secondary structure and tertiary fold, Grx3 and Grx1 display large functional differences in in vitro protein disulfide oxido-reduction reactions.In general, glutaredoxins (Grx) 1 and thioredoxins (Trx) are small (9 -12 kDa), well characterized proteins capable of catalyzing thiol-disulfide exchange reactions. Representatives of at least one of these two protein families have been found in all organisms studied, indicating that proteins of this type are essential for cellular functions (for reviews, cf. Gleason and Holmgren (1988) and Holmgren (1989)). In the cell, glutaredoxins and thioredoxins differ in the manner they are reduced. Glutaredoxins are reduced via the ubiquitous tripeptide glutathione (GSH), whereas thioredoxins are reduced directly by the specific flavoenzyme thioredoxin reductase. In both cases, reducing equivalents are ultimately derived from NADPH. In vitro, thioredoxins are found to be general reductants of a number of different protein disulfides, whereas glutaredoxins are less capable in this respect, but are known to readily reduce mixed disulfides between proteins (or low molecular weight thiol-containing compounds) and GSH (Gravina and Mieyal, 1993). This activity is measured conventionally using the ␤-hydroxyethylene disulfide (HED) reduction assay (Holmgren, 1979a). Glutaredoxins can also reduce some specific protein disulfides, such as the redox-active disulfide of ribonucl...