Penicillium chrysogenum is an important producer of penicillin antibiotics. A key step in their biosynthesis is the oxidative cyclization of 8-(L-01-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N by the enzyme isopenicillin N synthase (IPNS). bis-ACV, the oxidized disulfide form of ACV is, however, not a substrate for IPNS. We report here the characterization of a broad-range disulfide reductase from P. chrysogenum that efficiently reduces bis-ACV to the thiol monomer. When coupled in vitro with IPNS, it converts bis-ACV to isopenicillin N and may therefore play a role in penicillin biosynthesis. The disulfide reductase consists of two protein components, a 72-kDa NADPH-dependent reductase, containing two identical subunits, and a 12-kDa general disulfide reductant. The latter reduces disulfide bonds in low-molecular-weight compounds and in proteins. The genes coding for the reductase system were cloned and sequenced. Both possess introns. A comparative analysis of their predicted amino acid sequences showed that the 12-kDa protein shares 26 to 600% sequence identity with thioredoxins and that the 36-kDa protein subunit shares 44 to 49%o sequence identity with the two known bacterial thioredoxin reductases. In addition, the P. chrysogenum NADPH-dependent reductase is able to accept thioredoxin as a substrate. These results establish that the P. chrysogenum broad-range disulfide reductase is a member of the thioredoxin family of oxidoreductases. This is the first example of the cloning of a eucaryotic thioredoxin reductase gene.Penicillins are sulfur-containing f-lactam compounds that are produced by certain filamentous fungi and industrially by Penicillium chrysogenum. The initial steps in the biosynthesis of these antibiotics involve the condensation of a-aminoadipic acid, cysteine, and valine to form a tripeptide by the enzyme coupled with IPNS, it quantitatively converts bis-ACV to isopenicillin N. Because ACV structurally resembles glutathione, y-glutamyl-cysteinyl-glycine, the most common intracellular low-molecular-weight (LMW) thiol, we originally supposed that this disulfide reductase might be related to glutathione reductase. However, we have now shown that streptomycetes lack both glutathione and glutathione reductase (2, 35). Also, a biochemical characterization of the S. clavuligerus disulfide reductase revealed that, in contrast to glutathione reductase, the S. clavuligerus disulfide reductase reduces a wide range of disulfides in LMW compounds and in proteins and is composed of two nonidentical polypeptides. The high-molecular-weight (HMW) component is a 70-kDa flavoprotein containing two identical subunits. In the presence of NADPH, it catalyzes the transfer of electrons to the LMW component. The latter is a heat-stable 12-kDa protein that is a general disulfide reductant. These properties of the S. clavuligerus broad-range disulfide reductase led us to propose that it belongs to the thioredoxin-thioredoxin reductase class of flavoprotein disulfide oxidoreductases. This proposal was r...

The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of 13-lactam antibiotics. It consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coil thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region-Cys-Ala-Thr-Cys-and essentially the same flavin adenine dinucleotideand NADPH dinucleotide-binding sites as E. colt thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment-TrpCys-Gly-Pro-Cys-that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. colt methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (tixA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trx4 and txB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated.In a recent article, we described the characterization of a broad-range disulfide reductase from Streptomyces clavuligerus, a producer of penicillin and cephalosporin antibiotics (2). This study was prompted by the finding that the activity of isopenicillin-N-synthase, a key enzyme in the biosynthesis of these 3-lactam compounds, and its tripeptide substrate 8-(L-a-aminoadipyl)-L-cysteinyl-D-valine (ACV) are dependent on the redox state of their cysteine amino acid residues (3, 32). Thus, in vitro conversion of ACV to isopenicillin N occurs only if both enzyme and substrate are in their reduced-thiol state. Isopenicillin-N-synthase reactions are therefore carried out in the presence of an excess of a thiol reagent such as dithiothreitol. We looked for and identified in S. clavuligerus an enzymatic system that replaces the need for dithiothreitol in isopenicillin-N-synthase reactions. That system efficiently reduces bis-ACV, the oxidized disulfide form of ACV, to its thiol state, and modulates the activity of isopenicillin-N-synthase (2). Because ACV structurally resembles glutathione, y-glutamyl-cysteinyl-glycine, the most common intracellular lowmolecular-weight thiol, we r...

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