THIOREDOXIN-THIOREDOXIN REDUCTASE SYSTEM OF STREPTOMYCES-CLAVULIGERUS- SEQUENCES, EXPRESSION, AND ORGANIZATION OF THE GENES

The genes that encode thioredoxin and thioredoxin reductase of Strepto myces clavuligerus were cloned, and their DNA sequences were determine d. Previously, we showed that S. clavuligerus possesses a disulfide re ductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosy nthesis of beta-lactam antibiotics. It consists of two components, a 7 0-kDa NADPH-dependent flavoprotein disulfide reductase with two identi cal subunits and a 12-kDa heat-stable protein general disulfide reduct ant. In this study, we found, by comparative analysis of their predict ed amino acid sequences, that the 35-kDa protein is in fact thioredoxi n reductase; it shares 48.7% amino acid sequence identity with Escheri chia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, an d it shares 28 to 56% amino acid sequence identity with other thioredo xins. The streptomycete thioredoxin reductase has the identical cystei ne redox-active region-Cys-Ala-Thr-Cys-and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thiored oxin as a substrate. The streptomycete thioredoxin has the same cystei ne redox-active segment-Trp-Cys-Gly-Pro-Cys-that is present in virtual ly all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin red uctase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reducta se (trxB) genes are organized in a cluster. They are transcribed in th e same direction and separated by 33 nucleotides. In contrast, the trx A and trxB genes of E. coli, the only other organism in which both gen es have been characterized, are physically widely separated.