Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes

Cohen, Gerald; Yanko, Michaela; Mislovati, M; Argaman, A; Schreiber, Rachel; Av‐Gay, Yossef; Aharonowitz, Yair

doi:10.1128/jb.175.16.5159-5167.1993

Cited by 32 publications

(24 citation statements)

References 45 publications

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“…Unlike the vast majority of TrxR-Trx1 systems from other bacteria, the gene encoding TrxR is positioned just downstream from trx1 in the H. pylori genome. The clustering of trx1 and trxR in this order has also been observed in Streptomyces clavuligerus (14); however, in mycobacteria (72) and in C. jejuni (49), the opposite orientation, trxR and then trx1, is observed. In most other species, trxR and trx1 are widely separated on the chromosome.…”

Section: Resultsmentioning

confidence: 88%

Essential Thioredoxin-Dependent Peroxiredoxin System from Helicobacter pylori : Genetic and Kinetic Characterization

et al. 2001

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Helicobacter pylori, an oxygen-sensitive microaerophile, contains an alkyl hydroperoxide reductase homologue (AhpC, HP1563) that is more closely related to 2-Cys peroxiredoxins of higher organisms than to most other eubacterial AhpC proteins. Allelic replacement mutagenesis revealed ahpC to be essential, suggesting a critical role for AhpC in defending H. pylori against oxygen toxicity. Characterization of the ahpC promoter region divulged two putative regulatory elements and identified the transcription initiation site, which was mapped to 96 and 94 bp upstream of the initiation codon. No homologue of ahpF, which encodes the dedicated AhpC reductase in most eubacteria, was found in the H. pylori genome. Instead, homologues of Escherichia coli thioredoxin (Trx) reductase (TrxR, HP0825) and Trx (Trx1, HP0824) formed a reductase system for H. pylori AhpC. A second Trx homologue (Trx2, HP1458) was identified but was incapable of AhpC reduction, although Trx2 exhibited disulfide reductase activity with other substrates [insulin and 5,5-dithiobis(2-nitrobenzoic acid)]. AhpC interactions with each substrate, Trx1 and hydroperoxide, were bimolecular and nonsaturable (infinite V max and K m values) but rapid enough (at 1 ؋ 10 5 to 2 ؋ 10 5 M ؊1 s ؊1 ) to suggest an important role for AhpC in cellular peroxide metabolism. AhpC also exhibited a wide specificity for hydroperoxide substrates, which, taken together with the above results, suggests a minimal binding site for hydroperoxides composed of little more than the cysteinyl (Cys49) active site. H. pylori AhpC was not reduced by Salmonella typhimurium AhpF and was slightly more active with E. coli TrxR and Trx1 than was S. typhimurium AhpC, demonstrating the specialized catalytic properties of this peroxiredoxin.

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Section: Resultsmentioning

confidence: 88%

Essential Thioredoxin-Dependent Peroxiredoxin System from Helicobacter pylori : Genetic and Kinetic Characterization

et al. 2001

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“…DISCUSSION P. chrysogenum possesses an NADPH-dependent disulfide reductase that has broad substrate specificity and that is effective in reducing disulfides in LMW compounds and proteins. When coupled in vitro with IPNS, it converts bis-ACV to isopenicillin N. We previously proposed that because IPNS reactions occur in vitro only when ACV is in its reduced thiol form, 3-lactam-producing organisms may employ a disulfide reductase system to maintain intracellular ACV in its reduced state (2,6). The broad-range disulfide reductase described in this report may therefore play an important role in promoting penicillin production in P. chrysogenum.…”

Section: Resultsmentioning

confidence: 96%

“…The enzymes possess 334, 321, and 322 amino acids (counting the initiation amino acids), respectively, share 44 to 48.7% amino acid sequence identity, and contain essentially the same catalytic redox active site and FAD and NADPH dinucleotide binding domains. They clearly differ from other NADPH-dependent disulfide reductases, glutathione reductase, dihydrolipoamide dehydrogenase, trypanothione reductase, and mercuric reductase, which are much larger, share limited sequence similarity with each other and with the P. chrysogenum HMW reductase and the bacterial thioredoxin reductases, and have rather different redox active regions (6). The consensus dithiol redox active region of the former is -CV/LNVI/GC- (1,37), and that of the latter is -CAT/VC-(6, 37; this work).…”

Section: Resultsmentioning

confidence: 99%

“…Amino acid sequence comparison of the P. chrysogenum HMW protein with thioredoxin disulfide reductases. Figure 6 shows the alignment of the predicted amino acid sequence of the P. chrysogenum HMW reductase, from this work, with the sequences of the only other thioredoxin reductases that have been reported, those of E. coli (37) and S. clavuligerus (6) 30.6% identities, respectively). The P. chrysogenum HMW reductase has a redox active site (-CAVC-) similar to that present in the E. coli and S. clavuligerus thioredoxin reductase (-CATC-) and located in the same position in the polypeptide.…”

Section: Resultsmentioning

confidence: 99%

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The thioredoxin system of Penicillium chrysogenum and its possible role in penicillin biosynthesis

et al. 1994

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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...

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“…Similar sequences have been detected only in the related actinomycetes M. leprae and M. tuberculosis. orf205 converges on the genes encoding thioredoxin (trxA) and thioredoxin reductase (trxB) (9).…”

Section: Resultsmentioning

confidence: 99%

Partitioning of the Linear Chromosome during Sporulation of Streptomyces coelicolor A3(2) Involves an oriC -Linked parAB Locus

et al. 2000

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Candidate partitioning genes (parA and parB) for the linear chromosome of Streptomyces coelicolor were identified by DNA sequencing in a series of seven genes located between rnpA and trxA near the chromosomal replication origin. The most likely translation start point of parB overlapped the parA stop codon, suggestive of coregulation, and transcription analysis suggested that the two genes formed an operon. Deletion of part of parB had no effect on the growth or appearance of colonies but caused a deficiency in DNA partitioning during the multiple septation events involved in converting aerial hyphae into long chains of spores. At least 13% of spore compartments failed to inherit the normal DNA allocation. The same phenotype was obtained with a deletion removing a segment of DNA from both parA and parB. Reinforcing the idea of a special role for the par locus during sporulation, the stronger of two parAB promoters was greatly upregulated at about the time when sporulation septation was maximal in colonies. Three copies of a 14-bp inverted repeat (GTTTCACGT GAAAC) were found in or near the parAB genes, and at least 12 more identical copies were identified within 100 kb of oriC from the growing genome sequence database. Only one perfect copy of the 14-bp sequence was present in approximately 5 Mb of sequence available from the rest of the genome. The 14-bp sequence was similar to sequences identified as binding sites for Spo0J, a ParB homologue from Bacillus subtilis believed to be important for DNA partitioning

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Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes

Cited by 32 publications

References 45 publications

Essential Thioredoxin-Dependent Peroxiredoxin System from Helicobacter pylori : Genetic and Kinetic Characterization

Essential Thioredoxin-Dependent Peroxiredoxin System from Helicobacter pylori : Genetic and Kinetic Characterization

The thioredoxin system of Penicillium chrysogenum and its possible role in penicillin biosynthesis

Partitioning of the Linear Chromosome during Sporulation of Streptomyces coelicolor A3(2) Involves an oriC -Linked parAB Locus

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