Mycothiol [2-(N-acetylcysteinyl)amido-2-deoxy-␣-D-glucopyranosyl-(131)-myo-inositol] (MSH. Since this novel thiol is more resistant than glutathione to heavy-metal ion-catalyzed oxidation, it seems likely to be the antioxidant thiol used by aerobic gram-positive bacteria that do not produce glutathione (GSH). In the present study we sought to define the spectrum of organisms that produce MSH. GSH was absent in all actinomycetes and some of the other gram-positive bacteria studied. Surprisingly, the streptococci and enterococci contained GSH, and some strains appeared to synthesize it rather than import it from the growth medium. MSH was found at significant levels in most actinomycetes examined. Among the actinobacteria four Micrococcus species produced MSH, but MSH was not found in representatives of the Arthrobacter, Agromyces, or Actinomyces genera. Of the nocardioforms examined, Nocardia, Rhodococcus, and Mycobacteria spp. all produced MSH. In addition to the established production of MSH by streptomycetes, we found that Micromonospora, Actinomadura, and Nocardiopsis spp. also synthesized MSH. Mycothiol production was not detected in Propionibacterium acnes or in representative species of the Listeria, Staphylococcus, Streptococcus, Enterococcus, Bacillus, and Clostridium genera. Examination of representatives of the cyanobacteria, purple bacteria, and spirochetes also gave negative results, as did tests of rat liver, bonito, Candida albicans, Neurospora crassa, and spinach leaves. The results, which indicate that MSH production is restricted to the actinomycetes, could have significant implications for the detection and treatment of infections with actinomycetes, especially those caused by mycobacteria.

Several self-compatible species of higher plants, such as Arabidopsis thaliana, have recently been found to contain S-like RNases. These S-like RNases are homologous to the S-RNases that have been hypothesized to control selfincompatibility in Solanaceous species. However, the relationship of the S-like RNases to the S-RNases is unknown, and their roles in self-compatible plants are not understood. To address these questions, we have investigated the RNS2 gene, which encodes an S-like RNase (RNS2) of Arabidopsis. Amino acid sequence comparisons indicate that RNS2 and other S-like RNases make up a subclass within an RNase superfamily, which is distinct from the subclass formed by the S-RNases. RNS2 is most similar to RNase LE [Jost, W., Bak, H., Glund, K., Terpstra, P., Beintema, J. J. (1991) Eur. J. Biochem. 198,[1][2][3][4][5][6], an S-like RNase from Lycopersicon esculentum, a Solanaceous species. The fact that RNase LE is more similar to RNS2 than to the S-RNases from other Solanaceous plants indicates that the S-like RNases diverged from the S-RNases prior to speciation. Like the S-RNase genes, RNS2 is most highly expressed in flowers, but unlike the S-RNase genes, RNS2 is also expressed in roots, stems, and leaves of Arabidopsis. Moreover, the expression of RNS2 is increased in both leaves and petals of Arabidopsis during senescence. Phosphate starvation can also induce the expression ofRNS2. On the basis of these observations, we suggest that one role of RNS2 in Arabidopsis may be to remobilize phosphate, particularly when cells senesce or when phosphate becomes limiting.Fundamental insights into the relationship between protein structure and function and gene evolution have been gained from the study of members of the pancreatic RNase superfamily typified by RNase A (1). Another RNase family has recently been identified in the plant kingdom (2). RNases in this family are not homologous to the pancreatic RNase superfamily but rather share homology with a class of fungal RNases that includes RNase T2 ofAspergillus oryzae (3). The plant members of this family include the S-RNases, proteins associated with a self-recognition response known as selfincompatibility (SI) in certain species of higher plants. In Nicotiana alata and other members of the Solanaceae, pollen carrying a particular allele at the S locus, which controls SI, are unable to fertilize plants carrying the same S allele. The mechanism by which S-RNases may participate in the rejection of incompatible pollen is unknown; however, their genes cosegregate with the S locus (4).Initially it seemed plausible that the S-RNases were highly specialized polymorphic enzymes, without homologs in selfcompatible species. However, several recent studies have shown that this is not the case. Among these are dataThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.obtained from PCR experiments perform...

The human pathogen Staphylococcus aureus does not utilize the glutathione thiol/disulfide redox system employed by eukaryotes and many bacteria. Instead, this organism produces CoA as its major low molecular weight thiol. We report the identification and purification of the disulfide reductase component of this thiol/ disulfide redox system. Coenzyme A disulfide reductase (CoADR) catalyzes the specific reduction of CoA disulfide by NADPH. CoADR has a pH optimum of 7.5-8.0 and is a dimer of identical subunits of M r 49,000 each. The visible absorbance spectrum is indicative of a flavoprotein with a max ‫؍‬ 452 nm. The liberated flavin from thermally denatured enzyme was identified as flavin adenine dinucleotide. Steady-state kinetic analysis revealed that CoADR catalyzes the reduction of CoA disulfide by NADPH at pH 7.8 with a K m for NADPH of 2 M and for CoA disulfide of 11 M. In addition to CoA disulfide CoADR reduces 4,4-diphosphopantethine but has no measurable ability to reduce oxidized glutathione, cystine, pantethine, or H 2 O 2 . CoADR demonstrates a sequential kinetic mechanism and employs a single active site cysteine residue that forms a stable mixed disulfide with CoA during catalysis. These data suggest that S. aureus employs a thiol/disulfide redox system based on CoA/CoA-disulfide and CoADR, an unorthodox new member of the pyridine nucleotide-disulfide reductase superfamily.

Bovine pancreatic ribonuclease A (RNase A) has been the object of much landmark work in biological chemistry. Yet the application of the techniques of protein engineering to RNase A has been limited by problems inherent in the isolation and heterologous expression of its gene. A cDNA library was prepared from cow pancreas, and from this library the cDNA that codes for RNase A was isolated. This cDNA was inserted into expression plasmids that then directed the production of RNase A in Saccharomyces cerevisiae (fused to a modified alpha-factor leader sequence) or Escherichia coli (fused to the pelB signal sequence). RNase A secreted into the medium by S.cerevisiae was an active but highly glycosylated enzyme that was recoverable at 1 mg/l of culture. RNase A produced by E.coli was in an insoluble fraction of the cell lysate. Oxidation of the reduced and denatured protein produced active enzyme which was isolated at 50 mg/l of culture. The bacterial expression system is ideal for the large-scale production of mutants of RNase A. This system was used to substitute alanine, asparagine or histidine for Gln11, a conserved residue that donates a hydrogen bond to the reactive phosphoryl group of bound substrate. Analysis of the binding and turnover of natural and synthetic substrates by the wild-type and mutant enzymes shows that the primary role of Gln11 is to prevent the non-productive binding of substrate.

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A processive enzyme binds a polymeric substrate and catalyzes a series of similar chemical reactions along that polymer before releasing the fully modified polymer to solvent. Bovine pancreatic ribonuclease A (RNase A) is a nonprocessive endoribonuclease that binds the bases of adjacent RNA residues in three enzymic subsites: B1, B2, and B3. The B1 subsite binds only to residues having a pyrimidine base, while the B2 subsite prefers adenine and the B3 subsite prefers a purine base. RNase A mutants were created in which all natural amino acids were substituted for Thr45 or Phe120, two residues of the B1 subsite. These pools of mutant enzymes were screened for mutants that catalyze the cleavage of RNA after purine residues. The Ala45 and Gly45 enzymes cleave poly(A), poly(C), and poly(U) efficiently and with 10(3)-10(5)-fold increases in purine/pyrimidine specificity. Thus, substrate binding can be uncoupled from substrate turnover in catalysis by RNase A. In addition, both mutant enzymes cleave poly(A) processively. Our results provide a new paradigm: a processive enzyme has subsites, each specific for a repeating motif within a polymeric substrate. Further, we propose that processive enzymes bind more tightly to motifs that do repeat than to those that do not.

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The cdr gene encoding coenzyme A disulfide reductase (CoADR) from Staphylococcus aureus 8325-4 was cloned, sequenced, and overexpressed. The gene encodes a 438-amino acid polypeptide that has a calculated molecular weight of 49,200 and sequence similarity to the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes. The deduced primary structure contains consensus sequences for flavin adenine dinucleotide and NADPH-binding regions but lacks the catalytic disulfide signature sequence typical of the glutathione reductase family of disulfide reductases. The active site region of CoADR has only a single cysteine residue that is similar to that in the conserved SFXXC active site motif of NADH oxidase and NADH peroxidase from Enterococcus faecalis. CoADR is the first disulfide reductase reported having this active site region, and sequence comparisons of CoADR to representative members of the pyridine nucleotide-disulfide reductase superfamily placed CoADR in a distinct subfamily. CoADR was overexpressed in Escherichia coli using the pET expression system, and 5-10 mg of fully active recombinant enzyme were recovered per liter of E. coli cells.