[Show abstract][Hide abstract] ABSTRACT: Mycothiol (MSH) is the major low molecular weight thiol in mycobacteria. Two chemical mutants with low MSH and one with no MSH (strain 49) were produced in Mycobacterium smegmatis mc2155 to assess the role of MSH in mycobacteria. Strain 49 was shown to not produce 1-d-myo-inosityl-2-amino-2-deoxy-alpha-d-glucopyranoside (GlcN-Ins), an intermediate in MSH biosynthesis. Relative to the parent strain, mutant 49 formed colonies more slowly on solid media and was more sensitive to H2O2 and rifampin, but less sensitive to isoniazid. Complementation of mutant 49 with DNA from M. tuberculosis H37Rv partially restored production of GlcN-Ins and MSH, and resistance to H2O2, but largely restored colony growth rate and sensitivity to rifampin and isoniazid. The results indicate that MSH and GlcN-Ins are not essential for in vitro survival of mycobacteria but may play significant roles in determining the sensitivity of mycobacteria to environmental toxins.
Biochemical and Biophysical Research Communications 03/1999; 255(2):239-44. DOI:10.1006/bbrc.1999.0156 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 Mr 49,000 each. The visible absorbance spectrum is indicative of a flavoprotein with a lambdamax = 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 Km for NADPH of 2 muM and for CoA disulfide of 11 muM. In addition to CoA disulfide CoADR reduces 4,4'-diphosphopantethine but has no measurable ability to reduce oxidized glutathione, cystine, pantethine, or H2O2. 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Human pancreatic ribonuclease (HP-RNase) has considerable promise as a therapeutic agent. Structure-function analyses of HP-RNase have been impeded by the difficulty of obtaining the enzyme from its host. Here, a gene encoding HP-RNase was designed, synthesized, and inserted into two expression vectors that then direct the production of HP-RNase in Saccharomyces cerevisiae (fused to either an unmodified or a modified a-factor pre-pro segment) or Escherichia coli (fused to the pelB signal sequence). HP-RNase produced in S. cerevisiae was secreted into the medium as an active enzyme, isolable at 0.1-0.2 mg/liter of culture. This isolate was heterogeneous due to extensive glycosylation and incomplete maturation of the pre-pro segment. HP-RNase produced in E. coli with the pET expression system was purified from the insoluble fraction of the cell lysate. Renaturation of the reduced and denatured protein produced active, homogeneous enzyme recoverable at 1 mg/liter of culture. The N terminus of the HP-RNase produced from the bacterial expression system was processed fully in vivo. The yeast system, combined with techniques that allow detection of picograms of ribonuclease activity, offers a sensitive probe for studies of post-translational modification and secretory targeting in eukaryotic cells. The bacterial system enables studies both to reveal new structure-function relationships in ribonucleases and to evaluate the use of HP-RNase as a cytotoxin that is tolerated by the human immune system.
Protein Expression and Purification 06/1996; 7(3):253-61. DOI:10.1006/prep.1996.0036 · 1.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mycothiol [2-(N-acetylcysteinyl)amido-2-deoxy-alpha-D-glucopyranosyl- (1-->1)-myo-inositol] (MSH) has recently been identified as a major thiol in a number of actinomycetes (S. Sakuda, Z.-Y. Zhou, and Y. Yamada, Biosci. Biotech. Biochem. 58:1347-1348, 1994; H. S. C. Spies and D. J. Steenkamp, Eur. J. Biochem. 224:203-213, 1994; and G. L. Newton, C. A. Bewley, T. J. Dwyer, R. Horn, Y. Aharonowitz, G. Cohen, J. Davies, D. J. Faulkner, and R. C. Fahey, Eur. J. Biochem. 230:821-825, 1995). 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.
Journal of Bacteriology 04/1996; 178(7):1990-5. · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of the P-O5 bond of RNA after residues bound in the enzyme's B1 subsite. This subsite binds to cytidine 30-fold more tightly than to uridine and > 10(5)-fold more tightly than to adenine. Structural studies had suggested that the hydroxyl group of Thr45 can interact directly with the base of a bound nucleotide. In contrast, the carboxylate group of Asp83 cannot interact directly with bound substrate but can accept a hydrogen bond from the hydroxyl group of Thr45. To assess the role of the Thr45-Asp83 hydrogen bond in catalysis, T45G, D83A and T45G/D83A RNase A were prepared and their abilities to catalyze the cleavage of various substrates were determined. The results indicate that the side-chain of Asp83 enhances catalysis of reactions in which uridine is bound in the B1 subsite, but that this enhancement relies on the side-chain of Thr45. In contrast, the side-chain of Asp83 does not contribute to catalysis of reactions with cytidine in the B1 subsite. Thermodynamic cycles derived from kinetic parameters for the cleavage of poly(U) indicate that the Thr45-Asp83 interaction contributes 1.2 kcal/mol to transition state stabilization, which is 0.9 kcal/mol greater than its contribution to ground state stabilization. Thus, like many residue-substrate interactions, this residue to residue interaction enhances catalysis by becoming stronger as the reaction approaches the transition state.
[Show abstract][Hide abstract] ABSTRACT: 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.
Protein engineering 04/1995; 8(3):261-73. DOI:10.1093/protein/8.3.261
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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 self-incompatibility 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, 1-6.], 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 of RNS2. 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.
Proceedings of the National Academy of Sciences 07/1993; 90(11):5118-22. DOI:10.1073/pnas.90.11.5118 · 9.81 Impact Factor