Functions of Two Types of NADH Oxidases in Energy Metabolism and Oxidative Stress of Streptococcus mutans

We have previously identified two distinct NADH oxidases corresponding to H(2)O(2)-forming oxidase (Nox-1) and H(2)O-forming oxidase (Nox-2) induced in Streptococcus mutans. Sequence analyses indicated a strong similarity between Nox-1 and AhpF, the flavoprotein component of Salmonella typhimurium alkyl hydroperoxide reductase; an open reading frame upstream of nox-1 also showed homology to AhpC, the direct peroxide-reducing component of S. typhimurium alkyl hydroperoxide reductase. To determine their physiological functions in S. mutans, we constructed knockout mutants of Nox-1, Nox-2, and/or the AhpC homologue; we verified that Nox-2 plays an important role in energy metabolism through the regeneration of NAD(+) but Nox-1 contributes negligibly. The Nox-2 mutant exhibited greatly reduced aerobic growth on mannitol, whereas there was no significant effect of aerobiosis on the growth on mannitol of the other strains or growth on glucose of any of the strains. Although the Nox-2 mutants grew well on glucose aerobically, the end products of glucose fermentation by the Nox-2 mutant were substantially shifted to higher ratios of lactic acid to acetic acid compared with wild-type cells. The resistance to cumene hydroperoxide of Escherichia coli TA4315 (ahpCF-defective mutant) transformed with pAN119 containing both nox-1 and ahpC genes was not only restored but enhanced relative to that of E. coli K-12 (parent strain), indicating a clear function for Nox-1 as part of an alkyl hydroperoxide reductase system in vivo in combination with AhpC. Surprisingly, the Nox-1 and/or AhpC deficiency had no effect on the sensitivity of S. mutans to cumene hydroperoxide and H(2)O(2), implying that the existence of some other antioxidant system(s) independent of Nox-1 in S. mutans compensates for the deficiency.     

Characterization of two alkyl hydroperoxide reductase C homologs alkyl hydroperoxide reductase C_H1 and alkyl hydroperoxide reductase C_H2 in Bacillus subtilis

AIM: To identify alkyl hydroperoxide reductase subunit C(AhpC) homologs in Bacillus subtilis(B. subtilis) and to characterize their structural and biochemical properties. AhpC is responsible for the detoxification of reactive oxygen species in bacteria.METHODS: Two AhpC homologs(AhpC_H1 and AhpC_H2) were identified by searching the B. subtilis database; these were then cloned and expressed in Escherichia coli. AhpC mutants carrying substitutions of catalytically important Cys residues(C37S, C47 S, C166 S, C37/47 S, C37/166 S, C47/166 S, and C37/47/166 S for AhpC_H1; C52 S, C169 S, and C52/169 S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahp C genes were disrupted by the short flanking homology method, and the phenotypes of the resulting AhpC-deficient bacteria were examined.RESULTS: Comparative characterization of AhpC homologs indicates that AhpC_H1 contains an extra C37, which forms a disulfide bond with the peroxidatic C47, and behaves like an atypical 2-Cys AhpC, while AhpC_H2 functions like a typical 2-Cys AhpC. Tryptic digestion analysis demonstrated the presence of intramolecular Cys37-Cys47 linkage, which could be reduced by thioredoxin, resulting in the association of the dimer into higher-molecular-mass complexes. Peroxidase activity analysis of Cys→Ser mutants indicated that three Cys residues were involved in the catalysis. AhpC_H1 was resistant to inactivation by peroxide substrates, but had lower activity at physiological H2O2 concentrations compared to AhpC_H2, suggesting that in B. subtilis, the enzymes may be physiologically functional at different substrate concentrations. The exposure to organic peroxides induced AhpC_H1 expression, while AhpC_H1-deficient mutants exhibited growth retardation in the stationary phase, suggesting the role of AhpC_H1 as an antioxidant scavenger of lipid hydroperoxides and a stress-response factor in B. subtilis. CONCLUSION: AhpC_H1, a novel atypical 2-Cys AhpC, is functionally distinct from AhpC_H2, a typical 2-Cys AhpC.     

Mutant AhpC peroxiredoxins suppress thiol-disulfide redox deficiencies and acquire deglutathionylating activity

The bacterial peroxiredoxin AhpC, a cysteine-dependent peroxidase, can be converted through a single amino acid insertion to a disulfide reductase, AhpC*, active in the glutathione and glutaredoxin pathway. Here we show that, whereas AhpC* is inactive as a peroxidase, other point mutants in AhpC can confer the in vivo disulfide reductase activity without abrogating peroxidase activity. Moreover, AhpC* and several point mutants tested in vitro exhibit an enhanced reductase activity toward mixed disulfides between glutathione and glutaredoxin (Grx-S-SG), consistent with the in vivo requirements for these components. Remarkably, this Grx-S-SG reductase activity relies not on the peroxidatic cysteine but rather on the resolving cysteine that plays only a secondary role in the peroxidase mechanism. Furthermore, putative conformational changes, which impart this unusual Grx-S-SG reductase activity, are transmissible across subunits. Thus, AhpC and potentially other peroxiredoxins in this widespread family can elaborate a new reductase function that alleviates disulfide stress.     

Compensatory functions of two alkyl hydroperoxide reductases in the oxidative defense system of Legionella pneumophila

Legionella pneumophila expresses two catalase-peroxidase enzymes that exhibit strong peroxidatic but weak catalatic activities, suggesting that other enzymes participate in decomposition of hydrogen peroxide (H2O2). Comparative genomics revealed that L. pneumophila and its close relative Coxiella burnetii each contain two peroxide-scavenging alkyl hydroperoxide reductase (AhpC) systems: AhpC1, which is similar to the Helicobacter pylori AhpC system, and AhpC2 AhpD (AhpC2D), which is similar to the AhpC AhpD system of Mycobacterium tuberculosis. To establish a catalatic function for these two systems, we expressed L. pneumophila ahpC1 or ahpC2 in a catalase/peroxidase mutant of Escherichia coli and demonstrated restoration of H2O2 resistance by a disk diffusion assay. ahpC1::Km and ahpC2D::Km chromosomal deletion mutants were two- to eightfold more sensitive to H2O2, tert-butyl hydroperoxide, cumene hydroperoxide, and paraquat than the wild-type L. pneumophila, a phenotype that could be restored by trans-complementation. Reciprocal strategies to construct double mutants were unsuccessful. Mutant strains were not enfeebled for growth in vitro or in a U937 cell infection model. Green fluorescence protein reporter assays revealed expression to be dependent on the stage of growth, with ahpC1 appearing after the exponential phase and ahpC2 appearing during early exponential phase. Quantitative real-time PCR showed that ahpC1 mRNA levels were approximately 7- to 10-fold higher than ahpC2D mRNA levels. However, expression of ahpC2D was significantly increased in the ahpC1 mutant, whereas ahpC1 expression was unchanged in the ahpC2D mutant. These results indicate that AhpC1 or AhpC2D (or both) provide an essential hydrogen peroxide-scavenging function to L. pneumophila and that the compensatory activity of the ahpC2D system is most likely induced in response to oxidative stress.     

Cloning of a Corynebacterium diphtheriae iron-repressible gene that shares sequence homology with the AhpC subunit of alkyl hydroperoxide reductase of Salmonella typhimurium.

To understand how Corynebacterium diphtheriae responds to iron limitation, we compared the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles of both wild-type cells and iron uptake mutants grown in either high- or low-iron medium. The removal of iron by ethylene diamine di-(o-hydroxy-phenyl acetic acid) from the growth medium of wild-type cells resulted in induction of at least 14 polypeptides. DirA, a major iron-repressible polypeptide, was purified from wild-type cells by preparative SDS-PAGE, and the dirA structural gene was isolated from a genomic library of nontoxigenic C. diphtheriae. The nucleotide sequence of dirA was determined, and the deduced amino acid sequence of DirA revealed strong homologies with the AhpC subunit of Salmonella typhimurium alkyl hydroperoxide reductase and polypeptides of other microorganisms associated with oxidation reduction activity. Like AhpC, cloned DirA reduced the susceptibility of an Escherichia coli ahp mutant to cumene hydroperoxide, suggesting that DirA has alkyl hydroperoxide reductase activity.     

Attachment of the N-terminal domain of Salmonella typhimurium AhpF to Escherichia coli thioredoxin reductase confers AhpC reductase activity but does not affect thioredoxin reductase activity

AhpF of Salmonella typhimurium, the flavoprotein reductase required for catalytic turnover of AhpC with hydroperoxide substrates in the alkyl hydroperoxide reductase system, is a 57 kDa protein with homology to thioredoxin reductase (TrR) from Escherichia coli. Like TrR, AhpF employs tightly bound FAD and redox-active disulfide center(s) in catalyzing electron transfer from reduced pyridine nucleotides to the disulfide bond of its protein substrate. Homology of AhpF to the smaller (35 kDa) TrR protein occurs in the C-terminal part of AhpF; a stretch of about 200 amino acids at the N-terminus of AhpF contains an additional redox-active disulfide center and is required for catalysis of AhpC reduction. We have demonstrated that fusion of the N-terminal 207 amino acids of AhpF to full-length TrR results in a chimeric protein (Nt-TrR) with essentially the same catalytic efficiency (k(cat)/K(m)) as AhpF in AhpC reductase assays; both k(cat) and the K(m) for AhpC are decreased about 3-4-fold for Nt-TrR compared with AhpF. In addition, Nt-TrR retains essentially full TrR activity. Based on results from two mutants of Nt-TrR (C129, 132S and C342,345S), AhpC reductase activity requires both centers while TrR activity requires only the C-terminal-most disulfide center in Nt-TrR. The high catalytic efficiency with which Nt-TrR can reduce thioredoxin implies that the attached N-terminal domain does not block access of thioredoxin to the TrR-derived Cys342-Cys345 center of Nt-TrR nor does it impede the putative conformational changes that this part of Nt-TrR is proposed to undergo during catalysis. These studies indicate that the C-terminal part of AhpF and bacterial TrR have very similar mechanistic properties. These findings also confirm that the N-terminal domain of AhpF plays a direct role in AhpC reduction.     

Similarity between the amino-terminal portion of mammalian 58-kD sterol carrier protein (SCPx) and Escherichia coli acetyl-CoA acyltransferase: evidence for a gene fusion in SCPx.

A computer analysis of the amino acid sequences of rat and human 58-kD sterol carrier protein and Escherichia coli acetyl-CoA acyltransferase reveals that the two proteins have a segment of about 350 residues with strong sequence similarity. The ALIGN comparison scores for the rat and human sterol carrier proteins and the E. coli enzyme are 8.25 and 8.8 SD, respectively. The catalytically active cysteine of E. coli acetyl-CoA acyltransferase (cysteine 91) aligns with cysteine 93 and cysteine 94 on human and rat 58-kD sterol carrier protein, respectively.     

Identification of a putative isoform of the Na,K-ATPase beta subunit. Primary structure and tissue-specific expression

We have isolated cDNA clones from rat brain and human liver encoding a putative isoform of the Na,K-ATPase beta subunit. The rat brain cDNA contains an open reading frame of 870 nucleotides coding for a protein of 290 amino acids with a calculated molecular weight of 33,412. The corresponding amino acid sequence shows 98% identity with its human liver counterpart. The proteins encoded by the rat and human cDNAs exhibit a high degree of primary sequence and secondary structure similarity with the rat Na,K-ATPase beta subunit. We have therefore termed the polypeptides these cDNAs encode a beta 2 subunit with the previously characterized rat cDNA encoding a beta 1 subunit. Analysis of rat tissue RNA reveals that the beta 2 subunit gene encodes a 3.4-kilobase mRNA which is expressed in a tissue specific fashion distinct from that of rat beta 1 subunit mRNA. Cell lines derived from the rat central nervous system shown to lack beta 1 subunit mRNA sequences were found to express beta 2 subunit mRNA. These results suggest that different members of the Na,K-ATPase beta subunit family may have specialized functions.     

Multiple thioredoxin-mediated routes to detoxify hydroperoxides in Mycobacterium tuberculosis

Drug resistance and virulence of Mycobacterium tuberculosis are in part related to the pathogen's antioxidant defense systems. KatG(-) strains are resistant to the first line tuberculostatic isoniazid but need to compensate their catalase deficiency by alternative peroxidase systems to stay virulent. So far, only NADH-driven and AhpD-mediated hydroperoxide reduction by AhpC has been implicated as such virulence-determining mechanism. We here report on two novel pathways which underscore the importance of the thioredoxin system for antioxidant defense in M. tuberculosis: (i) NADPH-driven hydroperoxide reduction by AhpC that is mediated by thioredoxin reductase and thioredoxin C and (ii) hydroperoxide reduction by the atypical peroxiredoxin TPx that equally depends on thioredoxin reductase but can use both, thioredoxin B and C. Kinetic analyses with different hydroperoxides including peroxynitrite qualify the redox cascade comprising thioredoxin reductase, thioredoxin C, and TPx as the most efficient system to protect M. tuberculosis against oxidative and nitrosative stress in situ.     

Crystal structure of alkyl hydroperoxide-reductase (AhpC) from Helicobacter pylori

The AhpC protein from H. pylori, a thioredoxin (Trx)-dependent alkyl hydroperoxide-reductase, is a member of the ubiquitous 2-Cys peroxiredoxins family (2-Cys Prxs), a group of thiol-specific antioxidant enzymes. Prxs exert the protective antioxidant role in cells through their peroxidase activity, whereby hydrogen peroxide, peroxynitrite and a wide range of organic hydroperoxides (ROOH) are reduced and detoxified (ROOH + 2e(-)-->ROH + H2O). In this study AhpC has been cloned and overexpressed in E. coli. After purification to homogeneity, crystals of the recombinant protein were grown. They diffract to 2.95 A resolution using synchrotron radiation. The crystal structure of AhpC has been determined using the molecular replacement method (R = 23.6%, R(free) = 25.9%). The model, similar in the overall to other members of the 2-Cys Prx family crystallized as toroide-shaped complexes, consists of a pentameric arrangement of homodimers [(alpha2)5 decamer]. The model of AhpC from H. pylori presents significant differences with respect to other members of the family: apart from some loop regions, alpha5-helix and the C-terminus is shifted, preventing the C-terminal tail of the second subunit from extending toward this region of the molecule. Oligomerization properties of AhpC have been also characterized by gel filtration chromatography.     

Production of selenomethionine-labelled proteins using simplified culture conditions and generally applicable host/vector systems

The amino acid analogue selenomethionine (SeMet) is shown to be efficiently incorporated into recombinant proteins expressed in Escherichia coli grown in a simple minimal medium without the addition of synthetic amino acids. Furthermore, satisfactory SeMet incorporation is obtained with a methionine-prototrophic strain transformed with commonly used vector systems. As examples, purified tryparedoxin 1 from Crithidia fasciculata, alkylhydroperoxide reductase (AhpC) from Mycobacterium marinum and the 16-kDa antigen from M. tuberculosis are shown to be efficiently labelled with SeMet, using the culture conditions and the host/vector systems described here. Enzymatic analysis reveals no differences between native and SeMet-labelled tryparedoxin 1 enzyme. Both proteins yield crystals under similar conditions. The culture conditions and host vector systems described greatly facilitate selenium-labelling of proteins for 3-D structure determination.     

Compensatory increase in<Emphasis Type="Italic"> ahpC</Emphasis> gene expression and its role in protecting<Emphasis Type="Italic"> Burkholderia pseudomallei</Emphasis> against reactive nitrogen intermediates

In the human pathogen Burkholderia pseudomallei, katG encodes the antioxidant defense enzyme catalase-peroxidase. Interestingly, a B. pseudomallei mutant, disrupted in katG, is hyperresistant to organic hydroperoxide. This hyperresistance is due to the compensatory expression of the alkyl hydroperoxide reductase gene (ahpC) and depends on a global regulator OxyR. The KatG-deficient mutant is also highly resistant to reactive nitrogen intermediates (RNI). When overproduced, the B. pseudomallei AhpC protein, protected cells against killing by RNI. The levels of resistance to both organic peroxide and RNI returned to those of the wild-type when the katG mutant was complemented with katG. These studies establish the partially overlapping defensive activities of KatG and AhpC.     

Molecular cloning of rat liver 3α-hydroxysteroid dehydrogenase and identification of structurally related proteins from rat lung and kidney

3α-Hydroxysteroid dehydrogenase and related enzymes play important roles in the metabolism of endogenous compounds including androgens, corticosteroid, prostaglandins and bile acids, as well as drugs and xenobiotics such as benzo(a)pyrene. Complementary DNA clones encoding 3α-hydroxysteroid dehydrogenase were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. A full-length cDNA clone of 1286 base pairs contained an open reading frame encoding a protein of 322 amino acids with an estimated M(w) of 37 kD. When expressed in E. coli, the encoded protein migrated to the same position on SDS-polycrylamide gels as the enzyme in rat liver cytosols. The protein expressed in bacteria was highly active in androsterone oxidation in the presence of NAD as cofactor and this activity was inhibited by indomethacin, a potent inhibitor of 3α-hydroxysteroid dehydrogenase. The predicted amino acid sequence of 3α-hydroxysteroid d dehydrogenase was related to sequences of several other aldo-keto reductases such as bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase and frog lens epsilon-crystallin, suggesting that these proteins belong to the same gene family. Recently, we have found that monoclonal antibodies against 3α-hydroxysteroid dehydrogenase also recognized multiple antigenically related proteins in rat lung, kidney and testis. Further screening of liver, lung and kidney cDNA libraries using these monoclonal antibodies as probes resulted in the isolation of additional five different cDNAs encoding proteins with high degree of structural homology to rat liver 3α-hydroxysteroid dehydrogenase.     

Cloning and expression of human aldose reductase

The complete amino acid sequence of human retina and muscle aldose reductase was determined by nucleotide analysis of cDNA clones isolated using synthetic oligonucleotide probes based on partial amino acid sequences of purified human psoas muscle aldose reductase. The cDNA sequence differs substantially in the noncoding and coding regions of recently published sequences of this enzyme. The mRNA for aldose reductase was abundantly expressed in HeLa cells, but only scarcely in a neuroblastoma cell line. Recombinant baculovirus containing one of the muscle cDNA clones was constructed and used to infect Spodoptera frugiperda (SF9) cells. A prominent protein with an apparent molecular size of 36 kDa was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the culture medium as well as in the homogenate of SF9 cells after 2 days of infection. Culture medium or the supernatant fraction of cell homogenates containing this protein had high aldose reductase activity which showed characteristics of the reported human enzyme. These findings indicate that the amino acid sequence reported in this paper represents human retina and muscle aldose reductase and that functional human aldose reductase can be expressed in large amounts in a baculovirus expression system. The result should facilitate refined structural analysis and the development of new specific aldose reductase inhibitors for the treatment of diabetic complications.     

Identification of a new P450 expressed in human lung: complete cDNA sequence, cDNA-directed expression, and chromosome mapping

A cDNA coding for a P450 expressed in human lung was isolated from a lambda gt11 library constructed from human lung mRNA using a cDNA probe to rat P450 IVA1. The cDNA-deduced amino acid sequence of this P450, designated IVB1, consisted of 511 amino acids and had a calculated molecular weight of 59,558. The IVB1 amino acid sequence bore 51%, 53%, and 52% similarities to rat IVA1, IVA2, and rabbit P450p-2, respectively. Comparison of the primary amino acid sequence of human IVB1 with rat IVA and rabbit p-2 P450 sequences revealed a region of absolute sequence identity of 17 amino acids between residues 304 and 320. However, the functional significance of this conserved sequence is unknown. Human IVB1 also appears to be related to P450 isozyme 5 that has been extensively characterized in rabbits. The IVB1 cDNA was inserted into a vaccinia virus expression vector and the enzyme expressed in human cell lines. The expressed enzyme had an absorption spectrum with a lambda max at 450 nm when reduced and complexed with carbon monoxide, typical of other cytochrome P450s. Unlike rabbit P450 isozyme 5, however, human IVB1 was unable to activate the promutagen 2-aminofluorene. Human lung microsomal P450s were also unable to metabolize this compound despite the presence of IVB1 mRNA in three out of four human lungs analyzed. In contrast to its expression in lung, IVB1 mRNA was undetectable in livers from 14 individuals, including those from which the lungs were derived. IVB1-related mRNA was also expressed in rat lung and was undetectable in untreated rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and expression of rat liver 3 alpha-hydroxysteroid dehydrogenase

Complementary DNA clones encoding 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. The sizes of the cDNA inserts ranged from 1.3-2.3 kilobases. Sequence analysis indicated that variation in the DNA size was due to heterogeneity in the length of 3' noncoding sequences. A full-length cDNA clone of 1286 basepairs contained an open reading frame encoding a protein of 322 amino acids with an estimated mol wt of 37 kDa. When expressed in E. coli, the encoded protein migrated to the same position on sodium dodecyl sulfate-polyacrylamide gels as the enzyme purified from rat liver cytosols. The protein expressed in bacteria was highly active in androsterone reduction in the presence of NAD as cofactor, and this activity was inhibited by indomethacin, a potent inhibitor of 3 alpha HSD. The predicted amino acid sequence of 3 alpha HSD was related to sequences of several other enzymes, including bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase, and frog lens epsilon-crystalline, suggesting that these proteins belong to the same gene family.     

Streptococcus mutans H2O2-forming NADH oxidase is an alkyl hydroperoxide reductase protein

Nox-1 from Streptococcus mutans, the bacteria which cause dental caries, was previously identified as an H2O2-forming reduced nicotinamide adenine dinucleotide (NADH) oxidase. Nox-1 is homologous with the flavoprotein component, AhpF, of Salmonella typhimurium alkyl hydroperoxide reductase. A partial open reading frame upstream of nox1, homologous with the other (peroxidase) component, ahpC, from the S. typhimurium system, was also identified. We report here the complete sequence of S. mutans ahpC. Analyses of purified AhpC together with Nox-1 have verified that these proteins act as a cysteine-based peroxidase system in S. mutans, catalyzing the NADH-dependent reduction of organic hydroperoxides or H2O2 to their respective alcohols and/or H2O. These proteins also catalyze the four-electron reduction of O2 to H2O2, clarifying the role of Nox-1 as a protective protein against oxygen toxicity. Major differences between Nox-1 and AhpF include: (i) the absolute specificity of Nox-1 for NADH; (ii) lower amounts of flavin semiquinone and a more prominent FADH2 to NAD+ charge transfer absorbance band stabilized by Nox-1; and (iii) even higher redox potentials of disulfide centers relative to flavin for Nox-1. Although Nox-1 and AhpC from S. mutans were shown to play a protective role against oxidative stress in vitro and in vivo in Escherichia coli, the lack of a significant effect on deletion of these genes from S. mutans suggests the presence of additional antioxidant proteins in these bacteria.     

Molecular cloning, cDNA structure and tissue-specific expression of the human regulatory subunit RI beta of cAMP-dependent protein kinases.

Complementary DNA clones for the regulatory subunit RI beta of cAMP-dependent protein kinases were isolated from a human testis cDNA library using a mouse RI beta cDNA probe. One clone 2.4 kilobases (kb) in length contained an open reading frame of 1137 bases, and encoded a protein of 379 amino acids (excluding the initiator methionine). The human RI beta protein was one amino acid shorter than the corresponding protein in mouse and rat. The nucleotide similarity to mouse and rat sequences was 85.6% and 84.8%, respectively, while the amino acid similarity was 97.6% and 97.3%, respectively. Northern blot analyses revealed a 2.7 kb mRNA in human tissues and a 2.8 kb mRNA in mouse tissues. Both mouse and human RI beta mRNA were found to be expressed in most tissues, and not restricted to brain and testis as reported by others.