A cryptic oxidoreductase safeguards oxidative protein folding in Corynebacterium diphtheriae.

In many gram-positive Actinobacteria, including Actinomyces oris and Corynebacterium matruchotii, the conserved thiol-disulfide oxidoreductase MdbA that catalyzes oxidative folding of exported proteins is essential for bacterial viability by an unidentified mechanism. Intriguingly, in Corynebacterium diphtheriae, the deletion of mdbA blocks cell growth only at 37 °C but not at 30 °C, suggesting the presence of alternative oxidoreductase enzyme(s). By isolating spontaneous thermotolerant revertants of the mdbA mutant at 37 °C, we obtained genetic suppressors, all mapped to a single T-to-G mutation within the promoter region of tsdA, causing its elevated expression. Strikingly, increased expression of tsdA-via suppressor mutations or a constitutive promoter-rescues the pilus assembly and toxin production defects of this mutant, hence compensating for the loss of mdbA. Structural, genetic, and biochemical analyses demonstrated TsdA is a membrane-tethered thiol-disulfide oxidoreductase with a conserved CxxC motif that can substitute for MdbA in mediating oxidative folding of pilin and toxin substrates. Together with our observation that tsdA expression is upregulated at nonpermissive temperature (40 °C) in wild-type cells, we posit that TsdA has evolved as a compensatory thiol-disulfide oxidoreductase that safeguards oxidative protein folding in C. diphtheriae against thermal stress.

Structural basis for heme detoxification by an ATP-binding cassette-type efflux pump in gram-positive pathogenic bacteria.

Bacterial pathogens acquire heme from the host hemoglobin as an iron nutrient for their virulence and proliferation in blood. Concurrently, they encounter cytotoxic-free heme that escapes the heme-acquisition process. To overcome this toxicity, many gram-positive bacteria employ an ATP-binding cassette heme-dedicated efflux pump, HrtBA in the cytoplasmic membranes. Although genetic analyses have suggested that HrtBA expels heme from the bacterial membranes, the molecular mechanism of heme efflux remains elusive due to the lack of protein studies. Here, we show the biochemical properties and crystal structures of Corynebacterium diphtheriae HrtBA, alone and in complex with heme or an ATP analog, and we reveal how HrtBA extracts heme from the membrane and releases it. HrtBA consists of two cytoplasmic HrtA ATPase subunits and two transmembrane HrtB permease subunits. A heme-binding site is formed in the HrtB dimer and is laterally accessible to heme in the outer leaflet of the membrane. The heme-binding site captures heme from the membrane using a glutamate residue of either subunit as an axial ligand and sequesters the heme within the rearranged transmembrane helix bundle. By ATP-driven HrtA dimerization, the heme-binding site is squeezed to extrude the bound heme. The mechanism sheds light on the detoxification of membrane-bound heme in this bacterium.

An active site at work - the role of key residues in C. diphteriae coproheme decarboxylase.

Coproheme decarboxylases (ChdCs) are utilized by monoderm bacteria to produce heme b by a stepwise oxidative decarboxylation of the 2- and 4-propionate groups of iron coproporphyrin III (coproheme) to vinyl groups. This work compares the effect of hemin reconstitution versus the hydrogen peroxide-mediated conversion of coproheme to heme b in the actinobacterial ChdC from Corynebacterium diphtheriae (CdChdC) and selected variants. Both ferric and ferrous forms of wild-type (WT) CdChdC and its H118A, H118F, and A207E variants were characterized by resonance Raman and UV-vis spectroscopies. The heme b ligand assumes the same conformation in the WT active site for both the reconstituted and H2O2-mediated product, maintaining the same vinyl and propionate interactions with the protein. Nevertheless, it is important to note that the distal His118, which serves as a distal base, plays an important role in the stabilization of the cavity and for the heme b reconstitution. In fact, while the access of heme b is prevented by steric hindrance in the H118F variant, the substitution of His with the small apolar Ala residue favors the insertion of the heme b in the reversed conformation. The overall data strongly support that during decarboxylation, the intermediate product, a monovinyl-monopropionyl deuteroheme, rotates by 90o within the active site. Moreover, in the ferrous forms the frequency of the ν(Fe-Nδ(His)) stretching mode provides information on the strength of the proximal Fe-His bond and allows us to follow its variation during the two oxidative decarboxylation steps.

Characterisation of trehalose-6-phosphate phosphatases from bacterial pathogens.

The trehalose biosynthesis pathway has recently received attention for therapeutic intervention combating infectious diseases caused by bacteria, helminths or fungi. Trehalose-6-phosphate phosphatase (TPP) is a key enzyme of the most common trehalose biosynthesis pathway and a particularly attractive target owing to the toxicity of accumulated trehalose-6-phosphate in pathogens. Here, we characterised TPP-like proteins from bacterial pathogens implicated in nosocomial infections in terms of their steady-state kinetics as well as pH- and metal-dependency of their enzymatic activity. Analysis of the steady-state kinetics of recombinantly expressed enzymes from Acinetobacter baumannii, Corynebacterium diphtheriae and Pseudomonas stutzeri yielded similar kinetic parameters as those of other reported bacterial TPPs. In contrast to nematode TPPs, the divalent metal ion appears to be bound only weakly in the active site of bacterial TPPs, allowing the exchange of the resident magnesium ion with other metal ions. Enzymatic activity comparable to the wild-type enzyme was observed for the TPP from P. stutzeri with manganese, cobalt and nickel. Analysis of the enzymatic activity of S. maltophilia TPP active site mutants provides evidence for the involvement of four canonical aspartate residues as well as a strictly conserved histidine residue of TPP-like proteins from bacteria in the enzyme mechanism. That histidine residue is a member of an interconnected network of five conserved residues in the active site of bacterial TPPs which likely constitute one or more functional units, directly or indirectly cooperating to enhance different aspects of the catalytic activity.

Kinetics and Optimization of the Lysine-Isopeptide Bond Forming Sortase Enzyme from Corynebacterium diphtheriae.

Site-specifically modified protein bioconjugates have important applications in biology, chemistry, and medicine. Functionalizing specific protein side chains with enzymes using mild reaction conditions is of significant interest, but remains challenging. Recently, the lysine-isopeptide bond forming activity of the sortase enzyme that builds surface pili in Corynebacterium diphtheriae (CdSrtA) has been reconstituted in vitro. A mutationally activated form of CdSrtA was shown to be a promising bioconjugating enzyme that can attach Leu-Pro-Leu-Thr-Gly peptide fluorophores to a specific lysine residue within the N-terminal domain of the SpaA protein (NSpaA), enabling the labeling of target proteins that are fused to NSpaA. Here we present a detailed analysis of the CdSrtA catalyzed protein labeling reaction. We show that the first step in catalysis is rate limiting, which is the formation of the CdSrtA-peptide thioacyl intermediate that subsequently reacts with a lysine ε-amine in NSpaA. This intermediate is surprisingly stable, limiting spurious proteolysis of the peptide substrate. We report the discovery of a new enzyme variant (CdSrtAΔ) that has significantly improved transpeptidation activity, because it completely lacks an inhibitory polypeptide appendage ("lid") that normally masks the active site. We show that the presence of the lid primarily impairs formation of the thioacyl intermediate and not the recognition of the NSpaA substrate. Quantitative measurements reveal that CdSrtAΔ generates its cross-linked product with a catalytic turnover number of 1.4 ± 0.004 h-1 and that it has apparent KM values of 0.16 ± 0.04 and 1.6 ± 0.3 mM for its NSpaA and peptide substrates, respectively. CdSrtAΔ is 7-fold more active than previously studied variants, labeling >90% of NSpaA with peptide within 6 h. The results of this study further improve the utility of CdSrtA as a protein labeling tool and provide insight into the enzyme catalyzed reaction that underpins protein labeling and pilus biogenesis.

Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade.

Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122-Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122-Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation.

Structural basis for the promiscuous PAM recognition by Corynebacterium diphtheriae Cas9.

The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets bearing a protospacer adjacent motif (PAM) and complementarity to an RNA guide. Unlike other Cas9 orthologs, Corynebacterium diphtheriae Cas9 (CdCas9) recognizes the promiscuous NNRHHHY PAM. However, the CdCas9-mediated PAM recognition mechanism remains unknown. Here, we report the crystal structure of CdCas9 in complex with the guide RNA and its target DNA at 2.9 Å resolution. The structure reveals that CdCas9 recognizes the NNRHHHY PAM via a combination of van der Waals interactions and base-specific hydrogen bonds. Moreover, we find that CdCas9 exhibits robust DNA cleavage activity with the optimal 22-nucleotide length guide RNAs. Our findings highlight the mechanistic diversity of the PAM recognition by Cas9 orthologs, and provide a basis for the further engineering of the CRISPR-Cas9 genome-editor nucleases.

Protein Labeling via a Specific Lysine-Isopeptide Bond Using the Pilin Polymerizing Sortase from Corynebacterium diphtheriae.

Proteins that are site-specifically modified with peptides and chemicals can be used as novel therapeutics, imaging tools, diagnostic reagents and materials. However, there are few enzyme-catalyzed methods currently available to selectively conjugate peptides to internal sites within proteins. Here we show that a pilus-specific sortase enzyme from Corynebacterium diphtheriae (CdSrtA) can be used to attach a peptide to a protein via a specific lysine-isopeptide bond. Using rational mutagenesis we created CdSrtA3M, a highly activated cysteine transpeptidase that catalyzes in vitro isopeptide bond formation. CdSrtA3M mediates bioconjugation to a specific lysine residue within a fused domain derived from the corynebacterial SpaA protein. Peptide modification yields greater than >95% can be achieved. We demonstrate that CdSrtA3M can be used in concert with the Staphylococcus aureus SrtA enzyme, enabling dual, orthogonal protein labeling via lysine-isopeptide and backbone-peptide bonds.

Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii.

The actinobacterium Corynebacterium matruchotii has been implicated in nucleation of oral microbial consortia leading to biofilm formation. Due to the lack of genetic tools, little is known about basic cellular processes, including protein secretion and folding, in this organism. We report here a survey of the C. matruchotii genome, which encodes a large number of exported proteins containing paired cysteine residues, and identified an oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA (MdbACd). Crystallization studies uncovered that the 1.2-Å resolution structure of C. matruchotii MdbA (MdbACm) possesses two conserved features found in actinobacterial MdbA enzymes, a thioredoxin-like fold and an extended α-helical domain. By reconstituting the disulfide bond-forming machine in vitro, we demonstrated that MdbACm catalyzes disulfide bond formation within the actinobacterial pilin FimA. A new gene deletion method supported that mdbA is essential in C. matruchotii Remarkably, heterologous expression of MdbACm in the C. diphtheriae ΔmdbA mutant rescued its known defects in cell growth and morphology, toxin production, and pilus assembly, and this thiol-disulfide oxidoreductase activity required the catalytic motif CXXC. Altogether, the results suggest that MdbACm is a major thiol-disulfide oxidoreductase, which likely mediates posttranslocational protein folding in C. matruchotii by a mechanism that is conserved in ActinobacteriaIMPORTANCE The actinobacterium Corynebacterium matruchotii has been implicated in the development of oral biofilms or dental plaque; however, little is known about the basic cellular processes in this organism. We report here a high-resolution structure of a C. matruchotii oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA. By biochemical analysis, we demonstrated that C. matruchotii MdbA catalyzes disulfide bond formation in vitro Furthermore, a new gene deletion method revealed that deletion of mdbA is lethal in C. matruchotii Remarkably, C. matruchotii MdbA can replace C. diphtheriae MdbA to maintain normal cell growth and morphology, toxin production, and pilus assembly. Overall, our studies support the hypothesis that C. matruchotii utilizes MdbA as a major oxidoreductase to catalyze oxidative protein folding.

The glyceraldehyde-3-phosphate dehydrogenase GapDH of Corynebacterium diphtheriae is redox-controlled by protein S-mycothiolation under oxidative stress.

Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes and functions in post-translational thiol-modification by protein S-mycothiolation as emerging thiol-protection and redox-regulatory mechanism. Here, we have used shotgun-proteomics to identify 26 S-mycothiolated proteins in the pathogen Corynebacterium diphtheriae DSM43989 under hypochlorite stress that are involved in energy metabolism, amino acid and nucleotide biosynthesis, antioxidant functions and translation. The glyceraldehyde-3-phosphate dehydrogenase (GapDH) represents the most abundant S-mycothiolated protein that was modified at its active site Cys153 in vivo. Exposure of purified GapDH to H2O2 and NaOCl resulted in irreversible inactivation due to overoxidation of the active site in vitro. Treatment of GapDH with H2O2 or NaOCl in the presence of MSH resulted in S-mycothiolation and reversible GapDH inactivation in vitro which was faster compared to the overoxidation pathway. Reactivation of S-mycothiolated GapDH could be catalyzed by both, the Trx and the Mrx1 pathways in vitro, but demycothiolation by Mrx1 was faster compared to Trx. In summary, we show here that S-mycothiolation can function in redox-regulation and protection of the GapDH active site against overoxidation in C. diphtheriae which can be reversed by both, the Mrx1 and Trx pathways.

Conformational Control of UDP-Galactopyranose Mutase Inhibition.

UDP-galactopyranose mutase (Glf or UGM) catalyzes the formation of uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme is required for the production of Galf-containing glycans. UGM is absent in mammals, but members of the Corynebacterineae suborder require UGM for cell envelope biosynthesis. The need for UGM in some pathogens has prompted the search for inhibitors that could serve as antibiotic leads. Optimizing inhibitor potency, however, has been challenging. The UGM from Klebsiella pneumoniae (KpUGM), which is not required for viability, is more effectively impeded by small-molecule inhibitors than are essential UGMs from species such as Mycobacterium tuberculosis or Corynebacterium diphtheriae. Why KpUGM is more susceptible to inhibition than other orthologs is not clear. One potential source of difference is UGM ortholog conformation. We previously determined a structure of CdUGM bound to a triazolothiadiazine inhibitor in the open form, but it was unclear whether the small-molecule inhibitor bound this form or to the closed form. By varying the terminal tag (CdUGM-His6 and GSG-CdUGM), we crystallized CdUGM to capture the enzyme in different conformations. These structures reveal a pocket in the active site that can be exploited to augment inhibitor affinity. Moreover, they suggest the inhibitor binds the open form of most prokaryotic UGMs but can bind the closed form of KpUGM. This model and the structures suggest strategies for optimizing inhibitor potency by exploiting UGM conformational flexibility.

Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae.

The suborder Corynebacterineae encompasses species like Corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as Corynebacterium diphtheriae and Mycobacterium tuberculosis, which cause devastating human diseases. A distinctive component of the Corynebacterineae cell envelope is the mycolyl-arabinogalactan (mAG) complex. The mAG is composed of lipid mycolic acids, and arabinofuranose (Araf) and galactofuranose (Galf) carbohydrate residues. Elucidating microbe-specific differences in mAG composition could advance biotechnological applications and lead to new antimicrobial targets. To this end, we compare and contrast galactan biosynthesis in C. diphtheriae and M. tuberculosis In each species, the galactan is constructed from uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf), which is generated by the enzyme UDP-galactopyranose mutase (UGM or Glf). UGM and the galactan are essential in M. tuberculosis, but their importance in Corynebacterium species was not known. We show that small molecule inhibitors of UGM impede C. glutamicum growth, suggesting that the galactan is critical in corynebacteria. Previous cell wall analysis data suggest the galactan polymer is longer in mycobacterial species than corynebacterial species. To explore the source of galactan length variation, a C. diphtheriae ortholog of the M. tuberculosis carbohydrate polymerase responsible for the bulk of galactan polymerization, GlfT2, was produced, and its catalytic activity was evaluated. The C. diphtheriae GlfT2 gave rise to shorter polysaccharides than those obtained with the M. tuberculosis GlfT2. These data suggest that GlfT2 alone can influence galactan length. Our results provide tools, both small molecule and genetic, for probing and perturbing the assembly of the Corynebacterineae cell envelope.

A thiol-disulfide oxidoreductase of the Gram-positive pathogen Corynebacterium diphtheriae is essential for viability, pilus assembly, toxin production and virulence.

The Gram-positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane-bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein-folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol-disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin-like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature-sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae.

Corynebacterium diphtheriae methionine sulfoxide reductase a exploits a unique mycothiol redox relay mechanism.

Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen.

Role of active-site residues Tyr55 and Tyr114 in catalysis and substrate specificity of Corynebacterium diphtheriae C-S lyase.

In recent years, there has been increased interest in bacterial methionine biosynthesis enzymes as antimicrobial targets because of their pivotal role in cell metabolism. C-S lyase from Corynebacterium diphtheriae is a pyridoxal 5'-phosphate-dependent enzyme in the transsulfuration pathway that catalyzes the α,ß-elimination of sulfur-containing amino acids, such as L-cystathionine, to generate ammonia, pyruvate, and homocysteine, the immediate precursor of L-methionine. In order to gain deeper insight into the functional and dynamic properties of the enzyme, mutants of two highly conserved active-site residues, Y55F and Y114F, were characterized by UV-visible absorbance, fluorescence, and CD spectroscopy in the absence and presence of substrates and substrate analogs, as well as by steady-state kinetic studies. Substitution of Tyr55 with Phe apparently causes a 130-fold decrease in K(d)(PLP) at pH 8.5 providing evidence that Tyr55 plays a role in cofactor binding. Moreover, spectral data show that the mutant accumulates the external aldimine intermediate suggesting that the absence of interaction between the hydroxyl moiety and PLP-binding residue Lys222 causes a decrease in the rate of substrate deprotonation. Mutation of Tyr114 with Phe slightly influences hydrolysis of L-cystathionine, and causes a change in substrate specificity towards L-serine and O-acetyl-L-serine compared to the wild type enzyme. These findings, together with computational data, provide useful insights in the substrate specificity of C-S lyase, which seems to be regulated by active-site architecture and by the specific conformation in which substrates are bound, and will aid in development of inhibitors.

Engineered coryneform bacteria as a bio-tool for arsenic remediation.

Despite current remediation efforts, arsenic contamination in water sources is still a major health problem, highlighting the need for new approaches. In this work, strains of the nonpathogenic and highly arsenic-resistant bacterium Corynebacterium glutamicum were used as inexpensive tools to accumulate inorganic arsenic, either as arsenate (As(V)) or arsenite (As(III)) species. The assays made use of "resting cells" from these strains, which were assessed under well-established conditions and compared with C. glutamicum background controls. The two mutant As(V)-accumulating strains were those used in a previously published study: (i) ArsC1/C2, in which the gene/s encoding the mycothiol-dependent arsenate reductases is/are disrupted, and (ii) MshA/C mutants unable to produce mycothiol, the low molecular weight thiol essential for arsenate reduction. The As(III)-accumulating strains were either those lacking the arsenite permease activities (Acr3-1 and Acr3-2) needed in As(III) release or recombinant strains overexpressing the aquaglyceroporin genes (glpF) from Corynebacterium diphtheriae or Streptomyces coelicolor, to improve As(III) uptake. Both genetically modified strains accumulated 30-fold more As(V) and 15-fold more As(III) than the controls. The arsenic resistance of the modified strains was inversely proportional to their metal accumulation ability. Our results provide the basis for investigations into the use of these modified C. glutamicum strains as a new bio-tool in arsenic remediation efforts.

Tyrosine oxidation in heme oxygenase: examination of long-range proton-coupled electron transfer.

Heme oxygenase is responsible for the degradation of a histidine-ligated ferric protoporphyrin IX (Por) to biliverdin, CO, and the free ferrous ion. Described here are studies of tyrosyl radical formation reactions that occur after oxidizing Fe(III)(Por) to Fe(IV)=O(Por(·+)) in human heme oxygenase isoform-1 (hHO-1) and the structurally homologous protein from Corynebacterium diphtheriae (cdHO). Site-directed mutagenesis on hHO-1 probes the reduction of Fe(IV)=O(Por(·+)) by tyrosine residues within 11 Å of the prosthetic group. In hHO-1, Y58· is implicated as the most likely site of oxidation, based on the pH and pD dependent kinetics. The absence of solvent deuterium isotope effects in basic solutions of hHO-1 and cdHO contrasts with the behavior of these proteins in the acidic solution, suggesting that long-range proton-coupled electron transfer predominates over electron transfer.

Heme utilization by pathogenic bacteria: not all pathways lead to biliverdin.

The eukaryotic heme oxygenases (HOs) (E.C. 1.14.99.3) convert heme to biliverdin, iron, and carbon monoxide (CO) in three successive oxygenation steps. Pathogenic bacteria require iron for survival and infection. Extracellular heme uptake from the host plays a critical role in iron acquisition and virulence. In the past decade, several HOs required for the release of iron from extracellular heme have been identified in pathogenic bacteria, including Corynebacterium diphtheriae, Neisseriae meningitides, and Pseudomonas aeruginosa. The bacterial enzymes were shown to be structurally and mechanistically similar to those of the canonical eukaryotic HO enzymes. However, the recent discovery of the structurally and mechanistically distinct noncanonical heme oxygenases of Staphylococcus aureus and Mycobacterium tuberculosis has expanded the reaction manifold of heme degradation. The distinct ferredoxin-like structural fold and extreme heme ruffling are proposed to give rise to the alternate heme degradation products in the S. aureus and M. tuberculosis enzymes. In addition, several "heme-degrading factors" with no structural homology to either class of HOs have recently been reported. The identification of these "heme-degrading proteins" has largely been determined on the basis of in vitro heme degradation assays. Many of these proteins were reported to produce biliverdin, although no extensive characterization of the products was performed. Prior to the characterization of the canonical HO enzymes, the nonenzymatic degradation of heme and heme proteins in the presence of a reductant such as ascorbate or hydrazine, a reaction termed "coupled oxidation", served as a model for biological heme degradation. However, it was recognized that there were important mechanistic differences between the so-called coupled oxidation of heme proteins and enzymatic heme oxygenation. In the coupled oxidation reaction, the final product, verdoheme, can readily be converted to biliverdin under hydrolytic conditions. The differences between heme oxygenation by the canonical and noncanonical HOs and coupled oxidation will be discussed in the context of the stabilization of the reactive Fe(III)-OOH intermediate and regioselective heme hydroxylation. Thus, in the determination of heme oxygenase activity in vitro, it is important to ensure that the reaction proceeds through successive oxygenation steps. We further suggest that when bacterial heme degradation is being characterized, a systems biology approach combining genetics, mechanistic enzymology, and metabolite profiling should be undertaken.

New diphtheria toxin repressor types depicted in a Romanian collection of Corynebacterium diphtheriae isolates.

Corynebacterium diphtheriae is the etiological agent of diphtheria, a potential fatal disease caused by a corynephage toxin. The expression of this diphtheria toxin is controlled via an iron-dependent repressor with various functions (DtxR). Some mutations in the dtxR gene are associated with diminished activity or even with total loss of DtxR function. We conducted a molecular study to characterize the dtxR alleles harbored by 34 isolates of C. diphtheriae recovered from Romanian patients between 1961 and 2007. Three of the seven alleles identified in this study have not previously been described. Two new DtxR types were identified, one of which has an unusual polypeptide length. All the new DtxR types were found in toxigenic isolates, suggesting that they effectively regulate the expression of diphtheria toxin. Furthermore, one of the new DtxR identified was also found in a non-toxigenic isolate, making it a potential source of toxigenic isolates after lysogenic conversion.