Edward J. Crane

Borrelia burgdorferi bb0728 encodes a coenzyme A disulphide reductase whose function suggests a role in intracellular redox and the oxidative stress response

The cellular responses of Borrelia burgdorferiTo reactive oxygen species (ROS) encountered during the different stages of its infective cycle are poorly understood. Few enzymes responsible for protecting proteins, DNA/RNA and lipids from damage by ROS have been identified and characterized. Data presented here suggest that bb0728 encodes an enzyme involved in this process. Biochemical analyses on purified recombinant BB0728 indicated that it functioned as a coenzyme A disulphide reductase (CoADR) (specific activity ≈ 26 units per mg of protein). This enzyme was specific for coenzyme A (CoA) disulphide, required NADH and had no significant activity against other disulphides, such as oxidized glutathione or thioredoxin. The high intracellular concentration of reduced CoA (CoASH) in B. burgdorferi cells (∼1 mM) and absence of glutathione suggest that CoA is the major low‐molecular‐weight thiol in this spirochete. Interestingly, CoASH was able to reduce H2O2 and be regenerated by CoADR suggesting one role for the system may be to protect B. burgdorferi from ROS. Further, mobility‐shift assays and transcriptional fusion data indicated that bb0728 was positively regulated by the Borrelia oxidative stress response regulator, BosR. Taken together, these data suggest a role for BB0728 in intracellular redox and the oxidative stress response in B. burgdorferi.

Electron Transfer Reactivity of Type Zero Pseudomonas aeruginosa Azurin

Type zero copper is a hard-ligand analogue of the classical type 1 or blue site in copper proteins that function as electron transfer (ET) agents in photosynthesis and other biological processes. The EPR spectroscopic features of type zero Cu(II) are very similar to those of blue copper, although lacking the deep blue color, due to the absence of thiolate ligation. We have measured the rates of intramolecular ET from the pulse radiolytically generated C3-C26 disulfide radical anion to the Cu(II) in both type zero C112D/M121L and type 2 C112D Pseudomonas aeruginosa azurins in pH 7.0 aqueous solutions between 8 and 45 °C. We also have obtained rate/temperature (10-30 °C) profiles for ET reactions between these mutants and the wild-type azurin. Analysis of the rates and activation parameters for both intramolecular and intermolecular ET reactions indicates that the type zero copper reorganization energy falls in a range (0.9-1.1 eV) slightly above that for type 1 (0.7-0.8 eV), but substantially smaller than that for type 2 (>2 eV), consistent with XAS and EXAFS data that reveal minimal type zero site reorientation during redox cycling.

Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii

We have cloned NADH oxidase homologues from Pyrococcus horikoshii and P. furiosus, and purified the recombinant form of the P. horikoshii enzyme to homogeneity from Escherichia coli. Both enzymes (previously referred to as NOX2) have been shown to act as a coenzyme A disulfide reductases (CoADR: CoA‐S‐S‐CoA + NAD(P)H + H+→2CoA‐SH + NAD(P)+). The P. horikoshii enzyme shows a kcat app of 7.2 s−1 with NADPH at 75 °C. While the enzyme shows a preference for NADPH, it is able to use both NADPH and NADH efficiently, with both giving roughly equal kcats, while the Km for NADPH is roughly eightfold lower than that for NADH. The enzyme is specific for the CoA disulfide, and does not show significant reductase activity with other disulfides, including dephospho‐CoA. Anaerobic reductive titration of the enzyme with NAD(P)H proceeds in two stages, with an apparent initial reduction of a nonflavin redox center with the first reduction resulting in what appears to be an EH2 form of the enzyme. Addition of a second of NADPH results in the formation of an apparent FAD‐NAD(P)H complex. The behavior of this enzyme is quite different from the mesophilic staphylococcal version of the enzyme. This is only the second enzyme with this activity discovered, and the first from a strict anaerobe, an Archaea, or hyperthermophilic source. P. furiosus cells were assayed for small molecular mass thiols and found to contain 0.64 µmol CoA·g dry weight−1 (corresponding to 210 µm CoA in the cell) consistent with CoA acting as a pool of disulfide reducing equivalents.

Structure and substrate specificity of the pyrococcal coenzyme A disulfide reductases/polysulfide reductases (CoADR/Psr): implications for S(0)-based respiration and a sulfur-dependent antioxidant system in Pyrococcus.

FAD and NAD(P)H-dependent coenzyme A disulfide reductases/polysulfide reductases (CoADR/Psr) have been proposed to be important for the reduction of sulfur and disulfides in the sulfur-reducing anaerobic hyperthermophiles Pyrococcus horikoshii and Pyrococcus furiosus; however, the form(s) of sulfur that the enzyme actually reduces are not clear. Here we determined the structure for the FAD- and coenzyme A-containing holoenzyme from P. horikoshii to 2.7 Å resolution and characterized its substrate specificity. The enzyme is relatively promiscuous and reduces a range of disulfide, persulfide, and polysulfide compounds. These results indicate that the likely in vivo substrates are NAD(P)H and di-, poly-, and persulfide derivatives of coenzyme A, although polysulfide itself is also efficiently reduced. The role of the enzyme in the reduction of elemental sulfur (S(8)) in situ is not, however, ruled out by these results, and the possible roles of this substrate are discussed. During aerobic persulfide reduction, rapid recycling of the persulfide substrate was observed, which is proposed to occur via sulfide oxidation by O(2) and/or H(2)O(2). As expected, this reaction disappears under anaerobic conditions and may explain observations by others that CoADR is not essential for S(0) respiration in Pyrococcus or Thermococcus but appears to participate in oxidative defense in the presence of S(0). When compared to the homologous Npsr enzyme from Shewanella loihica PV-4 and homologous enzymes known to reduce CoA disulfide, the phCoADR structure shows a relatively restricted substrate channel leading into the sulfur-reducing side of the FAD isoalloxazine ring, suggesting how this enzyme class may select for specific disulfide substrates.

General acid catalysis of the cleavage of 2-(1-hydroxybenzyl)thiamin by a preassociation mechanism☆

Abstract Cleavage of racemic 2-(1-hydroxybenzyl)thiamin (HBT) to benzaldehyde and thiamin in aqueous solution, a retrograde aldol-type reaction, is catalyzed by substituted acetate ions and other oxygen-containing buffer bases at 40°C and ionic strength 1.0 m (KCl). The Bronsted β value is 0.61 for N(1′)-protonated HBT, but there is no significant solvent deuterium isotope effect for catalysis by acetate ion. The water and buffer base-catalyzed reactions are formulated as general acid catalysis of the departure of thiamin from the alcoholate anion (pKa′ROH = 10.7) of HBT (general base catalysis of thiamin attack in the reverse direction). It is concluded that this reaction proceeds by a concerted mechanism in aqueous solution that is determined by the short lifetime of the thiazolium C(2)-ylide even though the carbanion has a significant lifetime in aqueous solution and a stepwise pathway for the aldol-type addition reaction of the C(2)-ylide must exist. It is suggested that thiamin diphosphate-dependent enzymes could also use the lower energy, preassociation pathway.

Pyrobaculum igneiluti sp. nov., a novel anaerobic hyperthermophilic archaeon that reduces thiosulfate and ferric iron

A novel anaerobic, hyperthermophilic archaeon was isolated from a mud volcano in the Salton Sea geothermal system in southern California, USA. The isolate, named strain 521T, grew optimally at 90 °C, at pH 5.5-7.3 and with 0-2.0 % (w/v) NaCl, with a generation time of 10 h under optimal conditions. Cells were rod-shaped and non-motile, ranging from 2 to 7 µm in length. Strain 521T grew only in the presence of thiosulfate and/or Fe(III) (ferrihydrite) as terminal electron acceptors under strictly anaerobic conditions, and preferred protein-rich compounds as energy sources, although the isolate was capable of chemolithoautotrophic growth. 16S rRNA gene sequence analysis places this isolate within the crenarchaeal genus Pyrobaculum. To our knowledge, this is the first Pyrobaculum strain to be isolated from an anaerobic mud volcano and to reduce only either thiosulfate or ferric iron. An in silico genome-to-genome distance calculator reported <25 % DNA-DNA hybridization between strain 521T and eight other Pyrobaculum species. Due to its genotypic and phenotypic differences, we conclude that strain 521T represents a novel species, for which the name Pyrobaculum igneiluti sp. nov. is proposed. The type strain is 521T (=DSM 103086T=ATCC TSD-56T).

Retrograde aldol-type reactions involving thiamin in aqueous solution : evidence for changes in transition-state structure

Abstract Cleavage of racemic 2-(1-hydroxyethyl)- and 2-(1-hydroxyaryl)-3-R-4-methylthiazolium ions to the corresponding aldehyde and thiazolium ion in aqueous solution is catalyzed by oxygen-containing and amine buffer bases at 40°C and ionic strength 1.0 m (KCl). The buffer base-catalyzed reactions are formulated as general acid catalysis of the departure of thiazolium ion from the alcoholate anion of the substrate (general base catalysis of thiazolium ion attack on the aldehyde in the reverse direction). Bronsted α values decrease from 0.38 to 0.21 for general acid catalysis of the cleavage of (2-1-hydroxyethyl)-R-4-methylthiazolium ions (R = C6F5CH2, 4-aminopyrimidinyl, Bzl). The decrease in α with decreasing pKa of C(2)-H in the leaving thiazolium ion is described by a positive interaction coefficient p xy′ = ∂α ∂ p K lg = 0.3 ± 0.1 . Bronsted α values increase from 0.39 (R′ = Ph) to 0.48 (R′ = 4-CF3-Ph) for the corresponding reactions with 2-(HOCH(R′))thiamin. The increase in α as the carbon electrophile becomes less stable is described by a positive interaction coefficient p xy = sol∂α ∂σ para = 0.2 ± 0.1 . These positive interaction coefficients support a nonenforced concerted reaction mechanism with an important component of proton transfer in the transition state. Mechanistic implications for thiamin diphosphate-dependent enzymes are discussed.

A broader active site in Pyrococcus horikoshii CoA disulfide reductase accommodates larger substrates and reveals evidence of subunit asymmetry

Within the family of pyridine nucleotide disulfide oxidoreductase (PNDOR), enzymes are a group of single‐cysteine containing FAD‐dependent reductases that utilize a tightly bound coenzyme A to assist in the NAD(P)H‐dependent reduction of di‐, per‐, and polysulfide substrates in bacteria and archaea. For many of these homodimeric enzymes, it has proved difficult to determine the substrate specificity and metabolic function based on sequence and genome analysis alone. Coenzyme A‐disulfide reductase (CoADR) isolated from Pyrococcus horikoshii (phCoADR) reduces Co‐A per‐ and polysulfides, but, unlike other highly homologous members of this group, is a poor CoA disulfide reductase. The phCoADR structure has a narrower access channel for CoA substrates, which suggested that this restriction might be responsible for the enzymes poor activity toward the bulky CoA disulfide substrate. To test this hypothesis, the substrate channel was widened by making four mutations along the channel wall (Y65A, Y66A, P67G, and H367G). The structure of the quadruple mutant shows a widened substrate channel, which is supported by a fourfold increase in kcat for the NAD(P)H‐dependent reduction of CoA disulfide and enhanced activity toward the substrate at lower temperatures. Anaerobic titrations of the enzyme with NADH revealed a half‐site reactivity not observed with the wild‐type enzyme in which one subunit of the enzyme could be fully reduced to an EH4 state, while the other remained in an EH2 or EH2·NADH state. These results suggest that for these closely related enzymes, substrate channel morphology is an important determinant of substrate specificity, and homology modeling will be the preferred technique for predicting function among PNDORs.