Coenzyme A disulfide reductase from Pyrococcus horikoshii-OT3

The hyperthermophilic archaea are microorganisms that thrive at temperatures of >80^o C. Many of these organisms utilize sulfur as a final electron acceptor and are able to gain ATP by lithotropic sulfur respiration. While the mechanism for sulfur reduction in the bacterial genera Wollinella and Shewanella has been characterized, the mechanism of sulfur reduction in the archaea remains to be elucidated.

Microorganisms of the genus Pyrococcus are strictly anaerobic hyperthermophiles and are isolated from marine hydrothermal vents. The genomes of P. horikoshii, P. furiosus, and P. abyssii each contain at least two NADH oxidase or CoA disulfide reductase (CoADR) homologues. The characterization of one of these homologues (named NOX1, NADH OXidoreductase 1) from P. furiosus has been reported. The second homologue (previously described as NOX2 and now described as CoADR), isolated from P. horikoshii, demonstrated a slow NAD(P)H oxidase activity in the presence of high concentrations of substrate-level FAD. This FAD level is in addition to the enzyme-bound FAD cofactor. This enzyme is, therefore, most likely not an NADH oxidase in vivo. Instead, the enzyme has been shown to act as a CoADR and recent work has shown that it acts as an NADH and CoA dependent sulfur reductase. The general equation is:

CoA-S-S-CoA + NAD(P)H → 2CoASH + NAD(P)⁺

Project objectives

First, the physiological substrate for the CoADR from P. horikoshii will be elucidated.

Second, as there is a published crystal structure for the CoADR from Staphylococcus aureus (1YQZ), crystallographic comparisons will be made between this CoADR and that of P. horikoshii. Currently, crystals have been obtained and crystallographic data is being analyzed for CoADR. Of particular interest are crystals of P. horikoshii CoADR with substrate and inhibitors bound at the active site. The obtaining of the crystal structure would also determine the quaternary structure of the CoADR from P. horikoshii, which currently has not been unambiguously determined.

Finally, stopped-flow experiments will be conducted order to determine which of the proposed intermediates are kinetically competent and which steps in the catalytic cycle (reduction or oxidation) are rate limiting. These experiments will also allow the determination of microscopic rate constants within the oxidative and reductive half reactions.

CoADR from S. aureus (1YQZ)

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Diiron Enzymes

Carboxylate-bridgeddiiron proteins are found in almost all organisms and participate in a varietyof essential biochemical functions, including hydrocarbon and fatty acidhydroxylation, tyrosyl radical generation, oxidative stress protection, O₂transport and sensing, NO reduction, iron storage, fatty aciddesaturation, and ubiquinol oxidation in mitochondrial membranes. Some of theseproteins, like ribonucleotide reductase (RNR) and soluble methane monooxygenase(sMMO), have received significant attention because of their biomedical,industrial, and environmental importance. The diversity of these powerful O₂-utilizing dinuclear activesites rivals, if not surpasses, that of heme proteins, but diiron enzymes arefound less frequently in nature. Although most diiron proteins share severalstructural and mechanistic features, such as strikingly similar dinuclear ironunits that react with O₂ and traverse peroxo and/or superoxointermediates, it has been particularly challenging to reveal how the proteinscaffold around the metal center governs reactivity. My laboratory aims to investigate the structure/functionrelationships responsible for the chemistry and tuning of dinuclear iron activesites by 1) focusing on proteins that carry out novel reactions using uniquemetal coordination spheres and by 2) re-engineering well characterized systemsto perform new functions.

BacterialBiofilms

Mature bacterial cells can exist intwo states, as free-floating planktonic cells or as densely packed biofilms onthe surfaces of biological and abiotic materials. In their planktonic form,pathogenic bacteria species like Streptococcus pneumoniae, Staphalococcus aureus, Salmonella enterica and Pseudomonas aeruginosa are susceptible to antimicrobialagents. As biofilms, however,these bacteria are highly resistant to antimicrobials owing to a dense matrixof extracellular polysaccharides, proteins, and DNA known collectively as theextracellular polymeric substance (EPS). Although the general organization and function of the EPS matrix is notknown, it is proposed to promote adhesion between cells and host surfaces andoffer protection from hostile extracellular conditions. Because of the seemingly impenetratablenature of these films, chronic infections can result, such as in therespiratory and gastrointestinal tracts of patients with exposure toopportunistic pathogenic organisms. An understanding of biofilm development,composition and organization is essential for developing therapies aimed atdisrupting their formation.

Most investigations into bacterialbiofilms have focused on identifying the genetic, molecular, and physiologicaldeterminants of initiation and development. Genome-based microarray analysis and transposon mutagenesishave identified several intriguing protein targets, but, a universal set ofproteins responsible for this process have not been easy to identify sincedifferent bacterial species do not always use similar machinery for biofilmformation. Structural and biochemical analysis of biofilm related proteins inwell characterized model organisms will provide a clearer picture of the EPScomposition as well as new avenues to combat chronic infections in a variety ofpathogenic bacteria.

Funding

Camilleand Henry Dreyfus Faculty Start-up Award (2007)

Current Undergraduate Research Students