Kesen Ma

Key Role for Sulfur in Peptide Metabolism and in Regulation of Three Hydrogenases in the Hyperthermophilic Archaeon Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100 degrees C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S(0)). Growth rates were highest on media containing peptides and S(0), with or without maltose. Growth did not occur on the peptide medium without S(0). S(0) had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S(0) with or without maltose were the same, suggesting that S(0) is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S(0) in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S(0)-reducing enzyme in this organism and the mechanism of the S(0) dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.

[18] Hydrogenases I and II from Pyrococcus furiosus

Publisher Summary Hydrogenases catalyze the reversible oxidation of hydrogen (H 2 ) gas. They are widespread in the microbial world and enable organisms either to use H 2 as a source of energy and reductant or to dispose of reductant without the need for terminal electron acceptors other than protons. The hyperthermophilic archaeon Pyrococcus furiosus falls into the latter category. It grows optimally at 100° by the fermentation of carbohydrates and peptides, and the excess reductant generated by these oxidative pathways is used to generate H 2 as an end product. Two NADPH-dependent hydrogenases located in the cytoplasm are thought to be responsible for catalyzing H 2 production. This chapter describes the methods used for purification and characterization of the H 2 -evolving, S°-reducing hydrogenases from P. furiosus . The hydrogenase first reported from P. furiosus is referred to as hydrogenase I. This is responsible for about 90% of the H 2 evolution activity of the cytoplasmic fraction of P. furiosus cells. A second hydrogenase has been discovered in the cytoplasm of this organism and is termed hydrogenase II. The purification procedure described allows the purification of both hydrogenase I and hydrogenase II from the same batch of ceils.

In Vitro Reconstitution of an NADPH-Dependent Superoxide Reduction Pathway from Pyrococcus furiosus

ABSTRACT A scheme for the detoxification of superoxide in Pyrococcus furiosus has been previously proposed in which superoxide reductase (SOR) reduces (rather than dismutates) superoxide to hydrogen peroxide by using electrons from reduced rubredoxin (Rd). Rd is reduced with electrons from NAD(P)H by the enzyme NAD(P)H:rubredoxin oxidoreductase (NROR). The goal of the present work was to reconstitute this pathway in vitro using recombinant enzymes. While recombinant forms of SOR and Rd are available, the gene encoding P. furiosus NROR (PF1197) was found to be exceedingly toxic to Escherichia coli, and an active recombinant form (rNROR) was obtained via a fusion protein expression system, which produced an inactive form of NROR until cleavage. This allowed the complete pathway from NAD(P)H to the reduction of SOR via NROR and Rd to be reconstituted in vitro using recombinant proteins. rNROR is a 39.9-kDa protein whose sequence contains both flavin adenine dinucleotide (FAD)- and NAD(P)H-binding motifs, and it shares significant similarity with known and putative Rd-dependent oxidoreductases from several anaerobic bacteria, both mesophilic and hyperthermophilic. FAD was shown to be essential for activity in reconstitution assays and could not be replaced by flavin mononucleotide (FMN). The bound FAD has a midpoint potential of −173 mV at 23°C (−193 mV at 80°C). Like native NROR, the recombinant enzyme catalyzed the NADPH-dependent reduction of rubredoxin both at high (80°C) and low (23°C) temperatures, consistent with its proposed role in the superoxide reduction pathway. This is the first demonstration of in vitro superoxide reduction to hydrogen peroxide using NAD(P)H as the electron donor in an SOR-mediated pathway.

[4] Ferredoxin:NADP oxidoreductase from Pyrococcus furiosus

Publisher Summary Ferredoxin:NADP + oxidoreductase (FNOR) is a flavoenzyme that catalyzes electron transfer between the redox protein, ferredoxin, and the pyridine nucleotide coenzymes, NADP(H) and/or NAD(H). Enzymes of this type have been characterized from many organisms, including from both the bacterial and eukaryotic domains. However, only one such enzyme has been purified from the hypertherophilic archaea, that from Pyrococcus furiosus. The P. Furiosus enzyme not only functions as a very efficient FNOR, but also catalyzes a variety of reactions, including the reduction of polysulfide using NADPH as electron donor. This chapter describes the assay methods, purification procedure, and properties of P. furiosus FNOR.

Towards the crystal structure of glycerol dehydrogenase from Thermotoga maritima

The NAD(+)-dependent glycerol dehydrogenase (EC 1.1.1.6) from the extremely thermophilic bacterium Thermotoga maritima has been crystallized in the presence of glycerol by the hanging-drop vapour-diffusion method using 2-methyl-2,4-pentanediol (MPD) as the precipitating agent. Crystals of the enzyme complexed with NAD(+) have also been obtained. The crystals belong to the tetragonal system with space group I422 and unit-cell parameters a = 105.3, c = 134.5 A. They diffract to a maximum resolution of 1.4 A using synchrotron radiation (lambda = 0.838 A). Crystals of the enzyme-NAD(+) complex diffract to 2.5 A resolution using in-house Cu Kalpha radiation.

[6] NAD(P)H:rubredoxin oxidoreductase from Pyrococcus furiosus

Publisher Summary NAD(P)H:rubredoxin oxidoreductase (NROR) catalyzes the reduction of the redox protein rubredoxin with NAD(P)H as the electron donor. It has been purified from many anaerobic microorganisms, including the hyperthermophilic archaeon Pyrococcus furiosus. The physiological role of rubredoxin has not been firmly established, but it has been proposed to function as the electron donor to a new enzyme, superoxide reductase (SOR), which reduces superoxide to hydrogen peroxide. Unlike superoxide dismutase, the enzyme that protects aerobes from the toxic effects of oxygen, SOR does not catalyze the production of oxygen from superoxide and, therefore, confers a selective advantage to anaerobes. Hence, NROR may play a role in detoxifying reactive oxygen species by providing the reducing equivalents to SOR via rubredoxin. This chapter describes the methods used to purify, assay, and characterize P. furiosus NROR.

[16] Alcohol dehydrogenases from Thermococcus litoralis and Thermococcus strain ES-1

Publisher Summary Alcohol dehydrogenases catalyze the reversible interconversion of alcohols and aldehydes using NAD(P) as the electron carrier. Such enzymes are present in virtually all life forms and can be divided into three different groups based on their molecular properties, Group l contains the long-chain alcohol dehydrogenases represented by horse liver alcohol dehydrogenase, which is a zinc-containing enzyme, Group II contains short-chain alcohol dehydrogenases, such as the one from Drosophila melanogaster, and these lack metals. Group III contains a small number of iron-dependent alcohol dehydrogenases, which are represented by the second alcohol dehydrogenase, ADH2, purified from Zymomonas mobilis. Several alcohol dehydrogenases have been characterized from hyperthermophilic archaea. A zinc-containing group I enzyme was purified from Sulfolobus solfataricus , and a novel type of iron-containing alcohol dehydrogenase has been purified from Thermococcus litoralis and Thermococcus strain ES-1. This chapter describes the methods used to assay, purify, and characterize the iron-containing alcohol dehydrogenases from T. litoralis and Thermococcus strain ES-1.

Crystallization of the glutamate dehydrogenase from the hyperthermophilic archaeon Thermococcus litoralis

The NADP(+)-dependent glutamate dehydrogenase from Thermococcus litoralis has been crystallized by the hanging-drop method of vapour diffusion using an ammonium sulfate and PEG mixture as the precipitant. The crystals belong to the monoclinic system and are in space group C2 with unit-cell dimensions a = 142.7, b = 202.0, c = 125.8 A with beta = 113.1 degrees with a hexamer in the asymmetric unit. T. Litoralis, a hyperthermophilic organism, belongs to the family of Archaea and has a maximum growth temperature of about 370 K. The glutamate dehydrogenase isolated from this organism has a half-life of 2 h at 373 K and a comparison of this structure with that of other GluDHs from hyperthermophilic organisms and from mesophiles will contribute to an understanding of the molecular mechanisms which underlie thermostability.