Amy M. Grunden

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.

Hydrolysis of organophosphorus compounds by microbial enzymes

There are classes of microbial enzymes that have the ability to degrade harmful organophosphorus (OP) compounds that are present in some pesticides and nerve agents. To date, the most studied and potentially important OP-degrading enzymes are organophosphorus hydrolase (OPH) and organophosphorus acid anhydrolase (OPAA), which have both been characterized from a number of organisms. Here we provide an update of what is experimentally known about OPH and OPAA to include their structures, substrate specificity, and catalytic properties. Current and future potential applications of these enzymes in the hydrolysis of OP compounds are also addressed.

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.

Production of biofuels from synthesis gas using microbial catalysts.

World energy consumption is expected to increase 44% in the next 20 years. Today, the main sources of energy are oil, coal, and natural gas, all fossil fuels. These fuels are unsustainable and contribute to environmental pollution. Biofuels are a promising source of sustainable energy. Feedstocks for biofuels used today such as grain starch are expensive and compete with food markets. Lignocellulosic biomass is abundant and readily available from a variety of sources, for example, energy crops and agricultural/industrial waste. Conversion of these materials to biofuels by microorganisms through direct hydrolysis and fermentation can be challenging. Alternatively, biomass can be converted to synthesis gas through gasification and transformed to fuels using chemical catalysts. Chemical conversion of synthesis gas components can be expensive and highly susceptible to catalyst poisoning, limiting biofuel yields. However, there are microorganisms that can convert the CO, H(2), and CO(2) in synthesis gas to fuels such as ethanol, butanol, and hydrogen. Biomass gasification-biosynthesis processing systems have shown promise as some companies have already been exploiting capable organisms for commercial purposes. The discovery of novel organisms capable of higher product yield, as well as metabolic engineering of existing microbial catalysts, makes this technology a viable option for reducing our dependency on fossil fuels.

Expression of Pyrococcus furiosus Superoxide Reductase in Arabidopsis Enhances Heat Tolerance

Plants produce reactive oxygen species (ROS) in response to environmental stresses sending signaling cues, which, if uncontrolled, result in cell death. Like other aerobic organisms, plants have ROS-scavenging enzymes, such as superoxide dismutase (SOD), which removes superoxide anion radical (O2−) and prevents the production and buildup of toxic free radicals. However, increasing the expression of cytosolic SODs is complex, and increasing their production in vivo has proven to be challenging. To avoid problems with endogenous regulation of gene expression, we expressed a gene from the archaeal hyperthermophile Pyrococcus furiosus that reduces O2−. P. furiosus uses superoxide reductase (SOR) rather than SOD to remove superoxide. SOR is a thermostable enzyme that reduces O2− in a one-electron reduction without producing oxygen. We show that P. furiosus SOR can be produced as a functional enzyme in planta and that plants producing SOR have enhanced tolerance to heat, light, and chemically induced ROS. Stress tolerance in the SOR-producing plants correlates positively with a delayed increase in ROS-sensitive transcripts and a decrease in ascorbate peroxidase activity. The SOR plants provide a good model system to study the impact of cytosolic ROS on downstream signaling in plant growth and development. Furthermore, this work demonstrates that this synthetic approach for reducing cytosolic ROS holds promise as a means for improving stress tolerance in crop plants.

Bacterial biodegradation and bioconversion of industrial lignocellulosic streams.

Lignocellulose is a term for plant materials that are composed of matrices of cellulose, hemicellulose, and lignin. Lignocellulose is a renewable feedstock for many industries. Lignocellulosic materials are used for the production of paper, fuels, and chemicals. Typically, industry focuses on transforming the polysaccharides present in lignocellulose into products resulting in the incomplete use of this resource. The materials that are not completely used make up the underutilized streams of materials that contain cellulose, hemicellulose, and lignin. These underutilized streams have potential for conversion into valuable products. Treatment of these lignocellulosic streams with bacteria, which specifically degrade lignocellulose through the action of enzymes, offers a low-energy and low-cost method for biodegradation and bioconversion. This review describes lignocellulosic streams and summarizes different aspects of biological treatments including the bacteria isolated from lignocellulose-containing environments and enzymes which may be used for bioconversion. The chemicals produced during bioconversion can be used for a variety of products including adhesives, plastics, resins, food additives, and petrochemical replacements.

Biotechnological applications of halophilic lipases and thioesterases.

Lipases and esterases are enzymes which hydrolyze ester bonds between a fatty acid moiety and an esterified conjugate, such as a glycerol or phosphate. These enzymes have a wide spectrum of use in industrial applications where their high activity, broad substrate specificity, and stability under harsh conditions have made them integral in biofuel production, textile processing, waste treatment, and as detergent additives. To date, these industrial applications have mainly leveraged enzymes from mesophilic and thermophilic organisms. However, increasingly, attention has turned to halophilic enzymes as catalysts in environments where high salt stability is desired. This review provides a brief overview of lipases and esterases and examines specific structural motifs and evolutionary adaptations of halophilic lipases. Finally, we examine the state of research involving these enzymes and provide an in-depth look at an exciting algal-based biofuel production system. This system uses a recombinant halophilic lipase to increase oil production efficiency by cleaving algal fatty acids from the acyl carrier protein, which eliminates feedback inhibition of fatty acid synthesis.

Prolidase function in proline metabolism and its medical and biotechnological applications

Prolidase is a multifunctional enzyme that possesses the unique ability to degrade imidodipeptides in which a proline or hydroxyproline residue is located at the C‐terminal end. Prolidases have been isolated from archaea and bacteria, where they are thought to participate in proline recycling. In mammalian species, prolidases are found in the cytoplasm and function primarily to liberate proline in the final stage of protein catabolism, particularly during the biosynthesis and degradation of collagen. Collagen comprises nearly one‐third of the total protein in the body, and it is essential in maintaining tissue structure and integrity. Prolidase deficiency (PD), a rare autosomal recessive disorder in which mutations in the PEPD gene affect prolidase functionality, tends to have serious and sometimes life‐threatening clinical symptoms. Recombinant prolidases have many applications and have been investigated not only as a possible treatment for PD, but also as a part of anti‐cancer strategies, a component of biodecontamination cocktails and in the dairy industry. This review will serve to discuss the many in vivo functions of procaryotic and eucaryotic prolidases, as well as the most recent advances in therapeutic and biotechnological application of prolidases.

Basal Signaling Regulates Plant Growth and Development

The term signal transduction refers to the classical paradigm where an external stimulus is sensed and initiates an increase in second messengers. Each second messenger transmits and amplifies the signal by activating a subset of downstream pathways. This complex network of interwoven downstream

Characterization of the dinuclear metal center of Pyrococcus furiosus prolidase by analysis of targeted mutants.

Prolidases are dipeptidases specific for cleavage of Xaa‐Pro dipeptides. Pyrococcus furiosus prolidase is a homodimer having one Co‐bound dinuclear metal cluster per monomer with one tightly bound Co(II) site and the other loosely bound (K d 0.24 mM). To identify which Co site is tight‐binding and which is loose‐binding, site‐directed mutagenesis was used to modify amino acid residues that participate in binding the Co1 (E‐313 and H‐284), the Co2 site (D‐209) or the bidentate ligand (E‐327). Metal‐content, enzyme activity and CD‐spectra analyses of D209A‐, H284L‐, and E327L‐prolidase mutants show that Co1 is the tight‐binding and Co2 the loose‐binding metal center.