[Show abstract][Hide abstract]ABSTRACT: The active site of [NiFe] hydrogenase is an excellent catalyst for hydrogen conversion with an intriguing ability to recover from exposure to dioxygen. In their Communication on page 724 ff., W. Lubitz, S. P. Cramer, and co‐workers use the synchrotron technique of nuclear resonance vibrational spectroscopy to identify normal modes associated with active‐site FeCN and FeCO motion.
Full-text · Article · Jan 2013 · Angewandte Chemie International Edition
[Show abstract][Hide abstract]ABSTRACT: Nuclear inelastic scattering of (57) Fe labeled [NiFe] hydrogenase is shown to give information on different states of the enzyme. It was thus possible to detect and assign Fe-CO and Fe-CN bending and stretching vibrations of the active site outside the spectral range of the Fe-S cluster normal modes.
Full-text · Article · Jan 2013 · Angewandte Chemie International Edition
[Show abstract][Hide abstract]ABSTRACT: Blick ins Zentrum: Die Methode der inelastischen kernresonanten Streuung (NRVS) wurde genutzt, um 57Fe-markierte [NiFe]-Hydrogenase in unterschiedlichen Zuständen des Enzyms zu untersuchen. Damit gelang es, die Fe-CO- und Fe-CN-Beuge- und -Streckschwingung außerhalb des Spektralbereichs der Normalmoden der Fe-S-Cluster zu detektieren und zuzuordnen (siehe Spektrum).
Full-text · Article · Jan 2013 · Angewandte Chemie
[Show abstract][Hide abstract]ABSTRACT: C-1 carriers are essential cofactors in all domains of life, and in Archaea, these can be derivatives of tetrahydromethanopterin (H(4)-MPT) or tetrahydrofolate (H(4)-folate). Their synthesis requires 6-hydroxymethyl-7,8-dihydropterin diphosphate (6-HMDP) as the precursor, but the nature of pathways that lead to its formation were unknown until the recent discovery of the GTP cyclohydrolase IB/MptA family that catalyzes the first step, the conversion of GTP to dihydroneopterin 2',3'-cyclic phosphate or 7,8-dihydroneopterin triphosphate [El Yacoubi, B.; et al. (2006) J. Biol. Chem., 281, 37586-37593 and Grochowski, L. L.; et al. (2007) Biochemistry46, 6658-6667]. Using a combination of comparative genomics analyses, heterologous complementation tests, and in vitro assays, we show that the archaeal protein families COG2098 and COG1634 specify two of the missing 6-HMDP synthesis enzymes. Members of the COG2098 family catalyze the formation of 6-hydroxymethyl-7,8-dihydropterin from 7,8-dihydroneopterin, while members of the COG1634 family catalyze the formation of 6-HMDP from 6-hydroxymethyl-7,8-dihydropterin. The discovery of these missing genes solves a long-standing mystery and provides novel examples of convergent evolutions where proteins of dissimilar architectures perform the same biochemical function.
Full-text · Article · Aug 2012 · ACS Chemical Biology
[Show abstract][Hide abstract]ABSTRACT: The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters.
It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of
the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H2 production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI,
and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although
the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI
was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of
cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous
[NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production.
Preview · Article · Dec 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract]ABSTRACT: Hydrogen gas is an attractive alternative fuel as it is carbon neutral and has higher energy content per unit mass than fossil fuels. The biological enzyme responsible for utilizing molecular hydrogen is hydrogenase, a heteromeric metalloenzyme requiring a complex maturation process to assemble its O(2)-sensitive dinuclear-catalytic site containing nickel and iron atoms. To facilitate their utility in applied processes, it is essential that tools are available to engineer hydrogenases to tailor catalytic activity and electron carrier specificity, and decrease oxygen sensitivity using standard molecular biology techniques. As a model system we are using hydrogen-producing Pyrococcus furiosus, which grows optimally at 100°C. We have taken advantage of a recently developed genetic system that allows markerless chromosomal integrations via homologous recombination. We have combined a new gene marker system with a highly-expressed constitutive promoter to enable high-level homologous expression of an engineered form of the cytoplasmic NADP-dependent hydrogenase (SHI) of P. furiosus. In a step towards obtaining 'minimal' hydrogenases, we have successfully produced the heterodimeric form of SHI that contains only two of the four subunits found in the native heterotetrameric enzyme. The heterodimeric form is highly active (150 units mg(-1) in H(2) production using the artificial electron donor methyl viologen) and thermostable (t(1/2) ∼0.5 hour at 90°C). Moreover, the heterodimer does not use NADPH and instead can directly utilize reductant supplied by pyruvate ferredoxin oxidoreductase from P. furiosus. The SHI heterodimer and POR therefore represent a two-enzyme system that oxidizes pyruvate and produces H(2) in vitro without the need for an intermediate electron carrier.
[Show abstract][Hide abstract]ABSTRACT: We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study oxidized and reduced forms of the [4Fe-4S] cluster in the D14C variant ferredoxin from Pyrococcus furiosus (Pf D14C Fd). To assist the normal-mode assignments, we conducted NRVS with D14C ferredoxin samples with (36)S substituted into the [4Fe-4S] cluster bridging sulfide positions, and a model compound without ligand side chains, (Ph(4)P)(2)[Fe(4)S(4)Cl(4)]. Several distinct regions of NRVS intensity are identified, ranging from "protein" and torsional modes below 100 cm(-1), through bending and breathing modes near 150 cm(-1), to strong bands from Fe-S stretching modes between 250 and ∼400 cm(-1). The oxidized ferredoxin samples were also investigated by resonance Raman (RR) spectroscopy. We found good agreement between NRVS and RR frequencies, but because of different selection rules, the intensities vary dramatically between the two types of spectra. The (57)Fe partial vibrational densities of states for the oxidized samples were interpreted by normal-mode analysis with optimization of Urey-Bradley force fields for local models of the [4Fe-4S] clusters. Full protein model calculations were also conducted using a supplemented CHARMM force field, and these calculations revealed low-frequency modes that may be relevant to electron transfer with Pf Fd partners. Density functional theory (DFT) calculations complemented these empirical analyses, and DFT was used to estimate the reorganization energy associated with the [Fe(4)S(4)](2+/+) redox cycle. Overall, the NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.
[Show abstract][Hide abstract]ABSTRACT: GMPA scores/occurrences tables. GMPA scores for all metals and proteins studied. The number of fraction occurrences in the data set is listed for each protein as well.
[Show abstract][Hide abstract]ABSTRACT: In attempts to develop a method of introducing DNA into Pyrococcus furiosus, we discovered a variant within the wild-type population that is naturally and efficiently competent for DNA uptake. A pyrF gene deletion mutant was constructed in the genome, and the combined transformation and recombination frequencies of this
strain allowed marker replacement by direct selection using linear DNA. We have demonstrated the use of this strain, designated
COM1, for genetic manipulation. Using genetic selections and counterselections based on uracil biosynthesis, we generated
single- and double-deletion mutants of the two gene clusters that encode the two cytoplasmic hydrogenases. The COM1 strain
will provide the basis for the development of more sophisticated genetic tools allowing the study and metabolic engineering
of this important hyperthermophile.
Full-text · Article · Feb 2011 · Applied and Environmental Microbiology
[Show abstract][Hide abstract]ABSTRACT: Metal-containing proteins comprise a diverse and sizable category within the proteomes of organisms, ranging from proteins that use metals to catalyze reactions to proteins in which metals play key structural roles. Unfortunately, reliably predicting that a protein will contain a specific metal from its amino acid sequence is not currently possible. We recently developed a generally-applicable experimental technique for finding metalloproteins on a genome-wide scale. Applying this metal-directed protein purification approach (ICP-MS and MS/MS based) to the prototypical microbe Pyrococcus furiosus conclusively demonstrated the extent and diversity of the uncharacterized portion of microbial metalloproteomes since a majority of the observed metal peaks could not be assigned to known or predicted metalloproteins. However, even using this technique, it is not technically feasible to purify to homogeneity all metalloproteins in an organism. In order to address these limitations and complement the metal-directed protein purification, we developed a computational infrastructure and statistical methodology to aid in the pursuit and identification of novel metalloproteins.
We demonstrate that our methodology enables predictions of metal-protein interactions using an experimental data set derived from a chromatography fractionation experiment in which 870 proteins and 10 metals were measured over 2,589 fractions. For each of the 10 metals, cobalt, iron, manganese, molybdenum, nickel, lead, tungsten, uranium, vanadium, and zinc, clusters of proteins frequently occurring in metal peaks (of a specific metal) within the fractionation space were defined. This resulted in predictions that there are from 5 undiscovered vanadium- to 13 undiscovered cobalt-containing proteins in Pyrococcus furiosus. Molybdenum and nickel were chosen for additional assessment producing lists of genes predicted to encode metalloproteins or metalloprotein subunits, 22 for nickel including seven from known nickel-proteins, and 20 for molybdenum including two from known molybdo-proteins. The uncharacterized proteins are prime candidates for metal-based purification or recombinant approaches to validate these predictions.
We conclude that the largely uncharacterized extent of native metalloproteomes can be revealed through analysis of the co-occurrence of metals and proteins across a fractionation space. This can significantly impact our understanding of metallobiochemistry, disease mechanisms, and metal toxicity, with implications for bioremediation, medicine and other fields.
Full-text · Article · Feb 2011 · BMC Bioinformatics