Microbial hydrogen splitting in the presence of oxygen

2013 ◽  
Vol 41 (5) ◽  
pp. 1317-1324 ◽  
Author(s):  
Matthias Stein ◽  
Sandeep Kaur-Ghumaan
Keyword(s):  
Active Site ◽  
Large Subunit ◽  
Spectroscopic Studies ◽  
Small Subunit ◽  
Reduced Form ◽  
Cysteine Residues ◽  
Binding Motif ◽  
Oxygen Tolerance ◽  
Symmetry Approach ◽  
Long Time

The origin of the tolerance of a subclass of [NiFe]-hydrogenases to the presence of oxygen was unclear for a long time. Recent spectroscopic studies showed a conserved active site between oxygen-sensitive and oxygen-tolerant hydrogenases, and modifications in the vicinity of the active site in the large subunit could be excluded as the origin of catalytic activity even in the presence of molecular oxygen. A combination of bioinformatics and protein structural modelling revealed an unusual co-ordination motif in the vicinity of the proximal Fe–S cluster in the small subunit. Mutational experiments confirmed the relevance of two additional cysteine residues for the oxygen-tolerance. This new binding motif can be used to classify sequences from [NiFe]-hydrogenases according to their potential oxygen-tolerance. The X-ray structural analysis of the reduced form of the enzyme displayed a new type of [4Fe–3S] cluster co-ordinated by six surrounding cysteine residues in a distorted cubanoid geometry. The unusual electronic structure of the proximal Fe–S cluster can be analysed using the broken-symmetry approach and gave results in agreement with experimental Mößbauer studies. An electronic effect of the proximal Fe–S cluster on the remote active site can be detected and quantified. In the oxygen-tolerant hydrogenases, the hydride occupies an asymmetric binding position in the Ni-C state. This may rationalize the more facile activation and catalytic turnover in this subclass of enzymes.

scholarly journals Spectroscopic Insights into the Oxygen-tolerant Membrane-associated [NiFe] Hydrogenase of Ralstonia eutropha H16

2009 ◽  
Vol 284 (24) ◽  
pp. 16264-16276 ◽  
Author(s):  
Miguel Saggu ◽  
Ingo Zebger ◽  
Marcus Ludwig ◽  
Oliver Lenz ◽  
Bärbel Friedrich ◽  
...  
Keyword(s):  
Active Site ◽  
Ralstonia Eutropha ◽  
Large Subunit ◽  
Magnetic Coupling ◽  
Spectroscopic Characterization ◽  
Spectroscopic Properties ◽  
Small Subunit ◽  
Reduced Form ◽  
Strictly Anaerobic ◽  
Redox States

This study provides the first spectroscopic characterization of the membrane-bound oxygen-tolerant [NiFe] hydrogenase (MBH) from Ralstonia eutropha H16 in its natural environment, the cytoplasmic membrane. The H2-converting MBH is composed of a large subunit, harboring the [NiFe] active site, and a small subunit, capable in coordinating one [3Fe4S] and two [4Fe4S] clusters. The hydrogenase dimer is electronically connected to a membrane-integral cytochrome b. EPR and Fourier transform infrared spectroscopy revealed a strong similarity of the MBH active site with known [NiFe] centers from strictly anaerobic hydrogenases. Most redox states characteristic for anaerobic [NiFe] hydrogenases were identified except for one remarkable difference. The formation of the oxygen-inhibited Niu-A state was never observed. Furthermore, EPR data showed the presence of an additional paramagnetic center at high redox potential (+290 mV), which couples magnetically to the [3Fe4S] center and indicates a structural and/or redox modification at or near the proximal [4Fe4S] cluster. Additionally, significant differences regarding the magnetic coupling between the Nia-C state and [4Fe4S] clusters were observed in the reduced form of the MBH. The spectroscopic properties are discussed with regard to the unusual oxygen tolerance of this hydrogenase and in comparison with those of the solubilized, dimeric form of the MBH.

scholarly journals A model system for [NiFe] hydrogenase maturation studies: Purification of an active site-containing hydrogenase large subunit without small subunit

FEBS Letters ◽  
2005 ◽  
Vol 579 (20) ◽  
pp. 4292-4296 ◽  
Author(s):  
Gordon Winter ◽  
Thorsten Buhrke ◽  
Oliver Lenz ◽  
Anne Katherine Jones ◽  
Michael Forgber ◽  
...  
Keyword(s):  
Active Site ◽  
Large Subunit ◽  
Small Subunit ◽  
Model System ◽  
Hydrogenase Maturation

scholarly journals The Epstein-Barr Virus (EBV) Deubiquitinating Enzyme BPLF1 Reduces EBV Ribonucleotide Reductase Activity

2009 ◽  
Vol 83 (9) ◽  
pp. 4345-4353 ◽  
Author(s):  
Christopher B. Whitehurst ◽  
Shunbin Ning ◽  
Gretchen L. Bentz ◽  
Florent Dufour ◽  
Edward Gershburg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Ribonucleotide Reductase ◽  
Active Site ◽  
Epstein Barr Virus ◽  
Reductase Activity ◽  
Large Subunit ◽  
Small Subunit ◽  
Deubiquitinating Enzyme ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT A newly discovered virally encoded deubiquitinating enzyme (DUB) is strictly conserved across the Herpesviridae. Epstein-Barr virus (EBV) BPLF1 encodes a tegument protein (3,149 amino acids) that exhibits deubiquitinating (DUB) activity that is lost upon mutation of the active-site cysteine. However, targets for the herpesviral DUBs have remained elusive. To investigate a predicted interaction between EBV BPLF1 and EBV ribonucleotide reductase (RR), a functional clone of the first 246 N-terminal amino acids of BPLF1 (BPLF1 1-246) was constructed. Immunoprecipitation verified an interaction between the small subunit of the viral RR2 and BPLF1 proteins. In addition, the large subunit (RR1) of the RR appeared to be ubiquitinated both in vivo and in vitro; however, ubiquitinated forms of the small subunit, RR2, were not detected. Ubiquitination of RR1 requires the expression of both subunits of the RR complex. Furthermore, coexpression of RR1 and RR2 with BPLF1 1-246 abolishes ubiquitination of RR1. EBV RR1, RR2, and BPLF1 1-246 colocalized to the cytoplasm in HEK 293T cells. Finally, expression of enzymatically active BPLF1 1-246 decreased RR activity, whereas a nonfunctional active-site mutant (BPLF1 C61S) had no effect. These results indicate that the EBV deubiquitinating enzyme interacts with, deubiquitinates, and influences the activity of the EBV RR. This is the first verified protein target of the EBV deubiquitinating enzyme.

scholarly journals Molecular Analysis of a Novel Methanesulfonic Acid Monooxygenase from the Methylotroph Methylosulfonomonas methylovora

1999 ◽  
Vol 181 (7) ◽  
pp. 2244-2251 ◽  
Author(s):  
Paolo de Marco ◽  
Pedro Moradas-Ferreira ◽  
Timothy P. Higgins ◽  
Ian McDonald ◽  
Elizabeth M. Kenna ◽  
...  
Keyword(s):  
Large Subunit ◽  
Methanesulfonic Acid ◽  
Small Subunit ◽  
Methylotrophic Bacterium ◽  
Binding Motif ◽  
Chromosomal Dna ◽  
Mononuclear Iron ◽  
Degrading Bacterium ◽  
Genes Encoding ◽  
High Degree

ABSTRACT Methylosulfonomonas methylovora M2 is an unusual gram-negative methylotrophic bacterium that can grow on methanesulfonic acid (MSA) as the sole source of carbon and energy. Oxidation of MSA by this bacterium is carried out by a multicomponent MSA monooxygenase (MSAMO). Cloning and sequencing of a 7.5-kbp SphI fragment of chromosomal DNA revealed four tightly linked genes encoding this novel monooxygenase. Analysis of the deduced MSAMO polypeptide sequences indicated that the enzyme contains a two-component hydroxylase of the mononuclear-iron-center type. The large subunit of the hydroxylase, MsmA (48 kDa), contains a typical Rieske-type [2Fe–2S] center with an unusual iron-binding motif and, together with the small subunit of the hydroxylase, MsmB (20 kDa), showed a high degree of identity with a number of dioxygenase enzymes. However, the other components of the MSAMO, MsmC, the ferredoxin component, and MsmD, the reductase, more closely resemble those found in other classes of oxygenases. MsmC has a high degree of identity to ferredoxins from toluene and methane monooxygenases, which are enzymes characterized by possessing hydroxylases containing μ-oxo bridge binuclear iron centers. MsmD is a reductase of 38 kDa with a typical chloroplast-like [2Fe–2S] center and conserved flavin adenine dinucleotide- and NAD-binding motifs and is similar to a number of mono- and dioxygenase reductase components. Preliminary analysis of the genes encoding MSAMO from a marine MSA-degrading bacterium, Marinosulfonomonas methylotropha, revealed the presence of msm genes highly related to those found in Methylosulfonomonas, suggesting that MSAMO is a novel type of oxygenase that may be conserved in all MSA-utilizing bacteria.

Phylogenetic analysis of Rhizoclonium (Cladophoraceae, Cladophorales), and the description of Rhizoclonium subtile sp. nov. from China

Phytotaxa ◽  
2018 ◽  
Vol 383 (2) ◽  
pp. 147 ◽  
Author(s):  
ZHI-JUAN ZHAO ◽  
HUAN ZHU ◽  
GUO-XIANG LIU ◽  
ZHENG-YU HU
Keyword(s):  
Ribosomal Dna ◽  
Internal Transcribed Spacer ◽  
Large Subunit ◽  
Nuclear Gene ◽  
Morphological Characters ◽  
Small Subunit ◽  
Molecular Evidence ◽  
Gene Markers ◽  
Long Time ◽  
Small Subunit Ribosomal Dna

The genus Rhizoclonium (Cladophoraceae, Cladophorales) accommodates uniserial, unbranched filamentous algae, closely related to Cladophora and Chaetomorpha. Its taxonomy has been problematic for a long time due to the lack of diagnostic morphological characters. To clarify the species diversity and taxonomic relationships of this genus, we collected and analyzed thirteen freshwater Rhizoclonium specimens from China. The morphological traits of these specimens were observed and described in detail. Three nuclear gene markers small subunit ribosomal DNA (SSU), large subunit ribosomal DNA (LSU) and internal transcribed spacer 2 (ITS2) sequences were analyzed to elucidate their phylogenetic relationships. The results revealed that there were at least fifteen molecular species assignable to Rhizoclonium and our thirteen specimens were distributed in four clades. On the basis of morphological and molecular evidence we propose the new species, R. subtile sp. nov.

scholarly journals Genetic analysis of the large subunit of yeast transcription factor IIE reveals two regions with distinct functions.

1997 ◽  
Vol 17 (9) ◽  
pp. 5288-5298 ◽  
Author(s):  
N H Kuldell ◽  
S Buratowski
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Large Subunit ◽  
Small Subunit ◽  
Single Amino Acid ◽  
Binding Motif ◽  
Yeast Saccharomyces Cerevisiae ◽  
Pol Ii

Biochemical analysis of proteins necessary for transcription initiation by eukaryotic RNA polymerase II (pol II) has identified transcription factor IIE (TFIIE) as an essential component of the reaction. To better understand the role of TFIIE in transcription, we isolated conditional alleles of TFA1, the gene encoding the large subunit of TFIIE in the yeast Saccharomyces cerevisiae. The mutant Tfa1 proteins fall into two classes. The first class causes thermosensitive growth due to single amino acid substitutions of the cysteines comprising the Zn-binding motif. The second mutant class is made up of proteins that are C-terminally truncated and that cause a cold-sensitive growth phenotype. The behavior of these mutants suggests that Tfa1p possesses at least two domains with genetically distinct functions. The mutations in the Zn-binding motif do not affect the mutant protein's stability at the nonpermissive temperature or its ability to associate with the small subunit of TFIIE. Our studies further determined that wild-type TFIIE can bind to single-stranded DNA in vitro. However, this property is unaffected in the mutant TFIIE complexes. Finally, we have demonstrated the biological importance of TFIIE in pol II-mediated transcription by depleting the Tfa1 protein from the cells and observing a concomitant decrease in total poly(A)+ mRNA.

The importance of iron in the biosynthesis and assembly of [NiFe]-hydrogenases

2014 ◽  
Vol 5 (1) ◽  
pp. 55-70 ◽  
Author(s):  
Constanze Pinske ◽  
R. Gary Sawers
Keyword(s):  
Active Site ◽  
Large Subunit ◽  
Small Subunit ◽  
Transport Systems ◽  
Bacterial Cells ◽  
Carbamoyl Phosphate ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
Redox Active ◽  
Cofactor Biosynthesis

Abstract[NiFe]-hydrogenases (Hyd) are redox-active metalloenzymes that catalyze the reversible oxidation of molecular hydrogen to protons and electrons. These enzymes are frequently heterodimeric and have a unique bimetallic active site in their catalytic large subunit and possess a complement of iron sulfur (Fe-S) clusters for electron transfer in the small subunit. Depending on environmental and metabolic requirements, the Fe-S cluster relay shows considerable variation among the Hyd, even employing high potential [4Fe-3S] clusters for improved oxygen tolerance. The general iron sulfur cluster (Isc) machinery is required for small subunit maturation, possibly providing standard [4Fe-4S], which are then modified as required in situ. The [NiFe] cofactor in the active site also has an iron ion to which one CO and two CN- diatomic ligands are attached. Specific accessory proteins synthesize these ligands and insert the cofactor into the apo-hydrogenase large subunit. Carbamoyl phosphate is the precursor of the CN- ligands, and recent experimental evidence suggests that endogenously generated CO2 might be one precursor of CO. Recent advances also indicate how the machineries responsible for cofactor generation obtain iron. Several transport systems for iron into bacterial cells exist; however, in Escherichia coli, it is mainly the ferrous iron transporter Feo and the ferric-citrate siderphore system Fec that are involved in delivering the metal for Hyd biosynthesis. Genetic analyses have provided evidence for the existence of key checkpoints during cofactor biosynthesis and enzyme assembly that ensure correct spatiotemporal maturation of these modular oxidoreductases.

scholarly journals How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase

2014 ◽  
Vol 458 (3) ◽  
pp. 449-458 ◽  
Author(s):  
Lisa Bowman ◽  
Lindsey Flanagan ◽  
Paul K. Fyfe ◽  
Alison Parkin ◽  
William N. Hunter ◽  
...  
Keyword(s):  
Amino Acids ◽  
Large Subunit ◽  
Small Subunit ◽  
Oxygen Tolerance

A hydrogenase consists of two subunits: a large and a small subunit. In the present study, amino acids from the large subunit were found to influence a cofactor in the small subunit, such that they help to confer oxygen-tolerance to the enzyme.

scholarly journals Studies of the active site of m-calpain and the interaction with calpastatin

1993 ◽  
Vol 296 (1) ◽  
pp. 135-142 ◽  
Author(s):  
C Crawford ◽  
N R Brown ◽  
A C Willis
Keyword(s):  
Ion Exchange ◽  
Proteolytic Activity ◽  
Active Site ◽  
Binding Assay ◽  
Large Subunit ◽  
Gel Filtration ◽  
Small Subunit ◽  
Binding Domain ◽  
Competitive Binding Assay ◽  
Binding Domains

Calpain autolyses in the presence of Ca2+. In the case of m-calpain (80 + 30 kDa) the first product is an 80 + 18 kDa species which has an intact large subunit and the C-terminal Ca(2+)-binding domain of the small subunit. It was possible to bind E64 into the active site of calpain in the presence of Ca2+ before cleavage of either calpain subunit. This suggests that the active site is functional before any autolysis has occurred and that calpain is not a proenzyme. Prolonged autolysis generates several fragments including a 42 kDa active-site domain fragment that showed no proteolytic activity and Ca(2+)-binding domain fragments. Some of the Ca(2+)-binding domain fragments were found to exist as heterodimers (23 + 18 kDa and 22 + 18 kDa), with the Ca(2+)-binding domain of the large subunit interacting with the Ca(2+)-binding domain of the small subunit. These species were true heterodimers, as they showed co-elution of the two Ca(2+)-binding domains on ion-exchange and gel-filtration chromatography, and immunoprecipitation of both polypeptides with an antiserum specific for the small-subunit Ca(2+)-binding domain. The generation of the dimer species after only 15 min autolysis suggests that the interaction between the Ca(2+)-binding domains is present in the native calpain structure. The interaction of calpain with calpastatin was investigated using an assay based on binding to calpastatin-Sepharose and a competitive binding assay. Calpain, active-site-blocked calpain and calpain fragments generated by autolysis were studied. Calpain bound to calpastatin in the presence of Ca2+; however, the isolated active-site-containing 80 kDa large subunit (proteolytically inactive), a 42 kDa active-site-containing fragment (proteolytically inactive) and Ca(2+)-binding domain fragments of calpain did not. Active-site-blocked calpain bound to calpastatin, but with an affinity reduced by approximately two orders of magnitude when compared with native calpain.

Depletion and Reconstitution of Active E. Coli Ribosomes

1975 ◽  
Vol 33 ◽  
pp. 640-641
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Protein Biosynthesis ◽  
Large Subunit ◽  
High Resolution Electron Microscopy ◽  
Small Subunit ◽  
Structure And Function ◽  
Biologically Active ◽  
Biological Macromolecules ◽  
E Coli ◽  
Resolution Electron ◽  
And Function

Correlations between structure and function of biological macromolecules have been studied intensively for many years, mostly by indirect methods. High resolution electron microscopy is a unique tool which can provide such information directly by comparing the conformation of biopolymers in their biologically active and inactive state. We have correlated the structure and function of ribosomes, ribonucleoprotein particles which are the site of protein biosynthesis. 70S E. coli ribosomes, used in this experiment, are composed of two subunits - large (50S) and small (30S). The large subunit consists of 34 proteins and two different ribonucleic acid molecules. The small subunit contains 21 proteins and one RNA molecule. All proteins (with the exception of L7 and L12) are present in one copy per ribosome.This study deals with the changes in the fine structure of E. coli ribosomes depleted of proteins L7 and L12. These proteins are unique in many aspects.

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