Bacterial formate hydrogenlyase complex McDowall, Jennifer S; Murphy, Bonnie J; Haumann, Michael ...
Proceedings of the National Academy of Sciences - PNAS,
09/2014, Letnik:
111, Številka:
38
Journal Article
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Significance The isolation of an active formate hydrogenlyase is a breakthrough in understanding the molecular basis of bacterial hydrogen production. For over 100 years, Escherichia coli has been ...known to evolve H ₂ when cultured under fermentative conditions. Glucose is metabolized to formate, which is then oxidized to CO ₂ with the concomitant reduction of protons to H ₂ by a single complex called formate hydrogenlyase, which had been genetically, but never biochemically, characterized. In this study, innovative molecular biology and electrochemical experiments reveal a hydrogenase enzyme with the unique ability to sustain H ₂ production even under high partial pressures of H ₂. Harnessing bacterial H ₂ production offers the prospect of a source of fully renewable H ₂ energy, freed from any dependence on fossil fuel.
Under anaerobic conditions, Escherichia coli can carry out a mixed-acid fermentation that ultimately produces molecular hydrogen. The enzyme directly responsible for hydrogen production is the membrane-bound formate hydrogenlyase (FHL) complex, which links formate oxidation to proton reduction and has evolutionary links to Complex I, the NADH:quinone oxidoreductase. Although the genetics, maturation, and some biochemistry of FHL are understood, the protein complex has never been isolated in an intact form to allow biochemical analysis. In this work, genetic tools are reported that allow the facile isolation of FHL in a single chromatographic step. The core complex is shown to comprise HycE (a NiFe hydrogenase component termed Hyd-3), FdhF (the molybdenum-dependent formate dehydrogenase-H), and three iron-sulfur proteins: HycB, HycF, and HycG. A proportion of this core complex remains associated with HycC and HycD, which are polytopic integral membrane proteins believed to anchor the core complex to the cytoplasmic side of the membrane. As isolated, the FHL complex retains formate hydrogenlyase activity in vitro. Protein film electrochemistry experiments on Hyd-3 demonstrate that it has a unique ability among NiFe hydrogenases to catalyze production of H ₂ even at high partial pressures of H ₂. Understanding and harnessing the activity of the FHL complex is critical to advancing future biohydrogen research efforts.
The assembly of multi-protein complexes requires the concerted synthesis and maturation of its components and subsequently their co-ordinated interaction. The membrane-bound formate hydrogenlyase ...(FHL) complex is the primary hydrogen-producing enzyme in
and is composed of seven subunits mostly encoded within the
operon for NiFe-hydrogenase-3 (Hyd-3). The HycH protein is predicted to have an accessory function and is not part of the final structural FHL complex. In this work, a mutant strain devoid of HycH was characterised and found to have significantly reduced FHL activity due to the instability of the electron transfer subunits. HycH was shown to interact specifically with the unprocessed species of HycE, the catalytic hydrogenase subunit of the FHL complex, at different stages during the maturation and assembly of the complex. Variants of HycH were generated with the aim of identifying interacting residues and those that influence activity. The R70/71/K72, the Y79, the E81 and the Y128 variant exchanges interrupt the interaction with HycE without influencing the FHL activity. In contrast, FHL activity, but not the interaction with HycE, was negatively influenced by H37 exchanges with polar residues. Finally, a HycH Y30 variant was unstable. Surprisingly, an overlapping function between HycH with its homologous counterpart HyfJ from the operon encoding NiFe-hydrogenase-4 (Hyd-4) was identified and this is the first example of sharing maturation machinery components between Hyd-3 and Hyd-4 complexes. The data presented here show that HycH has a novel dual role as an assembly chaperone for a cytoplasmic NiFe-hydrogenase.
•Gene deletions identify the stable core complex of bacterial formate hydrogenlyase.•Synthetic biology approaches generated an active, stable fusion protein.•Attempts to engineer O2-tolerance into ...FHL were only partly successful.
The Escherichia coli formate hydrogenlyase (FHL) complex is produced under fermentative conditions and couples formate oxidation to hydrogen production. In this work, the architecture of FHL has been probed by analysing affinity-tagged complexes from various genetic backgrounds. In a successful attempt to stabilize the complex, a strain encoding a fusion between FdhF and HycB has been engineered and characterised. Finally, site-directed mutagenesis of the hycG gene was performed, which is predicted to encode a hydrogenase subunit important for regulating sensitivity to oxygen. This work helps to define the core components of FHL and provides solutions to improving the stability of the enzyme.
α-Synuclein (α-syn) is a cytosolic protein known for its association with neurodegenerative diseases, including Parkinson’s disease and other synucleinopathies. The potential cellular function of ...α-synuclein may be of consequence for understanding the pathogenesis of such diseases. Previous work has suggested that α-synuclein can catalyze the reduction of iron as a ferrireductase. We performed a detailed analysis of the steady-state kinetics of recombinant α-syn ferrireductase activity and for disease-associated variants. Our study illustrates that the ferrireductase activity we observed is clearly commensurate with bona fide enzyme activity and suggests a mechanistic rationale for the activity and the relationship to cellular regulation of the pool of Fe(III) and Fe(II). Using cell-based studies, we examined the functionally active conformation and found that the major catalytically active form is a putative membrane-associated tetramer. Using an artificial membrane environment with recombinant protein, we demonstrate that secondary structure folding of α-synuclein is insufficient to allow enzyme activity and the absolute specificity of the tertiary/quaternary structure is the primary requirement. Finally, we explored the steady-state kinetics of a range of disease α-synuclein variants and found that variants involved in neurodegenerative disease exhibited major changes in their enzymatic activity. We discuss these data in the context of a potential disease-associated mechanism for aberrant α-synuclein ferrireductase activity.
The normal cellular role of α-synuclein is of potential importance in understanding diseases in which an aggregated form of the protein has been implicated. A potential loss or change in the normal ...function of α-synuclein could play a role in the aetiology of diseases such as Parkinson's disease. Recently, it has been suggested that α-synuclein could cause the enzymatic reduction of iron and a cellular increase in Fe(II) levels. Experiments were carried out to determine if such activity could be measured in vivo. Experiments with rats overexpressing human α-synuclein in nigral dopaminergic neurons demonstrated a correlation between α-synuclein expression and ferrireductase activity. Furthermore, studies on tissue from Parkinson's disease patient brains showed a significant decrease in ferrireductase activity, possibly due to deposition of large amounts of inactive protein. Cellular studies suggest that increase ferrireductase activity results in increased levels of dopamine metabolites and increased sensitivity to the toxicity of DOPAL. These findings demonstrate that α-synuclein ferrireductase activity is present in vivo and its alteration may play a role in neuron loss in disease.
•Alpha-synuclein is a ferrireductase in an in vivo rat model.•Parkinson's disease patients show changes in ferrireductase activity in the striatum.•Increased ferrireductase activity increases HVA and DOPAC levels.•Increased ferrireductase causes increases sensitivity to DOPAL toxicity.
Two of the three NiFe-hydrogenases (Hyd) of Escherichia coli have a hydrogen-uptake function in anaerobic metabolism. While Hyd-2 is maximally synthesized when the bacterium grows by fumarate ...respiration, Hyd-1 synthesis shows a correlation with fermentation of sugar substrates. In an attempt to advance our knowledge on the physiological function of Hyd-1 during fermentative growth, we examined Hyd-1 activity and levels in various derivatives of E. coli K-12 MC4100 with specific defects in sugar utilization. MC4100 lacks a functional fructose phosphotransferase system (PTS) and therefore grows more slowly under anaerobic conditions in rich medium in the presence of d-fructose compared with d-glucose. Growth in the presence of fructose resulted in an approximately 10-fold increase in Hyd-1 levels in comparison with growth under the same conditions with glucose. This increase in the amount of Hyd-1 was not due to regulation at the transcriptional level. Reintroduction of a functional fruBKA-encoded fructose PTS into MC4100 restored growth on d-fructose and reduced Hyd-1 levels to those observed after growth on d-glucose. Reducing the rate of glucose uptake by introducing a mutation in the gene encoding the cAMP receptor protein, or consumption through glycolysis, by introducing a mutation in phosphoglucose isomerase, increased Hyd-1 levels during growth on glucose. These results suggest that the ability to oxidize hydrogen by Hyd-1 shows a strong correlation with the rate of carbon flow through glycolysis and provides a direct link between hydrogen, carbon and energy metabolism.
TheEscherichia coli formate hydrogenlyase (FHL) complex is produced under fermentative conditions and couples formate oxidation to hydrogen production. In this work, the architecture of FHL has been ...probed by analysing affinity‐tagged complexes from various genetic backgrounds. In a successful attempt to stabilize the complex, a strain encoding a fusion between FdhF and HycB has been engineered and characterised. Finally, site‐directed mutagenesis of thehycG gene was performed, which is predicted to encode a hydrogenase subunit important for regulating sensitivity to oxygen. This work helps to define the core components of FHL and provides solutions to improving the stability of the enzyme.
Gene deletions identify the stable core complex of bacterial formate hydrogenlyase.
Synthetic biology approaches generated an active, stable fusion protein.
Attempts to engineer O2‐tolerance into FHL were only partly successful.
InterPro protein classification McDowall, Jennifer; Hunter, Sarah
Methods in molecular biology (Clifton, N.J.),
2011, Letnik:
694
Journal Article
Improvements in nucleotide sequencing technology have resulted in an ever increasing number of nucleotide and protein sequences being deposited in databases. Unfortunately, the ability to manually ...classify and annotate these sequences cannot keep pace with their rapid generation, resulting in an increased bias toward unannotated sequence. Automatic annotation tools can help redress the balance. There are a number of different groups working to produce protein signatures that describe protein families, functional domains or conserved sites within related groups of proteins. Protein signature databases include CATH-Gene3D, HAMAP, PANTHER, Pfam, PIRSF, PRINTS, ProDom, PROSITE, SMART, SUPERFAMILY, and TIGRFAMs. Their approaches range from characterising small conserved motifs that can identify members of a family or subfamily, to the use of hidden Markov models that describe the conservation of residues over entire domains or whole proteins. To increase their value as protein classification tools, protein signatures from these 11 databases have been combined into one, powerful annotation tool: the InterPro database (http://www.ebi.ac.uk/interpro/) (Hunter et al., Nucleic Acids Res 37:D211-D215, 2009). InterPro is an open-source protein resource used for the automatic annotation of proteins, and is scalable to the analysis of entire new genomes through the use of a downloadable version of InterProScan, which can be incorporated into an existing local pipeline. InterPro provides structural information from PDB (Kouranov et al., Nucleic Acids Res 34:D302-D305, 2006), its classification in CATH (Cuff et al., Nucleic Acids Res 37:D310-D314, 2009) and SCOP (Andreeva et al., Nucleic Acids Res 36:D419-D425, 2008), as well as homology models from ModBase (Pieper et al., Nucleic Acids Res 37:D347-D354, 2009) and SwissModel (Kiefer et al., Nucleic Acids Res 37:D387-D392, 2009), allowing a direct comparison of the protein signatures with the available structural information. This chapter reviews the signature methods found in the InterPro database, and provides an overview of the InterPro resource itself.
A total of 30 Megasphaera elsdenii strains, selectively isolated from the feces of organically raised swine by using Me109 M medium, and one bovine strain were analyzed for tetracycline resistance ...genotypic and phenotypic traits. Tetracycline-resistant strains carried tet(O), tet(W), or a tet gene mosaic of tet(O) and tet(W). M. elsdenii strains carrying tet(OWO) genes exhibited the highest tetracycline MICs (128 to >256 microgram/ml), suggesting that tet(O)-tet(W) mosaic genes provide the selective advantage of greater tetracycline resistance for this species. Seven tet genotypes are now known for M. elsdenii, an archetype commensal anaerobe and model for tet gene evolution in the mammalian intestinal tract.
Human chromosome Xp11.3–Xp11.23 encompasses the map location for a growing number of diseases with a genetic basis or genetic component. These include several eye disorders, syndromic and ...nonsyndromic forms of X-linked mental retardation (XLMR), X-linked neuromuscular diseases and susceptibility loci for schizophrenia, type 1 diabetes, and Graves' disease. We have constructed an ∼2.7-Mb high-resolution physical map extending from DXS8026 to ELK1, corresponding to a genetic distance of ∼5.5 cM. A combination of chromosome walking and sequence-tagged site (STS)-content mapping resulted in an integrated framework and transcript map, precisely positioning 10 polymorphic microsatellites (one of which is novel), 16 ESTs, and 12 known genes (RP2, PCTK1, UHX1, UBE1, RBM10, ZNF157, SYN1, ARAF1, TIMP1, PFC, ELK1, UXT). The composite map is currently anchored with 89 STSs to give an average resolution of ∼1 STS every 30 kb. By a combination of EST database searches and in silico detection of UniGene clusters within genomic sequence generated from this template map, we have mapped several novel genes within this interval: a Na+/H+ exchanger (SLC9A7), at least two zincfinger transcription factors (KIAA0215 and Hs.68318), carbohydrate sulfotransferase-7 (CHST7), regucalcin (RGN), inactivation-escape-1 (INE1), the human ortholog of mouse neuronal protein 15.6, and four putative novel genes. Further genomic analysis enabled annotation of the sequence interval with 20 predicted pseudogenes and 21 UniGene clusters of unknown function. The combined PAC/BAC transcript map and YAC scaffold presented here clarifies previously conflicting data for markers and genes within the Xp11.3–Xp11.23 interval and provides a powerful integrated resource for functional characterization of this clonally unstable, yet gene-rich and clinically significant region of proximal Xp.
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