scholarly journals The dual-function chaperone HycH improves assembly of the formate hydrogenlyase complex

2017 ◽  
Vol 474 (17) ◽  
pp. 2937-2950 ◽  
Author(s):  
Ute Lindenstrauß ◽  
Philipp Skorupa ◽  
Jennifer S. McDowall ◽  
Frank Sargent ◽  
Constanze Pinske
Keyword(s):  
Escherichia Coli ◽  
Electron Transfer ◽  
Mutant Strain ◽  
Protein Complexes ◽  
Dual Function ◽  
Formate Hydrogenlyase ◽  
Accessory Function ◽  
Membrane Bound ◽  
Hydrogenase 3 ◽  
Influence Activity

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 Escherichia coli and is composed of seven subunits mostly encoded within the hycA-I 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.

scholarly journals A dual function of TMEM70 in OXPHOS: assembly of complexes I and V

2019 ◽  
Author(s):  
Laura Sánchez-Caballero ◽  
Dei M. Elurbe ◽  
Fabian Baertling ◽  
Sergio Guerrero-Castillo ◽  
Mariel van den Brand ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Congenital Defects ◽  
Dual Function ◽  
Inner Mitochondrial Membrane ◽  
Independent Evidence ◽  
Membrane Recruitment ◽  
Oxphos System ◽  
Membrane Bound ◽  
Assembly Intermediate ◽  
The Stability

AbstractProtein complexes from the oxidative phosphorylation (OXPHOS) system are assembled with the help of proteins called assembly factors. We here delineate the function of the inner mitochondrial membrane protein TMEM70, in which mutations have been linked to OXPHOS deficiencies, using a combination of BioID, complexome profiling and coevolution analyses. TMEM70 interacts with complex I and V and for both complexes the loss of TMEM70 results in the accumulation of an assembly intermediate followed by a reduction of the next assembly intermediate in the pathway. This indicates that TMEM70 has a role in the stability of membrane-bound subassemblies or in the membrane recruitment of subunits into the forming complex. Independent evidence for a role of TMEM70 in OXPHOS assembly comes from evolutionary analyses. The TMEM70/TMEM186/TMEM223 protein family, of which we show that TMEM186 and TMEM223 are mitochondrial in human as well, only occurs in species with OXPHOS complexes. Our results validate the use of combining complexomics with BioID and evolutionary analyses in elucidating congenital defects in protein complex assembly.

Differential effects of a molybdopterin synthase sulfurylase (moeB) mutation on Escherichia coli molybdoenzyme maturation

2002 ◽  
Vol 80 (4) ◽  
pp. 435-443 ◽  
Author(s):  
Damaraju Sambasivarao ◽  
Raymond J Turner ◽  
Peter T Bilous ◽  
Richard A Rothery ◽  
Gillian Shaw ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Mutant Strain ◽  
Physiological Function ◽  
Signal Sequence ◽  
Molybdenum Cofactor ◽  
Wild Type ◽  
Dmso Reductase ◽  
E Coli ◽  
Membrane Bound

We have generated a chromosomal mutant of moeB (moeBA228T) that demonstrates limited molybdenum cofactor (molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD)) availability in Escherichia coli and have characterized its effect on the maturation and physiological function of two well-characterized respiratory molybdoenzymes: the membrane-bound dimethylsulfoxide (DMSO) reductase (DmsABC) and the membrane-bound nitrate reductase A (NarGHI). In the moeBA228T mutant strain, E. coli F36, anaerobic respiratory growth is possible on nitrate but not on DMSO, indicating that cofactor insertion occurs into NarGHI but not into DmsABC. Fluorescence analyses of cofactor availability indicate little detectable cofactor in the moeBA228T mutant compared with the wild-type, suggesting that NarGHI is able to scavenge limiting cofactor, whereas DmsABC is not. MoeB functions to sulfurylate MoaD, and in the structure of the MoeB–MoaD complex, Ala-228 is located in the interface region between the two proteins. This suggests that the moeBA228T mutation disrupts the interaction between MoeB and MoaD. In the case of DmsABC, despite the absence of cofactor, the twin-arginine signal sequence of DmsA is cleaved in the moeBA228T mutant, indicating that maturation of the holoenzyme is not cofactor-insertion dependent.Key words: mdybdenum cofactor, DMSO reductase, nitrate reductase.

scholarly journals Involvement of hydrogenases in the formation of highly catalytic Pd(0) nanoparticles by bioreduction of Pd(II) using Escherichia coli mutant strains

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2630-2640 ◽  
Author(s):  
Kevin Deplanche ◽  
Isabelle Caldelari ◽  
Iryna P. Mikheenko ◽  
Frank Sargent ◽  
Lynne E. Macaskie
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Membrane Separation ◽  
Reductase Activity ◽  
Reduction Process ◽  
Hydrogenase Activity ◽  
Catalytic Subunits ◽  
E Coli ◽  
Economic Production ◽  
Membrane Bound

Escherichia coli produces at least three [NiFe] hydrogenases (Hyd-1, Hyd-2 and Hyd-3). Hyd-1 and Hyd-2 are membrane-bound respiratory isoenzymes with their catalytic subunits exposed to the periplasmic side of the membrane. Hyd-3 is part of the cytoplasmically oriented formate hydrogenlyase complex. In this work the involvement of each of these hydrogenases in Pd(II) reduction under acidic (pH 2.4) conditions was studied. While all three hydrogenases could contribute to Pd(II) reduction, the presence of either periplasmic hydrogenase (Hyd-1 or Hyd-2) was required to observe Pd(II) reduction rates comparable to the parent strain. An E. coli mutant strain genetically deprived of all hydrogenase activity showed negligible Pd(II) reduction. Electron microscopy suggested that the location of the resulting Pd(0) deposits was as expected from the subcellular localization of the particular hydrogenase involved in the reduction process. Membrane separation experiments established that Pd(II) reductase activity is membrane-bound and that hydrogenases are required to initiate Pd(II) reduction. The catalytic activity of the resulting Pd(0) nanoparticles in the reduction of Cr(VI) to Cr(III) varied according to the E. coli mutant strain used for the initial bioreduction of Pd(II). Optimum Cr(VI) reduction, comparable to that observed with a commercial Pd catalyst, was observed when the bio-Pd(0) catalytic particles were prepared from a strain containing an active Hyd-1. The results are discussed in the context of economic production of novel nanometallic catalysts.

Identification and partial characterization of an Escherichia coli mutant with altered hydrogenase activity

1980 ◽  
Vol 58 (4) ◽  
pp. 361-367 ◽  
Author(s):  
Bernard R. Glick ◽  
Patrick Y. Wang ◽  
Henry Schneider ◽  
William G. Martin
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Filter Paper ◽  
Specific Activity ◽  
Anaerobic Growth ◽  
Hydrogenase Activity ◽  
Wild Type ◽  
Coli Mutant ◽  
In The Wild ◽  
Membrane Bound

An Escherichia coli mutant strain with altered hydrogenase activity was isolated using a filter paper assay. This assay depends on the ability of hydrogenase-containing microorganisms to reduce methyl viologen impregnated in filter paper, producing purple-colored colonies in the presence of hydrogen. Membrane-bound and cytoplasmic hydrogenase activities of wild-type and mutant strains were compared by amperometric measurement of hydrogen production. The cytoplasmic activities of mutant and wild type were comparable. The membrane-bound activity was lower in the mutant than in the wild type. Upon addition of detergent to the membrane fraction the specific activity of the enzyme from the mutant strain increased so that it equalled that of the wild type. The mutant requires an exogenous electron acceptor for anaerobic growth providing evidence for the function of the hydrogenase in anaerobic growth.

scholarly journals An electron transfer competent structural ensemble of membrane-bound cytochrome P450 1A1 and cytochrome P450 oxidoreductase

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Goutam Mukherjee ◽  
Prajwal P. Nandekar ◽  
Rebecca C. Wade
Keyword(s):  
Electron Transfer ◽  
Cytochrome P450 ◽  
Drug Targets ◽  
Catalytic Domain ◽  
Cytochrome P450 1A1 ◽  
P450 Oxidoreductase ◽  
Distal Side ◽  
Cytochrome P450 Oxidoreductase ◽  
Membrane Bound ◽  
Transient Interactions

AbstractCytochrome P450 (CYP) heme monooxygenases require two electrons for their catalytic cycle. For mammalian microsomal CYPs, key enzymes for xenobiotic metabolism and steroidogenesis and important drug targets and biocatalysts, the electrons are transferred by NADPH-cytochrome P450 oxidoreductase (CPR). No structure of a mammalian CYP–CPR complex has been solved experimentally, hindering understanding of the determinants of electron transfer (ET), which is often rate-limiting for CYP reactions. Here, we investigated the interactions between membrane-bound CYP 1A1, an antitumor drug target, and CPR by a multiresolution computational approach. We find that upon binding to CPR, the CYP 1A1 catalytic domain becomes less embedded in the membrane and reorients, indicating that CPR may affect ligand passage to the CYP active site. Despite the constraints imposed by membrane binding, we identify several arrangements of CPR around CYP 1A1 that are compatible with ET. In the complexes, the interactions of the CPR FMN domain with the proximal side of CYP 1A1 are supplemented by more transient interactions of the CPR NADP domain with the distal side of CYP 1A1. Computed ET rates and pathways agree well with available experimental data and suggest why the CYP–CPR ET rates are low compared to those of soluble bacterial CYPs.

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