A whole-cell, high-throughput hydrogenase assay to identify factors that modulate [NiFe]-hydrogenase activity

Lacasse, Michael J.; Sebastiampillai, Stephanie; Côté, Jean-Philippe; Hodkinson, Nicholas; Brown, Eric D.; Zamble, Deborah B.

doi:10.1074/jbc.ra119.008101

Cited by 13 publications

(14 citation statements)

References 76 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recently, fhlA and the genes encoding the components of FHL-2, were found to be likely to contribute to mouse colonization in uropathogenic Proteus mirabilis by the genome-wide study 21 . In view that enzymes and the maturation machinery required for production of FHL hydrogenases are completely different from human proteins 22 , these pathways would be promising targets for the development of new antimicrobial strategies.…”

mentioning

confidence: 99%

Deacidification by FhlA-dependent hydrogenase is involved in urease activity and urinary stone formation in uropathogenic Proteus mirabilis

Lin

Liaw

2020

Sci Rep

View full text Add to dashboard Cite

Proteus mirabilis is an important uropathogen, featured with urinary stone formation. Formate hydrogenlyase (FHL), consisting of formate dehydrogenase H and hydrogenase for converting proton to hydrogen, has been implicated in virulence. In this study, we investigated the role of P. mirabilis FHL hydrogenase and the FHL activator, FhlA. fhlA and hyfG (encoding hydrogenase large subunit) displayed a defect in acid resistance. fhlA and hyfG mutants displayed a delay in medium deacidification compared to wild-type and ureC mutant failed to deacidify the medium. In addition, loss of fhlA or hyfG decreased urease activity in the pH range of 5–8. The reduction of urease activities in fhlA and hyfG mutants subsided gradually over the pH range and disappeared at pH 9. Furthermore, mutation of fhlA or hyfG resulted in a decrease in urinary stone formation in synthetic urine. These indicate fhlA- and hyf-mediated deacidification affected urease activity and stone formation. Finally, fhlA and hyfG mutants exhibited attenuated colonization in mice. Altogether, we found expression of fhlA and hyf confers medium deacidification via facilitating urease activity, thereby urinary stone formation and mouse colonization. The link of acid resistance to urease activity provides a potential strategy for counteracting urinary tract infections by P. mirabilis.

show abstract

mentioning

confidence: 99%

Deacidification by FhlA-dependent hydrogenase is involved in urease activity and urinary stone formation in uropathogenic Proteus mirabilis

Lin

Liaw

2020

Sci Rep

View full text Add to dashboard Cite

show abstract

“…However, current methods still have low throughput for measuring hydrogenase activity, which is a major limit for research studies and biotechnological applications. Lacasse et al [144] developed a whole-cell colorimetric hydrogenase activity assay suitable for high-throughput applications based on the reduction of benzyl viologen with a very high specificity. This assay is a promising method for future screenings of growth conditions and factors facilitating the heterologous production of active [NiFe] hydrogenases.…”

Section: Recombinant Hydrogenase Production In the Presence Of Specifmentioning

confidence: 99%

Heterologous Hydrogenase Overproduction Systems for Biotechnology—An Overview

Fan

Neubauer

Lenz

et al. 2020

IJMS

View full text Add to dashboard Cite

Hydrogenases are complex metalloenzymes, showing tremendous potential as H2-converting redox catalysts for application in light-driven H2 production, enzymatic fuel cells and H2-driven cofactor regeneration. They catalyze the reversible oxidation of hydrogen into protons and electrons. The apo-enzymes are not active unless they are modified by a complicated post-translational maturation process that is responsible for the assembly and incorporation of the complex metal center. The catalytic center is usually easily inactivated by oxidation, and the separation and purification of the active protein is challenging. The understanding of the catalytic mechanisms progresses slowly, since the purification of the enzymes from their native hosts is often difficult, and in some case impossible. Over the past decades, only a limited number of studies report the homologous or heterologous production of high yields of hydrogenase. In this review, we emphasize recent discoveries that have greatly improved our understanding of microbial hydrogenases. We compare various heterologous hydrogenase production systems as well as in vitro hydrogenase maturation systems and discuss their perspectives for enhanced biohydrogen production. Additionally, activities of hydrogenases isolated from either recombinant organisms or in vivo/in vitro maturation approaches were systematically compared, and future perspectives for this research area are discussed.

show abstract

“…Under laboratory conditions, the enzyme appeared to be neither transcribed nor enzymatically active [14][15][16], although there is some evidence for a physiological role in H 2 metabolism under some specific environmental conditions [17,18]. In addition, disruption of Hyd-4 genes alone did not affect overall H 2 production by E. coli [19,20], again indicating that cellular Hyd-4 activity was very low or absent under the conditions tested. It is also clear that the E. coli hyfABCDEFGHIJR-focB operon does not encode any homologue of HycI (Fig.…”

mentioning

confidence: 99%

Activation of a [NiFe]-hydrogenase-4 isoenzyme by maturation proteases

et al. 2020

View full text Add to dashboard Cite

Maturation of [NiFe]-hydrogenases often involves specific proteases responsible for cleavage of the catalytic subunits. Escherichia coli HycI is the protease dedicated to maturation of the Hydrogenase-3 isoenzyme, a component of formate hydrogenlyase-1. In this work, it is demonstrated that a Pectobacterium atrosepticum HycI homologue, HyfK, is required for hydrogenase-4 activity, a component of formate hydrogenlyase-2, in that bacterium. The P. atrosepticum ΔhyfK mutant phenotype could be rescued by either P. atrosepticum hyfK or E. coli hycI on a plasmid. Conversely, an E. coli ΔhycI mutant was complemented by either E. coli hycI or P. atrosepticum hyfK in trans. E. coli is a rare example of a bacterium containing both hydrogenase-3 and hydrogenase-4, however the operon encoding hydrogenase-4 has no maturation protease gene. This work suggests HycI should be sufficient for maturation of both E. coli formate hydrogenlyases, however no formate hydrogenlyase-2 activity was detected in any E. coli strains tested here.

show abstract

A whole-cell, high-throughput hydrogenase assay to identify factors that modulate [NiFe]-hydrogenase activity

Cited by 13 publications

References 76 publications

Deacidification by FhlA-dependent hydrogenase is involved in urease activity and urinary stone formation in uropathogenic Proteus mirabilis

Deacidification by FhlA-dependent hydrogenase is involved in urease activity and urinary stone formation in uropathogenic Proteus mirabilis

Heterologous Hydrogenase Overproduction Systems for Biotechnology—An Overview

Activation of a [NiFe]-hydrogenase-4 isoenzyme by maturation proteases

Contact Info

Product

Resources

About