Biosynthesis of Salmonella enterica [NiFe]-hydrogenase-5: probing the roles of system-specific accessory proteins

Abstract

Nonstandard abbreviationsHyd-1, hydrogenase-1; Hyd-2, hydrogenase-2; Hyd-5, hydrogenase-5; MB, methylene blue; MBH, membrane bound hydrogenase(s); Rh. leguminosarum, Rhizobium leguminosarum; R. eutropha, Ralstonia eutrophaThe final publication is available at Springer via http://dx.doi.org/10.1007/s00775-016-1385- As well as structural genes, the Hyd-5 operon also contains several accessory genes that are predicted to be involved in different stages of biosynthesis of the enzyme. In this work, deletions in the hydF, hydG and hydH genes have been constructed. The hydF gene encodes a protein related to Ralstonia eutropha HoxO, which is known to interact with the small subunit of a[NiFe]-hydrogenase. HydG is predicted to be a fusion of the R. eutropha HoxQ and HoxR proteins, both of which have been implicated in the biosynthesis of an O2-tolerant hydrogenase, and HydH is a homologue of R. eutropha HoxV, which is a scaffold for [NiFe] cofactor assembly. It is shown here that HydG and HydH play essential roles in Hyd-5 biosynthesis. Hyd-5 can be isolated and characterised from a hydF strain, indicating that HydF may not play the same vital role as the orthologous HoxO. This study therefore emphasises differences that can be observed when comparing the function of hydrogenase maturases in different biological systems.

Molecular Hydrogen Metabolism: a Widespread Trait of Pathogenic Bacteria and Protists

“…1a). The operon encoding this enzyme (hydABCDEFGHI) encodes several accessory proteins essential for hydrogenase maturation (244); these include two proteins implicated in synthesizing the [4Fe3S] cluster under oxic conditions (244-246) (Fig. 5).…”

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

“…Furthermore, hydrogenases are the subject of extensive research because of potential applications as catalysts in a hydrogen-fueled economy (2). Despite ongoing efforts to characterize these enzymes and their associated pathways, progress is hindered because current methods to measure hydrogenase activity in cells or extracts are laborious, are not amenable to high-throughput methods, and require specialized equipment (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). A rapid low-cost hydrogenase activity screening method would allow for the collection of system-level data and facilitate studies of the mechanisms of production, optimization of activity, and identification of inhibitors.…”

“…A variety of methods to monitor hydrogenase activity have been reported. These include spectrophotometric analysis of small-molecule dyes that are oxidized or reduced by hydrogenases, including benzyl viologen and methyl viologen (5)(6)(7)(8)(9)(10)17); electrochemical methods that measure the reduction potentials or molecular hydrogen concentrations (11)(12)(13)(14); and GC to detect changes in the levels of hydrogen gas (15,16). The growth conditions, sample processing, and specialized equipment required for these methods are not amenable to highthroughput screening, which is typically performed on multiwell plates by robotic equipment.…”

“…For example, hydrogenase activity is often analyzed in cell lysates or with purified proteins prepared using multistep protocols from bacteria grown under anaerobic conditions. Similarly, recently described bacterial assays involve a centrifugation step and/or buffers saturated with externally sourced hydrogen gas and artificial exclusion of oxygen (11,31).…”