Heterologous complementation studies in Escherichia coli with the Hyp accessory protein machinery from Chloroflexi provide insight into [NiFe]-hydrogenase large subunit recognition by the HypC protein family

Abstract

Six Hyp maturation proteins (HypABCDEF) are conserved in micro-organisms that synthesize [NiFe]-hydrogenases (Hyd). Of these, the HypC chaperones interact directly with the apo-form of the catalytically active large subunit of Hyd enzymes and are believed to transfer the Fe(CN) 2 CO moiety of the bimetallic cofactor from the Hyp machinery to this large subunit. In E. coli, HypC is specifically required for maturation of Hyd-3 while its paralogue, HybG, is specifically required for Hyd-2 maturation; either HypC or HybG can mature Hyd-1.In this study, we demonstrate that the products of the hypABFCDE operon from the deeply branching hydrogen-dependent and obligate organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1 were capable of maturing and assembling active Hyd-1, Hyd-2 and Hyd-3 in an E. coli hyp mutant. Maturation of Hyd-1 was less efficient, presumably because HypB of E. coli was necessary to restore optimal enzyme activity. In a reciprocal maturation study, the highly O 2 -sensitive H 2 -uptake HupLS [NiFe]-hydrogenase from D. mccartyi CBDB1 was also synthesized in an active form in E. coli. Together, these findings indicated that HypC from D. mccartyi CBDB1 exhibits promiscuity in its large subunit interaction in E. coli. Based on these findings, we generated amino acid variants of E. coli HybG capable of partial recovery of Hyd-3-dependent H 2 production in a hypC hybG double null mutant. Together, these findings identify amino acid regions in HypC accessory proteins that specify interaction with the large subunits of hydrogenase and demonstrate functional compatibility of Hyp accessory protein machineries. INTRODUCTION[NiFe]-hydrogenases are widespread amongst archaeal and bacterial species (Vignais & Billoud, 2007). These enzymes can either oxidize H 2 to generate a chemiosmotic proton gradient via a membrane-based electron transfer chain, as well as provide a source of reducing power, or dissipate accumulated intracellular reductant by reducing protons to produce H 2 ; some perform both functions under certain physiological conditions (Lubitz et al., 2014;Pinske et al., 2015;Vignais & Billoud, 2007 (Ogata et al., 2015). The biosynthesis of this cofactor is complicated, requiring the combined activities of six Hyp accessory proteins, whose functions include synthesis and insertion of an Fe(CN) 2 CO moiety into the apo-catalytic subunit followed by introduction of the nickel ion (Böck et al., 2006;Forzi & Sawers, 2007). Subsequent to successful cofactor insertion, a C-terminal peptide present on the large subunit of most [NiFe]-hydrogenases is cleaved by a hydrogenase-specific protease and further assembly of the enzyme can then be completed (Böck et al., 2006;Pinske & Sawers, 2014).The Hyp proteins include HypA and the GTPase HypB, which, together with the peptidyl-prolyl cis/trans isomerase SlyD (Zhang et al., 2005), deliver the nickel ion; HypC, which is a small iron-and CO 2 -binding protein (Soboh et al., 2013); the FeS cluster protein HypD, which acts as a scaffold for assembl...

The Extended C-Terminal α-Helix of the HypC Chaperone Restricts Recognition of Large Subunit Precursors by the Hyp-Scaffold Machinery during [NiFe]-Hydrogenase Maturation in Escherichia coli

“…Importantly, these truncated variants of HypC retained their ability to mature Hyd-1 and Hyd-3. This finding also explains why the short (71 amino acids) HypC from D. mccartyi, when heterologously produced in the E. coli hypChybG double-null mutant SSH228, showed promiscuous maturation behaviour in that it was able to mature all 3 E. coli enzymes [Hartwig et al, 2015]. A C-terminal extension on the HypC orthologue, HupF, which co-exists with HypC in the cells of the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum, has been shown to stabilize maturation of the target hydrogenase large subunit towards oxygen [Albareda et al, 2012], suggesting that modification or extension of the C-terminus can add functional specificity to these chaperones.…”

“…2a), highlights the 3 variable regions, VR1 (amino acids 13-18 with reference to HypC Ec ), VR2 (33-43), and VR3 (75-90); the amino acid range of VR1 and of VR3 has been restricted somewhat compared to the previous definition [Hartwig et al, 2015]. The solution structure of HypC from E. coli has been determined [Wang et al, 2007] and the locations of VR1, VR2, and VR3 in the protein are shown in Fig.…”

“…A recent study identified 3 variable regions in the HypC and HybG proteins that could potentially be involved in the recognition of the Hyd large subunit precursor [Hartwig et al, 2015]. Amino acid residues in these variable regions of HybG that conferred upon it the ability to mature Hyd-3, at least partially, could be identified [Hartwig et al, 2015].…”

“…In VR3, 2 C-terminally truncated variants, HypC(Gstop) and HypC(Vstop), which removed 9 and 14 amino acids, respectively, from the C-terminus, were constructed to deliver proteins of a length similar to HybG (82 amino acids) and HypC (71 amino acids) from D. mccartyi, respectively. Genes encoding these variants were introduced into the ΔhypCΔhybG double null mutant, SHH228 [Hartwig et al, 2015], and after anaerobic fermentative growth in glucose minimal medium, Hyd-containing protein complexes in detergent-solubilized crude extracts derived from these strains were separated in non-denaturing polyacrylamide gel electrophoresis (PAGE), and subsequently stained for H 2 -oxidizing enzyme activity (Fig. 3a).…”

“…Amino acid residues in these variable regions of HybG that conferred upon it the ability to mature Hyd-3, at least partially, could be identified [Hartwig et al, 2015]. In this study, we have undertaken a detailed analysis of these 3 variable regions in HypC and analyzed their influence on the protein's ability to distinguish between the precursors of the large subunits of Hyd-1 (HyaB), Hyd-2 (HybC), and Hyd-3 (HycE).…”