Identification and structure of a novel archaeal HypB for [NiFe] hydrogenase maturation

Sasaki, Daisuke; Watanabe, Satoshi; Matsumi, Rie; Shoji, Toshitaka; Yasukochi, Ayako; Tagashira, Kenta; Fukuda, Wakao; Kanai, Tamotsu; Atomi, Haruyuki; Imanaka, Tadayuki; Miki, Kunio

doi:10.1107/s0108767312096742

Cited by 4 publications

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“…Because nanomolar affinity is sufficient for specific binding for the Ni ion, HypB AT appears to function as a metallochaperone enhancer that increases the metal-binding affinity of the target metallochaperone (HypA). The concept of metallochaperone enhancer can account for the previous generic observations that disruption of the hypB AT gene resulted in a significant defect in [NiFe]-hydrogenase maturation (26). Without HypB AT , HypA cannot adopt the high-affinity form for Ni ions and, therefore, fails to acquire and transfer Ni ions.…”

Section: Discussionmentioning

confidence: 94%

“…HypB consists of a common GTPase domain and a less conserved metal-binding region (23)(24)(25). Recently, ATPase-type HypB (HypB AT , previously abbreviated as mmHypB) proteins were identified from Thermococcales (26). GTPase and ATPase types of HypB belong to the SIMIBI class NTPase family and share a similar architecture, despite their low sequence similarity (27).…”

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confidence: 99%

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Structural basis of a Ni acquisition cycle for [NiFe] hydrogenase by Ni-metallochaperone HypA and its enhancer

Watanabe

Kawashima

Nishitani

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The Ni atom at the catalytic center of [NiFe] hydrogenases is incorporated by a Ni-metallochaperone, HypA, and a GTPase/ATPase, HypB. We report the crystal structures of the transient complex formed between HypA and ATPase-type HypB (HypB AT ) with Ni ions. Transient association between HypA and HypB AT is controlled by the ATP hydrolysis cycle of HypB AT, which is accelerated by HypA. Only the ATP-bound form of HypB AT can interact with HypA and induces drastic conformational changes of HypA. Consequently, upon complex formation, a conserved His residue of HypA comes close to the N-terminal conserved motif of HypA and forms a Ni-binding site, to which a Ni ion is bound with a nearly square-planar geometry. The Ni binding site in the HypAB AT complex has a nanomolar affinity (K d = 7 nM), which is in contrast to the micromolar affinity (K d = 4 μM) observed with the isolated HypA. The ATP hydrolysis and Ni binding cause conformational changes of HypB AT , affecting its association with HypA. These findings indicate that HypA and HypB AT constitute an ATP-dependent Ni acquisition cycle for [NiFe]-hydrogenase maturation, wherein HypB AT functions as a metallochaperone enhancer and considerably increases the Ni-binding affinity of HypA.X-ray crystallography | metalloprotein | transient complex | metallochaperone A pproximately one-half of all cellular proteins require specific metal ions for proper function, which are delivered by specific metallochaperones (1, 2). However, the mechanisms of correct acquisition and delivery to target proteins of many metallochaperones remain poorly understood.[NiFe] hydrogenases harbor a complex metal cofactor, NiFe(CN) 2 CO, in their active sites (3). This cofactor catalyzes reversible H 2 production. The Ni atom in the NiFe(CN) 2 CO cofactor is bound to four thiolate groups, two of which also bridge the Fe(CN) 2 CO group (4, 5). NiFe(CN) 2 CO biosynthesis requires specific maturation machinery, in which six Hyp proteins (HypA-HypF) play key roles (6, 7). Four Hyp proteins (HypC-HypF) are involved in the biosynthesis and incorporation of the Fe(CN) 2 CO group (8-15). After Fe insertion, HypA and HypB insert the Ni ion into the hydrogenase large subunit (16).HypA is a Ni-metallochaperone that binds to a Ni ion with micromolar affinity (17-19), and its structure consists of a Ni-binding domain (NiBD) and a Zn-binding domain (ZnBD) (20, 21). The NiBD contains a highly conserved MHE motif that is essential for Ni binding at the N terminus (20,22). HypB consists of a common GTPase domain and a less conserved metal-binding region (23)(24)(25). Recently, ATPase-type HypB (HypB AT , previously abbreviated as mmHypB) proteins were identified from Thermococcales (26). GTPase and ATPase types of HypB belong to the SIMIBI class NTPase family and share a similar architecture, despite their low sequence similarity (27). HypA and HypB form a transient complex in the Ni insertion process (17,22,28,29). In the Escherichia coli system, Ni transfer occurs from HypB to HypA (30). However, the functi...

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Section: Discussionmentioning

confidence: 94%

mentioning

confidence: 99%

Structural basis of a Ni acquisition cycle for [NiFe] hydrogenase by Ni-metallochaperone HypA and its enhancer

Watanabe

Kawashima

Nishitani

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…HypB is involved in the latter stages of [NiFe]-hydrogenase maturation where it functions together with HypA and the peptidyl-prolyl cis/trans isomerase SlyD to deliver the nickel ion to the active site (Maier et al, 1993;Waugh & Boxer, 1986;Zhang et al, 2005). A recent structural study indicates that HypA proteins likely deliver nickel directly to the hydrogenase large subunit, as exemplified for T. kodakarensis (Watanabe et al, 2015); however, Functional conservation of the Hyp machinery T. kodakarensis lacks a classical HypB protein and instead has a functional homologue (Sasaki et al, 2013) that has an 'enhancer'-like role in facilitating delivery of nickel to the active site by the HypA protein. The data presented here nevertheless suggest that the HypB protein might recognize specific amino acid residues on the apo-large subunits of hydrogenases during maturation, and that D. mccartyi CBDB1 HypB lacks the ability to interact effectively with HyaB, the large subunit of Hyd-1.…”

Section: Discussionmentioning

confidence: 99%

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

Hartwig¹,

Thomas²,

Krumova³

et al. 2015

Microbiology

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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...

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“…The structures of HypB from T. kodakarensis, H. pylori, and Methanocaldococcus jannaschii have been determined by X-ray crystallography, and all three reveal a homodimer with the nucleotide-binding site near the interface (Figure 5). 65,92,95,96 Nucleotide and metal promote HypB homodimer formation, which has been observed in solution with micromolar dissociation constants. 95,97−99 The generation of a HypB mutant unable to form dimers in vitro reduces hydrogenase production in bacteria but does not abolish it entirely, so the specific importance of the HypB dimer in [NiFe]-hydrogenase biosynthesis is still not clear.…”

Section: ■ Iron Insertionmentioning

confidence: 93%

[NiFe]-Hydrogenase Maturation

2016

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[NiFe]-hydrogenases catalyze the reversible conversion of hydrogen gas into protons and electrons and are vital metabolic components of many species of bacteria and archaea. At the core of this enzyme is a sophisticated catalytic center comprising nickel and iron, as well as cyanide and carbon monoxide ligands, which is anchored to the large hydrogenase subunit through cysteine residues. The production of this multicomponent active site is accomplished by a collection of accessory proteins and can be divided into discrete stages. The iron component is fashioned by the proteins HypC, HypD, HypE, and HypF, which functionalize iron with cyanide and carbon monoxide. Insertion of the iron center signals to the metallochaperones HypA, HypB, and SlyD to selectively deliver the nickel to the active site. A specific protease recognizes the completed metal cluster and then cleaves the C-terminus of the large subunit, resulting in a conformational change that locks the active site in place. Finally, the large subunit associates with the small subunit, and the complete holoenzyme translocates to its final cellular position. Beyond this broad overview of the [NiFe]-hydrogenase maturation process, biochemical and structural studies are revealing the fundamental underlying molecular mechanisms. Here, we review recent work illuminating how the accessory proteins contribute to the maturation of [NiFe]-hydrogenase and discuss some of the outstanding questions that remain to be resolved.

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Identification and structure of a novel archaeal HypB for [NiFe] hydrogenase maturation

Cited by 4 publications

References 4 publications

Structural basis of a Ni acquisition cycle for [NiFe] hydrogenase by Ni-metallochaperone HypA and its enhancer

Structural basis of a Ni acquisition cycle for [NiFe] hydrogenase by Ni-metallochaperone HypA and its enhancer

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

[NiFe]-Hydrogenase Maturation

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