Effects of Metal on the Biochemical Properties of Helicobacter pylori HypB, a Maturation Factor of [NiFe]-Hydrogenase and Urease

Abstract

The biosyntheses of the [NiFe]-hydrogenase and urease enzymes in Helicobacter pylori require several accessory proteins for proper construction of the nickel-containing metallocenters. The hydrogenase accessory proteins HypA and HypB, a GTPase, have been implicated in the nickel delivery steps of both enzymes. In this study, the metal-binding properties of H. pylori HypB were characterized, and the effects of metal binding on the biochemical behavior of the protein were examined. The protein can bind stoichiometric amounts of Zn(II) or Ni(II), each with nanomolar affinity. Mutation of Cys106 and His107, which are located between two major GTPase motifs, results in undetectable Ni(II) binding, and the Zn(II) affinity is weakened by 2 orders of magnitude. These two residues are also required for the metal-dependent dimerization observed in the presence of Ni(II) but not Zn(II). The addition of metals to the protein has distinct impacts on GTPase activity, with zinc significantly reducing GTP hydrolysis to below detectable levels and nickel only slightly altering the k cat and K m of the reaction. The regulation of HypB activities by metal binding may contribute to the maturation of the nickel-containing enzymes.

Relationship between Ni(II) and Zn(II) Coordination and Nucleotide Binding by the Helicobacter pylori [NiFe]-Hydrogenase and Urease Maturation Factor HypB

“…HpHypB Expression and Purification-The construction of the WT HpHypB-pET24b expression vector was described previously (30). The H107A and C142S mutations were introduced into the HpHypB-pET24b construct by QuikChange PCR mutagenesis (Stratagene) with Pfu polymerase using the following primers: HpHypB C142S forward, 5Ј-CGTGGGGAATTTGGTTTcCCCCTCAAGCTATAATCTAG-3Ј; HpHypB C142S reverse, 5Ј-CTAGATTATAGCTTGAGGGGgAAACCAAA-TTCCCCACG-3Ј; HpHypB H107A forward, 5Ј-CCACCGGCGAAGCATGCgcTTTGGAAGCGAGC-3Ј; HpHypB H107A reverse, 5Ј-GCTCGCTTCCAAAgcGCATGCTTCGCCGGT-GG-3Ј.…”

“…All plasmids were sequenced (ACGT Corp., Toronto, Ontario, Canada) in the forward and reverse directions by using the T7 promoter and terminator primers. The proteins were overexpressed and purified as reported previously (30). A sample of each protein was sent for electrospray ionization mass spectrometry (Department of Chemistry, University of Toronto), and the determined molecular masses of the WT, H107A, and C142S proteins corresponded to their calculated molecular masses (MM) Preparation of Proteins-Reduced apoprotein was produced by incubating HpHypB with 12.5 mM EDTA and 30 mM TCEP in a Coy anaerobic glovebox at 4°C for 48 h. The TCEP and EDTA were removed by passing the protein sequentially over two PD-10 gel filtration columns (GE Healthcare) equilibrated with protein buffer (25 mM HEPES, pH 7.6 and 100 mM KCl) in the glove box.…”

“…Mutation of several of the conserved residues that serve as metal ligands in Escherichia coli HypB (EcHypB) 2 abrogates hydrogenase production in this organism, indicating the critical role of this metal-binding site of HypB during metallocenter assembly (31). This is the only metal site identified in H. pylori HypB (HpHypB) that can bind one nickel ion with a K d of 150 nM (30). However, zinc binds with an affinity that is about 2 orders of magnitude tighter, prompting speculation about the identity of the physiologically relevant metal (30).…”

“…This is the only metal site identified in H. pylori HypB (HpHypB) that can bind one nickel ion with a K d of 150 nM (30). However, zinc binds with an affinity that is about 2 orders of magnitude tighter, prompting speculation about the identity of the physiologically relevant metal (30). The only crystal structure of metal-loaded HypB is of the protein from Methanocaldococcus jannaschii, which revealed a dinuclear asymmetric zinc site at the interface of a GTP␥S-bound protein dimer (32).…”

“…The only crystal structure of metal-loaded HypB is of the protein from Methanocaldococcus jannaschii, which revealed a dinuclear asymmetric zinc site at the interface of a GTP␥S-bound protein dimer (32). The observation that Zn(II) binding to HpHypB abolishes any detectable GTPase activity whereas Ni(II) binding has a more subtle impact prompted speculation that this metal site serves a regulatory role during hydrogenase maturation (30), but little was known how these key cofactors of HypB affect each other.…”