Assembly of Preactivation Complex for Urease Maturation in Helicobacter pylori

Fong, Y.H.; Wong, Ho Chun; Chuck, Chi Pang; Chen, Yu Wai; Sun, Hongzhe; Wong, Kam-Bo

doi:10.1074/jbc.m111.296830

Cited by 42 publications

(86 citation statements)

References 41 publications

(50 reference statements)

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“…Target protein expression, extraction, and purification in the bacterial system were performed as described (21) except that isopropyl β-D-thiogalactopyranoside (IPTG) with a final concentration of 1.0 mM was added when OD 600 reached 0.6, and 350 mM imidazole in lysis buffer was used to elute the target protein. The HisSUMO tag was removed with SUMO protease SenP1C (22).…”

Section: Methodsmentioning

confidence: 99%

ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses

Cheung

Miao

et al. 2016

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

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G proteins are involved in almost all aspects of the cellular regulatory pathways through their ability to bind and hydrolyze GTP. The YchF subfamily, interestingly, possesses the unique ability to bind both ATP and GTP, and is possibly an ancestral form of G proteins based on phylogenetic studies and is present in all kingdoms of life. However, the biological significance of such a relaxed ligand specificity has long eluded researchers. Here, we have elucidated the different conformational changes caused by the binding of a YchF homolog in rice (OsYchF1) to ATP versus GTP by X-ray crystallography. Furthermore, by comparing the 3D relationships of the ligand position and the various amino acid residues at the binding sites in the crystal structures of the apo-bound and ligand-bound versions, a mechanism for the protein's ability to bind both ligands is revealed. Mutation of the noncanonical G4 motif of the OsYchF1 to the canonical sequence for GTP specificity precludes the binding/hydrolysis of ATP and prevents OsYchF1 from functioning as a negative regulator of plant-defense responses, while retaining its ability to bind/hydrolyze GTP and its function as a negative regulator of abiotic stress responses, demonstrating the specific role of ATP-binding/hydrolysis in disease resistance. This discovery will have a significant impact on our understanding of the structure-function relationships of the YchF subfamily of G proteins in all kingdoms of life.G TP-binding proteins (G proteins) play important roles in diverse fundamental biological processes in living organisms, including signal transduction, cell division, development, intracellular transport, translation, and others (1, 2). The functions and working mechanisms of some ancestral G proteins, which are believed to play essential roles in cellular processes, are still unknown. One such group is the Obg family, which features an N-terminal glycine-rich sequence, followed by a G domain for nucleotide binding and a C-terminal TGS (ThrRS, GTPase, and SpoT) domain that has nucleic acid binding affinity (3).A unique subgroup within the Obg family, the YchF proteins, exhibit relaxed nucleotide-binding specificities (4-6). All G proteins contain a G domain (composed of the G1-G5 motifs) for GTP binding and hydrolysis (4). The G4 motif (canonical sequence: NKxD) confers the specificity for binding GTP (4). In YchFs, the noncanonical G4 sequence (NxxE) is correlated with the loss of nucleotide-binding specificities (4, 5). In some cases, ATP is the preferred ligand (4, 5). However, the physiological significance of the ancient and evolutionarily conserved YchF proteins having the relaxed binding specificities for both ATP and GTP is still unknown.Only a few reports touch on the biological functions of YchF proteins and most focus on human and microorganisms (7-9). The YchF proteins may play a role in iron utilization and regulation of the Ton system in Brucella melitensis (7), and in the growth of the procyclic forms of Trypanosoma cruzi (5). The human YchF homolog hOLA...

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Section: Methodsmentioning

confidence: 99%

ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses

Cheung

Miao

et al. 2016

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

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“…Heterologous expression of ureD or ureH in E. coli yields insoluble products (24,25); however, these problems were circumvented by different approaches. For the K. aerogenes protein, a maltose-binding protein (MBP) 2 fusion variant of UreD is soluble and functionally replaces the native protein (26).…”

Section: Ured/ureh: Scaffold For Recruitment Of Other Accessory Protementioning

confidence: 99%

“…For the K. aerogenes protein, a maltose-binding protein (MBP) 2 fusion variant of UreD is soluble and functionally replaces the native protein (26). In the case of H. pylori ureH, solubilization is achieved by coexpression with ureF, which provides a UreH:UreF complex (25). The structure of this complex (Protein Data Bank code 3SF5) and that of a UreH: UreF:UreG complex 3 reveal a novel ␤-helical fold for UreH, with 17 ␤-strands and two ␣-helices (Fig.…”

Section: Ured/ureh: Scaffold For Recruitment Of Other Accessory Protementioning

confidence: 99%

Biosynthesis of the Urease Metallocenter

Farrugia

Macomber

Hausinger

2013

Journal of Biological Chemistry

114

101

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Metalloenzymes often require elaborate metallocenter assembly systems to create functional active sites. The medically important dinuclear nickel enzyme urease provides an excellent model for studying metallocenter assembly. Nickel is inserted into the urease active site in a GTP-dependent process with the assistance of UreD/UreH, UreE, UreF, and UreG. These accessory proteins orchestrate apoprotein activation by delivering the appropriate metal, facilitating protein conformational changes, and possibly providing a requisite post-translational modification. The activation mechanism and roles of each accessory protein in urease maturation are the subject of ongoing studies, with the latest findings presented in this minireview.Metallocenters serve essential biological functions such as transferring electrons, stabilizing biomolecules, binding substrates, and catalyzing desirable reactions. Synthesis of these sites must be tightly controlled because simple competition between metals may lead to misincorporation with loss of function and because excess cytoplasmic concentrations of free metal ions can have toxic cellular effects. In many cases, cells have evolved elaborate metallocenter assembly systems that sequester metal cofactors from the cellular milieu, thus offering protection from adventitious reactions while ensuring the fidelity of metal insertion. In addition to maintaining metal homeostasis, these assembly systems facilitate protein conformational changes and active site modifications that are required for full enzymatic activity. Several metalloproteins have been investigated as models to understand the mechanisms and dynamics of active site assembly and the complex orchestrations of their metallocenter assembly systems. In this minireview, we discuss recent findings related to maturation of the nickel-containing enzyme urease.

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“…Metal chaperones are also used to control availability of nickel and cobalt (17), although the amount of these metals in the cell is kept low to avoid interference with iron. Anabaena has specific nickel chaperones for urease (UreE, alr0733) and hydrogenase (HypA/HupA, alr0699) biosynthesis (7,11,32). There are also several systems that buffer the intracellular iron pool in bacteria (1).…”

Section: Figmentioning

confidence: 99%

Zinc Starvation Response in a Cyanobacterium Revealed

Nies¹

2012

Journal of Bacteriology

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T he cyanobacterium Anabaena needs a variety of metals for its cellular biochemistry. The general problem is to supply all the different metals to the right enzymes, thereby avoiding toxic side reactions and interference between these metals. Anabaena has to deal with surplus metals but also with metal starvation, and this problem has to be solved for many environments, each with a different availability of metal cations.In this issue, Napolitano et al. (34) report a study in which they unraveled the zinc starvation response in Anabaena. Zinc is an important essential trace element in all organisms. More than 100 zinc-dependent or -binding proteins are known in Escherichia coli (39), e.g., a fructose-bisphosphate aldolase, DNA primase, carbonic anhydrase, alkaline phosphatase, and RNA polymerase. In mammals, including humans, zinc is required for proper brain function and for insulin and semen release and acts as cellular signal (30).The story told by Napolitano et al. (34) is wonderful to read. They identified the main transcriptional regulator to cope with zinc deficiency in Anabaena, Zur, and dozens of genes under Zur control. Most interesting among those were putative zinc-binding metallochaperones, other regulatory proteins, and, surprisingly, TonB-dependent outer membrane proteins, which may be involved in active transport of zinc or zinc chelates across the outer membrane. Anabaena might even synthesize and excrete a zinc chelator (a "zincophore"?), reminiscent of siderophores for iron or chalkophores for copper acquisition. To place this work (34) in a proper context, it is important to reiterate what is known about Anabaena, how this bacterium grows, why it needs metal cations, how zinc homeostasis in the context of general metal homeostasis might function, and what is new in the report by Napolitano et al. (34) (Fig. 1).Anabaena sp. PCC 7120 is a cyanobacterium. Without these organisms, life on this planet would be different. They are a major phylum of the superkingdom Bacteria, and their ecological niche is a physiological one, namely, oxygenic photosynthesis. Cyanobacteria are the only organisms able to perform this reaction, either as free-living organisms or as plastid endosymbionts (or slaves?) of eukaryotic cells. The product of oxygenic photosynthesis is molecular oxygen. With a half-cell potential (E o =) of ϩ816 mV (61), transfer of electrons from NADH (E o = ϭ Ϫ320 mV) releases about Ϫ238 kJ/mol under standard conditions (molar concentrations; pH ϭ 7), enough to conserve 3 ATP per electron pair transferred. No other respiratory electron acceptor allows such a high energy conservation by electron transfer-dependent phosphorylation. Production of molecular oxygen by cyanobacteria led to two major oxygenation events about 2.4 billion and 700 million years ago, which may have sparked evolution of eukaryotes and multicellular organisms, respectively (22,25,45). Thus, cyanobacteria have efficiently changed the biogeochemistry of our planet.Anabaena needs transition metals to perform oxygenic photos...

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Assembly of Preactivation Complex for Urease Maturation in Helicobacter pylori

Cited by 42 publications

References 41 publications

ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses

ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses

Biosynthesis of the Urease Metallocenter

Zinc Starvation Response in a Cyanobacterium Revealed

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