Cloning, purification and preliminary crystallographic analysis of the complex of<i>Helicobacter pylori</i>α-carbonic anhydrase with acetazolamide

Modak, Joyanta K.; Revitt-Mills, Sarah A.; Roujeinikova, Anna

doi:10.1107/s1744309113026146

Cited by 9 publications

(8 citation statements)

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“…We have previously shown that when subjected to gel-filtration, HpαCA eluted as a single peak with an apparent molecular weight of approximately 50 kDa, indicating that HpαCA forms a dimer in solution [ 29 ], in line with previous reports of the dimeric state of αCAs from S . yellowstonense [ 35 ] and N .…”

Section: Resultssupporting

confidence: 85%

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Structural Basis for the Inhibition of Helicobacter pylori α-Carbonic Anhydrase by Sulfonamides

et al. 2015

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Periplasmic α-carbonic anhydrase of Helicobacter pylori (HpαCA), an oncogenic bacterium in the human stomach, is essential for its acclimation to low pH. It catalyses the conversion of carbon dioxide to bicarbonate using Zn(II) as the cofactor. In H. pylori, Neisseria spp., Brucella suis and Streptococcus pneumoniae this enzyme is the target for sulfonamide antibacterial agents. We present structural analysis correlated with inhibition data, on the complexes of HpαCA with two pharmacological inhibitors of human carbonic anhydrases, acetazolamide and methazolamide. This analysis reveals that two sulfonamide oxygen atoms of the inhibitors are positioned proximal to the putative location of the oxygens of the CO2 substrate in the Michaelis complex, whilst the zinc-coordinating sulfonamide nitrogen occupies the position of the catalytic water molecule. The structures are consistent with acetazolamide acting as site-directed, nanomolar inhibitors of the enzyme by mimicking its reaction transition state. Additionally, inhibitor binding provides insights into the channel for substrate entry and product exit. This analysis has implications for the structure-based design of inhibitors of bacterial carbonic anhydrases.

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Section: Resultssupporting

confidence: 85%

“…HpαCA is a homo-dimer in solution with a subunit mass of approximately 27 kDa and was prepared as described [ 29 ]. AAZ and MZA were purchased from Sigma-Aldrich.…”

Section: Methodsmentioning

confidence: 99%

Structural Basis for the Inhibition of Helicobacter pylori α-Carbonic Anhydrase by Sulfonamides

et al. 2015

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“…In addition to cag ‐related structures, the crystal structures of the H. pylori UreF/UreG/UreH urease accessory complex , of one of its major adhesins SabA , of DprA, a DNA‐binding protein , and a preliminary structure of the carboanhydrase enzyme became available lately, which broaden our molecular knowledge on colonization and pathogenesis‐associated factors of H. pylori . The SabA structure, which shares similarity with tetratricopeptide protein folds, covers the extracellularly exposed domain of the adhesin SabA which links H. pylori to the human sialylated Lewis X cell surface glycan.…”

Section: Structural Biology Of Helicobacter Pylori Pathogenesismentioning

confidence: 99%

Pathogenesis of Helicobacter pylori Infection

Bernard

Josenhans

2014

Helicobacter

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Helicobacter pylori relies on multiple colonization and virulence factors to persist in the human stomach for life. In addition, these factors can be modulated and vary to suit the ever-changing environment within the host individual. This article outlines the novel developments in this field of research during the past year, highlighting the cag pathogenicity island, VacA, c-glutamyl-transpeptidase as well as including recent advances in protein structure, bacteria-host interaction, and the role of stomach microbiota. Helicobacter pylori ColonizationIt is well established that flagella are essential for the motility of Helicobacter pylori and are particularly crucial at early stages of infection. In a recent study [1], seeking to learn more about the proteins that, as VacA, are secreted via the autotransporter (Type V) pathway, it has been demonstrated that one of the three VacA-like proteins, namely flagella-associated autotransporter A (FaaA), is enriched in the sheath covering the flagella filament and their terminal bulb, unlike the other two that localize on the surface of the bacteria. Interestingly, mutants lacking the faaA gene show a mislocalization of flagella, which also appear more fragile. This characteristic probably depends on the effect that FaaA exerts on the stability of the major flagellar protein FlaA that, despite an increased mRNA expression, is reduced in faaA deletion mutants. The latter show a reduced motility in vitro but, more importantly, a reduced ability in colonizing the stomach of mice [1].In conjunction with motility, bacterial shape is an important persistence factor. Recently, novel determinants for H. pylori cell shape, which, among others, depend on factors involved in peptidoglycan (PG) biosynthesis and degradation, were identified using a transposon mutagenesis-based approach coupled to flow cytometry [2]. Enriched clones contained insertions in known PG endopeptidases and novel genes involved in PG biosynthesis initiation and PG cleavage.A second step in colonization is supposed to be adherence. Earlier on, it had been reported that H. pylori can bind to trefoil factor 1 (TFF1), one of several peptides released during tissue trauma. Novel data on TFF1 now suggest that H. pylori binding to TFF1 dimer and to TFF1-overproducing gastric cells is dependent on copper availability in the environment [3]. It remains unclear how these in vitro findings translate to the in vivo situation in the human stomach.Similarly, further investigations are required to demonstrate the role in vivo of the recently identified twinarginine translocation system (Tat) of H. pylori [4]. In H. pylori, this system was predicted to be composed of three proteins, TatA, TatB, and TatC, and to permit the translocation of four substrates: the catalase accessory protein, KapA; the hydrogenase small-subunit protein HydA; a putative biotin sulfoxide reductase BisC; and the cytochrome oxidase Rieske subunit protein FbcF. tatB deletion was found to be compatible with H. pylori survival, while deletion o...

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“…In this context, the design of selective inhibitors for HpCA could help to better clarify the effective role played by this enzyme in the control of pH and could eventually open the way to improved classes of therapeutics against the bacterium. The crystallization of HpCA with acetazolamide in a orthorhombic crystal form (Modak et al, 2013) has been published and its structure complexed with the clinically used compound sulfonamide has recently been published (Modakh et al, 2015). In this paper, the crystal structure of HpCA is presented and the possible implications for its function are discussed.…”

Section: Introductionmentioning

confidence: 99%

Structure of α-carbonic anhydrase from the human pathogenHelicobacter pylori

Compostella

Berto

Vallese

et al. 2015

Acta Crystallogr F Struct Biol Commun

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The crystal structure of α-carbonic anhydrase, an enzyme present in the periplasm of Helicobacter pylori, a bacterium that affects humans and that is responsible for several gastric pathologies, is described. Two enzyme monomers are present in the asymmetric unit of the monoclinic space group P21, forming a dimer in the crystal. Despite the similarity of the enzyme structure to those of orthologues from other species, the H. pylori protein has adopted peculiar features in order to allow the bacterium to survive in the difficult environment of the human stomach. In particular, the crystal structure shows how the bacterium has corrected for the mutation of an essential amino acid important for catalysis using a negative ion from the medium and how it localizes close to the inner membrane in the periplasm. Since carbonic anhydrase is essential for the bacterial colonization of the host, it is a potential target for antibiotic drugs. The definition of the shape of the active-site entrance and cavity constitutes a basis for the design of specific inhibitors.

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Cloning, purification and preliminary crystallographic analysis of the complex ofHelicobacter pyloriα-carbonic anhydrase with acetazolamide

Cited by 9 publications

References 40 publications

Structural Basis for the Inhibition of Helicobacter pylori α-Carbonic Anhydrase by Sulfonamides

Structural Basis for the Inhibition of Helicobacter pylori α-Carbonic Anhydrase by Sulfonamides

Pathogenesis of Helicobacter pylori Infection

Structure of α-carbonic anhydrase from the human pathogenHelicobacter pylori

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