Identification of residues involved in nucleotidyltransferase activity of JHP933 from helicobacter pyloriby site-directed mutagenesis

Helicobacter pylori is a well-known bacterial pathogen involved in the development of peptic ulcer, gastric adenocarcinoma and other forms of gastric cancer. Evidence has suggested that certain strain-specific genes in the plasticity region may play key roles in the pathogenesis of H. pylori-associated gastroduodenal diseases. Therefore there is considerable interest in the strain-specific genes located in the plasticity regions of H. pylori. JHP933 is encoded by the gene in the plasticity region of H. pylori strain J99. Recently, the crystal structure of JHP933 has confirmed it as a nucleotidyltransferase (NTase) superfamily protein and a putative active site has been proposed. However, no evidence from direct functional assay has been presented to confirm the active site and little is known about the functional mechanism of JHP933. Here, through superimposition with Cid1/NTP complex structures, we modelled the complex structures of JHP933 with different NTPs. Based on the models and using rational site-directed mutagenesis combined with enzymatic activity assays, we confirm the active site and identify several residues important for the nucleotidyl transferring function of JHP933. Furthermore, mutations of these active site residues result in the abolishment of the nucleotidyltransferase activity of JHP933. This work provides preliminary insight into the molecular mechanism underlying the pathophysiological role in H. pylori infection of JHP933 as a novel NTase superfamily protein

Crystal structure of thermostable alkylsulfatase SdsAP from Pseudomonas sp. S9

Correspondence: Yunkun Wu ([email protected]) A novel alkylsulfatase from bacterium Pseudomonas sp. S9 (SdsAP) was identified as a thermostable alkylsulfatases (type III), which could hydrolyze the primary alkyl sulfate such as sodium dodecyl sulfate (SDS). Thus, it has a potential application of SDS biodegradation. The crystal structure of SdsAP has been solved to a resolution of 1.76Å and reveals that SdsAP contains the characteristic metallo-β-lactamase-like fold domain, dimerization domain, and C-terminal sterol carrier protein type 2 (SCP-2)-like fold domain. Kinetic characterization of SdsAP to SDS by isothermal titration calorimetry (ITC) and enzymatic activity assays of constructed mutants demonstrate that Y246 and G263 are important residues for its preference for the hydrolysis of 'primary alkyl' chains, confirming that SdsAP is a primary alkylsulfatase

Active site conservation and substrate binding of JHP933, LinA and LinB.

<p>The C atoms of active site residues are shown in ball-and-stick representation and distinctively colored: lime for JHP933, magenta for LinA (4E8J), and cyan for LinB (3JZ0). The substrate Mg²⁺ ions, as cyan spheres, AMPCPP and clindamycin, in yellow, are from LinB complex structure.</p

Sequence and secondary structure comparison of JHP933 with structurally related LinA.

<p>The secondary structures of JHP93 (top row) are labeled in lime and LinA from <i>S. haemolyticu</i> (bottom row) in magenta. The conserved active site motifs involved in catalysis ([DE]h[DE]h, h[DE]h) and substrate binding (hG) of NTase superfamily are shadowed in gray.</p

Overall structure of JHP933.

<p>Ribbon diagram of the JHP933 structure, N-terminal core domain is colored in lime and C-terminal tail domain in cyan. α-helices are labelled with α, β-strands are labelled with β, and 3₁₀ helices are labelled with η.</p

Crystal Structure Confirmation of JHP933 as a Nucleotidyltransferase Superfamily Protein from <i>Helicobacter pylori</i> Strain J99

<div><p><i>Helicobacter pylori</i> is a well-known pathogen involved in the development of peptic ulcer, gastric adenocarcinoma and other forms of gastric cancer. Recently, there has been more considerable interest in strain-specific genes located in plasticity regions with great genetic variability. However, little is known about many of these genes. Studies suggested that certain genes in this region may play key roles in the pathogenesis of <i>H. pylori</i>-associated gastroduodenal diseases. JHP933, a conserved putative protein of unknown function, is encoded by the gene in plasticity region of <i>H. pylori</i> strain J99. Here we have determined the structure of JHP933. Our work demonstrates that JHP933 is a nucleotidyltransferase superfamily protein with a characteristic αβαβαβα topology. A superposition demonstrates overall structural homology of the JHP933 N-terminal fragment with lincosamide antibiotic adenylyltransferase LinA and identifies a possible substrate-binding cleft of JHP933. Furthermore, through structural comparison with LinA and LinB, we pinpoint conservative active site residues which may contribute to divalent ion coordination and substrate binding.</p></div

Data collection and refinement statistics.

<p>Numbers in parentheses refer to the highest-resolution shell.</p

Putative substrate binding site of JHP933.

<p>Ribbon diagram and surface representation of JHP933 are colored in lime, the modelled substrate lincomycin of the superimposed LinA/lincomycin complex is shown in ball-and-stick representation and colored in magenta (LinA protein not shown).</p

The superposition of JHP933 and LinA/Lincomycin complex (4E8J) structures.

<p>Ribbon diagram of JHP933/LinA, with JHP933 is colored in lime and LinA in magenta, and substrate lincomycin of LinA is shown in ball-and-stick representation.</p