X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine

Ásgeirsson, Bjarni; Markússon, Sigurbjörn; Hlynsdóttir, Sigríður Stefanía; Helland, Ronny; Hjörleifsson, Jens Guðmundur

doi:10.1016/j.bbrep.2020.100830

Cited by 7 publications

(3 citation statements)

References 61 publications

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“…We have previously crystallized the wild-type VAP construct (PDB:6 T26), and the crystal structure contained tightly bound phosphate in both active sites, likely originating from overexpression in E. coli. 30 In an attempt to remove the bound phosphate for cocrystallization with chloride, the enzyme was subjected to extensive dialysis (∼48 h at 4 °C, with frequent renewal of buffer) against 20 mM CAPS pH 10, 500 mM NaCl, 10 mM MgCl 2 , and 15% (v/v) ethylene glycol, as VAP affinity toward phosphate is reduced by an order of magnitude at alkaline pH. 14 The protein buffer was then exchanged back to the pH 8.0 buffer through dilution prior to concentration and crystallization.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Structural Characterization of Functionally Important Chloride Binding Sites in the Marine Vibrio Alkaline Phosphatase

et al. 2022

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Enzyme stability and function can be affected by various environmental factors, such as temperature, pH, and ionic strength. Enzymes that are located outside the relatively unchanging environment of the cytosol, such as those residing in the periplasmic space of bacteria or extracellularly secreted, are challenged by more fluctuations in the aqueous medium. Bacterial alkaline phosphatases (APs) are generally affected by ionic strength of the medium, but this varies substantially between species. An AP from the marine bacterium Vibrio splendidus (VAP) shows complex pH-dependent activation and stabilization in the 0−1.0 M range of halogen salts and has been hypothesized to specifically bind chloride anions. Here, using X-ray crystallography and anomalous scattering, we have located two chloride binding sites in the structure of VAP, one in the active site and another one at a peripheral site. Further characterization of the binding sites using sitedirected mutagenesis and small-angle X-ray scattering showed that upon binding of chloride to the peripheral site, structural dynamics decreased locally, resulting in thermal stabilization of the VAP active conformation. Binding of the chloride ion in the active site did not displace the bound inorganic phosphate product, but it may promote product release by facilitating rotational stabilization of the substrate-binding Arg129. Overall, these results reveal the complex nature and dynamics of chloride binding to enzymes through long-range modulation of electronic potential in the vicinity of the active site, resulting in increased catalytic efficiency and stability.

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Section: ■ Materials and Methodsmentioning

confidence: 99%

Structural Characterization of Functionally Important Chloride Binding Sites in the Marine Vibrio Alkaline Phosphatase

et al. 2022

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“…Protein crystallization and structure refinement. We have previously crystallized the wildtype VAP construct (PDB:6T26), and the crystal structure contained tightly bound phosphate in both active sites, likely originating from overexpression in E. coli 30 . In an attempt to remove the bound phosphate for co-crystallization with chloride, the enzyme was subjected to extensive dialysis (~48 h at 4 °C, with frequent renewal of buffer) against 20 mM CAPS pH 10, 500 mM NaCl, 10 mM MgCl2 and 15% (v/v) ethylene glycol, as VAP affinity towards phosphate is reduced by an order of magnitude at alkaline pH 14 .…”

Section: Methodsmentioning

confidence: 99%

Structural characterization of functionally important chloride binding sites in the marine Vibrio alkaline phosphatase

Markússon

Hjörleifsson

Kursula

et al. 2022

Preprint

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Enzyme stability and function can be affected by various environmental factors, such as temperature, pH and ionic strength. Enzymes that are located outside the relatively unchanging environment of the cytosol, such as those residing in the periplasmic space of bacteria or extracellularly secreted, are challenged by more fluctuations in the aqueous medium. Bacterial alkaline phosphatases (APs) are generally affected by ionic strength of the medium, but this varies substantially between species. An AP from the marine bacterium Vibrio splendidus (VAP) shows complex pH-dependent activation and stabilization in the 0 – 1.0 M range of halogen salts and has been hypothesized to specifically bind chloride anions. Here, using X-ray crystallography and anomalous scattering, we have located two chloride binding sites in the structure of VAP, one in the active site and another one at a peripheral site. Further characterization of the binding sites using site-directed mutagenesis and small angle X-ray scattering (SAXS) showed that upon binding of chloride to the peripheral site, structural dynamics decreased locally, resulting in thermal stabilization of the VAP active conformation. Binding of the chloride ion in the active site did not displace the bound inorganic phosphate product, but it may promote product release by facilitating rotational stabilization of the substrate-binding Arg129. Overall, these results reveal the complex nature and dynamics of chloride binding to enzymes through long-range modulation of electronic potential in the vicinity of the active site, resulting in increased catalytic efficiency and stability.

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“…Interestingly, the crown domain was not conserved across the bacterial APs. The most distinctive crown domain structure across reported APs was Vibrio-AP (PDB ID: 3E2D), essential for the protein's stability and function (Helland et al, 2009;Hjörleifsson et al, 2021;Ásgeirsson et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Structural and Substrate Interaction Properties of Alkaline Phosphatase from Paenibacillus sp. PSB04: In-Silico Analysis

Lindang¹,

Budiman²

2022

OnLine Journal of Biological Sciences

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A Phosphate-Solubilising Bacterium (PSB) of Paenibacillus sp. PSB04 was previously isolated from the Sabah tropical rainforest in Malaysia. Interestingly, the genome sequence of the PSB04 strain harbored an Alkaline Phosphatase (AP) (EC 3.1.3.1) gene and was hypothesized to have unique structural characteristics. Therefore, this study aims to determine the AP three-Dimensional (3D) model and catalytic mechanism from Paenibacillus sp. PSB04 (PAP). To address this, the 3D model of this protein was built and docked into a model substrate of p-nitrophenyl phosphate. As a result, the best complex was shown to have the lowest binding energy of -5.9 kcal/moL. Furthermore, the complex showed the atomic coordination of catalytic residues of PAP and the substrate was similar to that of AP from Escherichia coli (ECAP), which implies that both APs shared a similar catalytic mechanism. In this mechanism, Ser 94 of PAP acted as nucleophilic residues, which were activated by the Zn ion. Arg 145 is predicted to be mobile due to its location in the loop region, which allows this residue to stabilize the substrate through direction or watermediated secondary interaction. Docking simulation of pNPP indicated that the putative residues involved in the catalysis mainly are Ser 94, Ser 141, Ala 146, Thr 147, Pro 148, Asp 293, and Glu 294. Glu 294 is considered a unique residue corresponding to Lys 328 ECAP, allowing the PAP to have a better affinity to stabilize the substrate in the binding cavity. Accordingly, a unique catalytic mechanism of PAP was proposed.

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X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine

Cited by 7 publications

References 61 publications

Structural Characterization of Functionally Important Chloride Binding Sites in the Marine Vibrio Alkaline Phosphatase

Structural Characterization of Functionally Important Chloride Binding Sites in the Marine Vibrio Alkaline Phosphatase

Structural characterization of functionally important chloride binding sites in the marine Vibrio alkaline phosphatase

Structural and Substrate Interaction Properties of Alkaline Phosphatase from Paenibacillus sp. PSB04: In-Silico Analysis

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