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

Abstract

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.

An LPS-dephosphorylating alkaline phosphatase of PhoA family from the marine bacteriumCobetia amphilectiKMM 296 and multiplicity of alkaline phosphatase families inCobetiaspp

“…The PhoA structural family of ALPs is predominantly associated with marine heterotrophic bacteria of Bacteroidetes and γ- Proteobacteria neighboring cyanobacteria populations [13,22], as well as the microorganisms isolated from plant and animal microbiomes, such as a symbiont (pathogen) Vibrio spp. of planktonic crustaceans (Copepods ) , a symbiont (facultative pathogen) of human E. coli , a sugarcane root endophyte Enterobacter roggenkampii [29,50,51], marine fish pathogens V. splendidus [52], and Moritella sp. [53].…”

LPS-Dephosphorylating Cobetia amphilecti Alkaline Phosphatase of PhoA Family Divergent from the Multiple Homologues of Cobetia spp.

“…The PhoA structural family of ALPs is predominantly associated with marine heterotrophic bacteria of Bacteroidetes and γ- Proteobacteria -neighbouring cyanobacteria populations [ 13 , 22 ], as well as microorganisms isolated from plant and animal microbiomes, such as symbiont (or pathogen) Vibrio spp. of planktonic crustaceans (Copepods), a symbiont (facultative pathogen) of human E. coli , a sugarcane root endophyte Enterobacter roggenkampii [ 29 , 63 , 64 ], and marine fish pathogens Vibrio splendidus [ 65 ] and Moritella sp. [ 66 ].…”

Methods to Characterise Enzyme Kinetics with Biological and Medicinal Substrates: The Case of Alkaline Phosphatase

“…Ultraviolet‐visible (UV‐Vis) absorbance spectroscopy can monitor this reaction in real‐time. For example, pNPP was recently employed in a study of the pH‐dependent binding of chloride ions to a marine bacterial AP from Vibrio splendidus [19] . Analogues of pNPP that release the same pNP product can be employed to characterise other activities of APs (e. g., diesterase, triesterase, and sulfatase) [20] .…”

“…For example, pNPP was recently employed in a study of the pH-dependent binding of chloride ions to a marine bacterial AP from Vibrio splendidus. [19] Analogues of pNPP that release the same pNP product can be employed to characterise other activities of APs (e. g., diesterase, triesterase, and sulfatase). [20] Similarly to pNPP, AP converts 4-methylumbelliferylphosphate (4MUP) to P i and fluorescent 4-methylumbelliferone (4MU).…”

“…[31] Of course, substrates for use with other approaches are available too. These include bioluminescence, [32] electrochemistry, [33] UV-Vis spectroscopy, [34] Raman spectroscopy and surface-enhanced (resonance) Raman spectroscopy (SERS and SERRS), [35] chemical exchange saturation transfer magnetic resonance imaging (CEST MRI), [36] electron paramagnetic resonance (EPR) spectroscopy, [37] and 19 F nuclear magnetic resonance (NMR) spectroscopy. [38] Although many options are available, a limitation shared by these methods is their reliance on the inherent properties of a particular substrate or its product to generate a signal change depending on the instrument being used to follow the reaction.…”