RESUMEN
Fructokinase (FRK) catalyzes the first step of fructose metabolism i.e., D-fructose to D-fructose-6-phosphate (F6P), however, the mechanistic insights of this reaction are elusive yet. Here we demonstrate that the putative Vibrio cholerae fructokinase (VcFRK) exhibit strong fructose-6-kinase activity allosterically modulated by K+/Cs+. We have determined the crystal structures of apo-VcFRK and its complex with fructose, fructose-ADP-Ca2+, fructose-ADP-Ca2+-BeF3-. Collectively, we propose the catalytic mechanism and allosteric activation of VcFRK in atomistic details explaining why K+/Cs+ are better activator than Na+. Structural results suggest that apo VcFRK allows entry of fructose in the active site, sequester it through several conserved H-bonds and attains a closed form through large scale conformational changes. A double mutant (H108C/T261C-VcFRK), that arrests the closed form but unable to reopen for F6P release, is catalytically impotent highlighting the essentiality of this conformational change. Negative charge accumulation around ATP upon fructose binding, is presumed to redirect the γ-phosphate towards fructose for efficient phosphotransfer. Reduced phosphotransfer rate of the mutants E205Q and E110Q supports this view. Atomic resolution structure of VcFRK-fructose-ADP-Ca2+-BeF3-, reported first time for any sugar kinase, suggests that BeF3- moiety alongwith R176, Ca2+ and 'anion hole' limit the conformational space for γ-phosphate favoring in-line phospho-transfer.
Asunto(s)
Fructoquinasas/química , Fructoquinasas/metabolismo , Modelos Moleculares , Conformación Molecular , Azúcares/química , Azúcares/metabolismo , Vibrio cholerae/metabolismo , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Dominio Catalítico , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de ProteínaAsunto(s)
Proteínas Bacterianas/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Multimerización de Proteína , Estructura Secundaria de Proteína , Vibrio cholerae/enzimología , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Secuencia de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carbohidratos/química , Dominio Catalítico , Cationes Monovalentes/química , Cationes Monovalentes/metabolismo , Cationes Monovalentes/farmacología , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Unión Proteica , Ribosa/química , Ribosa/metabolismo , Homología de Secuencia de Aminoácido , Vibrio cholerae/genéticaRESUMEN
Ribokinase (RK) is one of the principal enzymes in carbohydrate metabolism, catalyzing the reaction of D-ribose and adenosine triphosphate to produce ribose-5-phosphate and adenosine diphosphate (ADP). To provide further insight into the catalytic mechanism, the rbsK gene from Vibrio cholerae O395 encoding ribokinase was cloned and the protein was overexpressed in Escherichia coli BL21 (DE3) and purified using Ni(2+)-NTA affinity chromatography. Crystals of V. cholerae RK (Vc-RK) and of its complex with ribose and ADP were grown in the presence of polyethylene glycol 6000 and diffracted to 3.4 and 1.75â Å resolution, respectively. Analysis of the diffraction data showed that both crystals possess symmetry consistent with space group P1. In the Vc-RK crystals, 16 molecules in the asymmetric unit were arranged in a spiral fashion, leaving a large empty space inside the crystal, which is consistent with its high Matthews coefficient (3.9â Å(3)â Da(-1)) and solvent content (68%). In the Vc-RK co-crystals four molecules were located in the asymmetric unit with a Matthews coefficient of 2.4â Å(3)â Da(-1), corresponding to a solvent content of 50%.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Vibrio cholerae/enzimología , Secuencia de Bases , Cristalización , Cristalografía por Rayos X , Cartilla de ADN , Reacción en Cadena de la PolimerasaRESUMEN
Fructokinase (FK), one of the crucial enzymes for sugar metabolism in bacterial systems, catalyses the unidirectional phosphorylation reaction from fructose to fructose 6-phosphate, thereby allowing parallel entry of fructose into glycolysis beside glucose. The cscK gene from Vibrio cholerae O395 coding for the enzyme FK has been cloned, overexpressed in Escherichia coli BL21 (DE3) and purified using Ni-NTA affinity chromatography. Crystals of V. cholerae FK (Vc-FK) and its cocrystal with fructose, adenosine diphosphate (ADP) and Mg2+ were grown in the presence of polyethylene glycol 6000 and diffracted to 2.45 and 1.75â Å resolution, respectively. Analysis of the diffraction data showed that both crystal forms have symmetry consistent with space group P2(1)2(1)2, but with different unit-cell parameters. Assuming the presence of two molecules in the asymmetric unit, the Matthews coefficient for the apo Vc-FK crystals was estimated to be 2.4â Å3â Da(-1), which corresponds to a solvent content of 48%. The corresponding values for the ADP- and sugar-bound Vc-FK crystals were 2.1â Å3â Da(-1) and 40%, respectively, assuming the presence of one molecule in the asymmetric unit.