Structural and Biochemical Investigations on the Catalytic Mechanism of Pyridoxal Kinase (PdxK) from Salmonella Typhimurium and its Interactions with PLP-Dependent Enzymes

Abstract

Pyridoxal kinase (PdxK), a member of ribokinase superfamily of enzymes, is involved in pyridoxal 5’ phosphate (PLP) synthesis by a salvage pathway. PdxK ensures availability of PLP to a large number of enzymes involved in amino acid and sugar metabolism that use PLP as a cofactor and is regarded as a potential drug target. We have determined the crystal structure of PdxK from Salmonella typhimurium (stPLK) in its unliganded form as well as in complex with Mg ATP and Mg ATP-PLP at 2.6A, 1.9A and 2.5A resolutions, respectively. The protomeric structure of the dimeric enzyme consisting of three layered αβα structure is similar to those of other ribokinase family proteins. A segment of residues 134-142 constituting a flexible loop undergoes a large conformational change from an open form to a closed form upon ligand binding and guards the active site from solvent exposure and prevents premature hydrolysis of ATP. During catalysis, the substrate PL moves by a distance of ∼6A to interact with the γ phosphate of ATP bound near an anion hole constituted by residues 234-237 (GTGD). As reported for the E. coli enzyme, kinetic studies show that stPLK has higher activity in the presence of Mg2+ when compared to other divalent metal ions. High concentration of PLP was found to inhibit stPLK and the structure of crystals obtained in presence of excess PLP reveals that PLP is covalently attached as an internal aldimine to Lys233. Surface Plasmon resonance (SPR ) and ELISA studies show that stPLK specifically interacts with diaminopropionate ammonia lyase, a fold type II PLP dependent enzyme, suggesting probable direct transfer of the product PLP from PdxK to the apo form of PLP-dependent enzymes.