Abstract
The first steps of gluconate metabolism in Escherichia coli are the transport of gluconate into the cell by a gluconate transport system, followed by phosphorylation to gluconate-6- phosphate by gluconate kinase. There exist two such different pathways, Grill and GntIl, respectively. The predominant pathway, GntI, contains a high- and a low-affinity gluconate transporter and a gluconate kinase, GntK. Gluconate-6-phosphate is further metabolized via the Entner-Doudoroff pathway.
The three dimensional structure of gluconate kinase from Escherichia coli, containing a P- loop binding motif, GXXXXGK[TS], has been determined by X-ray crystallography. The structure of the apo enzyme was determined by multiple-wavelength anomalous diffraction and refined to 2.0 Å (P212121), 2.1 Å (C2) and 2.6 Å (P21212) resolution, respectively. Each subunit is built up from a central five-stranded parallel P-sheet and seven alpha- helices. The structure can be divided into three parts, the NMPbind domain (residues 40-61), the LID (residues 123-130) and the CORE domain (residues 1-39, 62-122, 131-175). The structure of gluconate kinase belongs to the structural family of nucleoside monophosphate (NMP) kinases. In all space groups there is one common dimer interface, indicating that the dimer is the catalytic form of GntK in solution.
The structures of three complexes of GntK with ATP, AMPPCP or gluconate-6- phosphate bound in the active site have also been determined in space group P21 to 2.3 A, 2.8 A and 2.4 A resolution, respectively. There is a conformational change upon binding of ATP. The NMPbind and LID move away from the active site rather than closing into the active site to protect the ATP from hydrolysis as normally observed in this family of enzymes. The interactions of bound Mg2+ - ATP are similar to other P-loop containing enzymes and the binding of gluconate-6-phosphate is consistent with an in line phosphoryl transfer from ATP to gluconate.
