Crystal Structures ofEscherichia coliATP-Dependent Glucokinase and Its Complex with Glucose

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Abstract
Intracellular glucose inEscherichia colicells imported by phosphoenolpyruvate-dependent phosphotransferase system-independent uptake is phosphorylated by glucokinase by using ATP to yield glucose-6-phosphate. Glucokinases (EC 2.7.1.2) are functionally distinct from hexokinases (EC 2.7.1.1) with respect to their narrow specificity for glucose as a substrate. While structural information is available for ADP-dependent glucokinases fromArchaea, no structural information exists for the large sequence family of eubacterial ATP-dependent glucokinases. Here we report the first structure determination of a microbial ATP-dependent glucokinase, that fromE. coliO157:H7. The crystal structure ofE. coliglucokinase has been determined to a 2.3-Å resolution (apo form) and refined to finalRwork/Rfreefactors of 0.200/0.271 and to 2.2-Å resolution (glucose complex) with finalRwork/Rfreefactors of 0.193/0.265.E. coliGlK is a homodimer of 321 amino acid residues. Each monomer folds into two domains, a small α/β domain (residues 2 to 110 and 301 to 321) and a larger α+β domain (residues 111 to 300). The active site is situated in a deep cleft between the two domains.E. coliGlK is structurally similar toSaccharomyces cerevisiaehexokinase and human brain hexokinase I but is distinct from the ADP-dependent GlKs. Bound glucose forms hydrogen bonds with the residues Asn99, Asp100, Glu157, His160, and Glu187, all of which, except His160, are structurally conserved in human hexokinase 1. Glucose binding results in a closure of the small domains, with a maximal Cα shift of ∼10 Å. A catalytic mechanism is proposed that is consistent with Asp100 functioning as the general base, abstracting a proton from the O6 hydroxyl of glucose, followed by nucleophilic attack at the γ-phosphoryl group of ATP, yielding glucose-6-phosphate as the product.