Affiliation:
1. Biotechnology Research Institute, National Research Council of Canada, and Montreal Joint Centre for Structural Biology, Montreal, Quebec, Canada
Abstract
ABSTRACT
Intracellular glucose in
Escherichia coli
cells 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 from
Archaea
, 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 from
E. coli
O157:H7. The crystal structure of
E. coli
glucokinase has been determined to a 2.3-Å resolution (apo form) and refined to final
R
work
/
R
free
factors of 0.200/0.271 and to 2.2-Å resolution (glucose complex) with final
R
work
/
R
free
factors of 0.193/0.265.
E. coli
GlK 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. coli
GlK is structurally similar to
Saccharomyces cerevisiae
hexokinase 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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
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