CRYSTAL STRUCTURE OF THE BIOSENSOR GLUCOSE DEHYDROGENASE

Arthur Oubrie, Henriëtte Rozeboom, Kor Kalk and Bauke Dijkstra

Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.

Keywords: biosensor, crystal structure, diabetes, glucose dehydrogenase, protein engineering.

For diabetes mellitus patients, a reliable and quick glucose detection is crucial for the management of plasma glucose concentrations. To achieve this goal, biosensors and test strips have been equipped with the soluble quinoprotein glucose dehydrogenase (s-GDH) from the bacterium Acinetobacter calcoaceticus as the glucose sensing element. The substrate specificity and lifetime of the biosensors and test strips may still be improved by protein engineering of s-GDH. For these purposes, however, knowledge of the three-dimensional structure of s-GDH is essential. The crystal structure of s-GDH has been determined in a complexed form with the cofactor pyrroloquinoline quinone (PQQ) to a resolution of 2.2 A resolution and in a cofactor-deficient form to a resolution of 1.7 A. s-GDH adopts a b-propeller or superbarrel fold, which is composed of six four-stranded anti-parallel b-sheets. The holo-enzyme is a dimer. Each monomer binds three Ca²⁺-ions, two of which are located in the dimerization interface. A third Ca²⁺-ion binds PQQ in the active site near the top of the b-propeller. The binding of PQQ to the enzyme is predominantly governed by polar/charged interactions. The active site Ca2+-ion is liganded by one of the two carbonyl oxygens of the reactive orthoquinone group of the cofactor, suggesting a catalytic role for Ca²⁺ in s-GDH. His144 is located close to the ortho-quinone group, and its orientation is stabilized by a hydrogen bond with Asp163. In this dyad system, the His144 Ne2 atom may be preserved in an unprotonated state, which would allow His144 to act as the catalytic base that abstracts a proton from the C1-hydroxyl group of the substrate. Two possible sugar binding sites have been identified close to the prosthetic group. X-ray analysis at 1.5 A of the covalently attached inhibitor methylhydrazine provides new insight into the reactivity of PQQ in s-GDH. A plausible reaction mechanism may be based on these results.