Soluble Aldose Sugar Dehydrogenase from Escherichia coli: A HIGHLY EXPOSED ACTIVE SITE CONFERRING BROAD SUBSTRATE SPECIFICITY

Stacey M Southall,Justin J Doel,David J Richardson,Arthur Oubrie

doi:10.1074/jbc.m601783200

Abstract

A water-soluble aldose sugar dehydrogenase (Asd) has been purified for the first time from Escherichia coli. The enzyme is able to act upon a broad range of aldose sugars, encompassing hexoses, pentoses, disaccharides, and trisaccharides, and is able to oxidize glucose to gluconolactone with subsequent hydrolysis to gluconic acid. The enzyme shows the ability to bind pyrroloquinoline quinone (PQQ) in the presence of Ca2+ in a manner that is proportional to its catalytic activity. The x-ray structure has been determined in the apo-form and as the PQQ-bound active holoenzyme. The beta-propeller fold of this protein is conserved between E. coli Asd and Acinetobacter calcoaceticus soluble glucose dehydrogenase (sGdh), with major structural differences lying in loop and surface-exposed regions. Many of the residues involved in binding the cofactor are conserved between the two enzymes, but significant differences exist in residues likely to contact substrates. PQQ is bound in a large cleft in the protein surface and is uniquely solvent-accessible compared with other PQQ enzymes. The exposed and charged nature of the active site and the activity profile of this enzyme indicate possible factors that underlie a low affinity for glucose but generic broad substrate specificity for aldose sugars. These structural and catalytic properties of the enzymes have led us to propose that E. coli Asd provides a prototype structure for a new subgroup of PQQ-dependent soluble dehydrogenases that is distinct from the A. calcoaceticus sGdh subgroup.

Highlights

Proteins that require the prosthetic group pyrroloquinoline quinone (PQQ)3 for catalysis form the largest and best-characterized subclass of quinoproteins, catalyzing the conversion of a wide range of different alcohol- and aldehyde-containing com
The significant catalytic and structural differences between the enzymes lead us to propose that the E. coli yliI gene product provides the first structure for a new subtype of soluble PQQdependent dehydrogenases, with substrate selectivity and structural features that distinguish them from A. calcoaceticus soluble glucose dehydrogenase (sGdh) enzyme type and which we propose to call soluble aldose sugar dehydrogenase (Asd)
From this we deduce that one molecule of PQQ binds per molecule of YliI, similar to that shown with sGdh from A. calcoaceticus [28]

Summary

Introduction

Proteins that require the prosthetic group pyrroloquinoline quinone (PQQ) for catalysis form the largest and best-characterized subclass of quinoproteins, catalyzing the conversion of a wide range of different alcohol- and aldehyde-containing com-. The A. calcoaceticus sGdh has been successfully incorporated into successful diagnostic systems to monitor glucose levels in the blood of diabetics (14 –16) This system has the advantage of being extremely rapid due to the high catalytic activity of sGdh and of being oxygen-independent. In many cases the bacterial and archaeal homologues of sGdh identified previously [1] have rather low amino acid sequence identity with the A. calcoaceticus sGdh and their specificity and activity toward glucose and maltose cannot be predicted. The gene product is predicted to be a PQQ-binding protein that has a low 18% identity with the A. calcoaceticus sGdh. Our biochemical analysis shows this enzyme to have a low affinity for both glucose and maltose but a generic promiscuity toward mono-, di-, and trisaccharide aldose sugars. The significant catalytic and structural differences between the enzymes lead us to propose that the E. coli yliI gene product provides the first structure for a new subtype of soluble PQQdependent dehydrogenases, with substrate selectivity and structural features that distinguish them from A. calcoaceticus sGdh enzyme type and which we propose to call soluble aldose sugar dehydrogenase (Asd)

Methods

Results

Conclusion