Crystal Structure of the Deglycating Enzyme Fructosamine Oxidase (Amadoriase II)

François Collard,Jianye Zhang,Ina Nemet,Kaustubha R Qanungo,Vincent M Monnier,Vivien C Yee

doi:10.1074/jbc.m804885200

Abstract

Fructosamine oxidases (FAOX) catalyze the oxidative deglycation of low molecular weight fructosamines (Amadori products). These proteins are of interest in developing an enzyme to deglycate proteins implicated in diabetic complications. We report here the crystal structures of FAOX-II from the fungi Aspergillus fumigatus, in free form and in complex with the inhibitor fructosyl-thioacetate, at 1.75 and 1.6A resolution, respectively. FAOX-II is a two domain FAD-enzyme with an overall topology that is most similar to that of monomeric sarcosine oxidase. Active site residues Tyr-60, Arg-112 and Lys-368 bind the carboxylic portion of the fructosamine, whereas Glu-280 and Arg-411 bind the fructosyl portion. From structure-guided sequence comparison, Glu-280 was identified as a signature residue for FAOX activity. Two flexible surface loops become ordered upon binding of the inhibitor in a catalytic site that is about 12A deep, providing an explanation for the very low activity of FAOX enzymes toward protein-bound fructosamines, which would have difficulty accessing the active site. Structure-based mutagenesis showed that substitution of Glu-280 and Arg-411 eliminates enzyme activity. In contrast, modification of other active site residues or of amino acids in the flexible active site loops has little effect, highlighting these regions as potential targets in designing an enzyme that will accept larger substrates.

Highlights

Diabetes and taken to be in part responsible for diabetic complications
All these enzymes catalyze the detachment of the sugar moiety from the amine of the fructosamine, leading to its “deglycation.” Those three families of enzymes use different catalytic mechanisms and have different physiological roles; fructosamine 3-kinase is an ATP-dependent protein-repair enzyme that clears fructosamines as they form on proteins in the cell, whereas the members of the last two families, fructoselysine-6-phosphate deglycase and fructosamine oxidases (FAOX), are microbial enzymes enabling the utilization of glycated amino acids as energy source
In the free FAOX-II structure, no continuous electron density was seen for residues 58 – 66 and 110 – 116, revealing that in the absence of ligand bound in the active site, these two surface regions are flexible

Summary

Introduction

Diabetes and taken to be in part responsible for diabetic complications. Fructosamines are relatively unstable compounds and are precursors for advanced glycation end products (AGEs),5 some of which cause proteins cross-linking, extracellular matrix stiffening, and activation of the receptor for AGEs (RAGEs) [1, 2]. Crystallization and Cryoprotection—Crystals of wild-type and selenomethionine FAOX-II were obtained by the sitting drop vapor diffusion method at 20 °C by mixing 1 ␮l of the protein solution (15 mg/ml in 10 mM Tris buffer, pH 8.0) in the presence or absence of a 3 mM concentration of the inhibitor fructosyl thioacetate (FSA) with 1 ␮l of the reservoir containing 0.1 M Hepes, pH 7.4, 10% isopropanol, and 18% polyethylene glycol 4000.

Results

Conclusion