Crystal structure of the deglycating enzyme fructosamine oxidase (amadoriase II).
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.
Collard
François
F
Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Zhang
Jianye
J
Nemet
Ina
I
Qanungo
Kaustubha R
KR
Monnier
Vincent M
VM
Yee
Vivien C
VC
D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites / Q000502:physiology; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D019270:Fructosamine / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D020125:Mutation, Missense; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D013379:Substrate Specificity / Q000502:physiology
D005656:Fungal Proteins; D005182:Flavin-Adenine Dinucleotide; D019270:Fructosamine; D000594:Amino Acid Oxidoreductases; C103012:amadoriase