title: Crystal structure of exo-inulinase from Aspergillus awamori: the enzyme fold and structural determinants of substrate recognition.
abstract: Exo-inulinases hydrolyze terminal, non-reducing 2,1-linked and 2,6-linked beta-d-fructofuranose residues in inulin, levan and sucrose releasing beta-d-fructose. We present the X-ray structure at 1.55A resolution of exo-inulinase from Aspergillus awamori, a member of glycoside hydrolase family 32, solved by single isomorphous replacement with the anomalous scattering method using the heavy-atom sites derived from a quick cryo-soaking technique. The tertiary structure of this enzyme folds into two domains: the N-terminal catalytic domain of an unusual five-bladed beta-propeller fold and the C-terminal domain folded into a beta-sandwich-like structure. Its structural architecture is very similar to that of another member of glycoside hydrolase family 32, invertase (beta-fructosidase) from Thermotoga maritima, determined recently by X-ray crystallography The exo-inulinase is a glycoprotein containing five N-linked oligosaccharides. Two crystal forms obtained under similar crystallization conditions differ by the degree of protein glycosylation. The X-ray structure of the enzyme:fructose complex, at a resolution of 1.87A, reveals two catalytically important residues: Asp41 and Glu241, a nucleophile and a catalytic acid/base, respectively. The distance between the side-chains of these residues is consistent with a double displacement mechanism of reaction. Asp189, which is part of the Arg-Asp-Pro motif, provides hydrogen bonds important for substrate recognition.
authors.affiliation: Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador São-carlense 400, CEP 13560-970, São Carlos, SP, Brazil.
authors.forename: R A P
authors.initials: RA
authors.lastname: Nagem
authors.forename: A L
authors.initials: AL
authors.lastname: Rojas
authors.forename: A M
authors.initials: AM
authors.lastname: Golubev
authors.forename: O S
authors.initials: OS
authors.lastname: Korneeva
authors.forename: E V
authors.initials: EV
authors.lastname: Eneyskaya
authors.forename: A A
authors.initials: AA
authors.lastname: Kulminskaya
authors.forename: K N
authors.initials: KN
authors.lastname: Neustroev
authors.forename: I
authors.initials: I
authors.lastname: Polikarpov
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001230:Aspergillus / Q000201:enzymology*; D020134:Catalytic Domain; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D005632:Fructose / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000302:isolation & purification; D006031:Glycosylation; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry
chemical_list: D009844:Oligosaccharides; D014867:Water; D005973:Glutamine; D005632:Fructose; D001224:Aspartic Acid; D006026:Glycoside Hydrolases; C023086:inulinase