title: Biochemical and structural features of a novel cyclodextrinase from cow rumen metagenome.
abstract: A novel enzyme, RA.04, belonging to the alpha-amylase family was obtained after expression of metagenomic DNA from rumen fluid (Ferrer et al.: Environ. Microbiol. 2005, 7, 1996-2010). The purified RA.04 has a tetrameric structure (280 kDa) and exhibited maximum activity (5000 U/mg protein) at 70 degrees C and was active within an unusually broad pH range from 5.5 to 9.0. It maintained 80% activity at pH 5.0 and 9.5 and 75 degrees C. The enzyme hydrolyzed alpha-D-(1,4) bonds 13-fold faster than alpha-D-(1,6) bonds to yield maltose and glucose as the main products, and it exhibited transglycosylation activity. Its preferred substrates, in the descending order, were maltooligosaccharides (C3-C7), cyclomaltoheptaose (beta-CD), cyclomaltohexaose (alpha-CD), cyclomaltooctaose (gamma-CD), soluble starch, amylose, pullulan and amylopectin. The biochemical properties and amino acid sequence alignments suggested that this enzyme is a cyclomaltodextrinase. However, despite the similarity in the catalytic module (with Glu359 and Asp331 being the catalytic nucleophile and substrate-binding residues, respectively), the enzyme bears a shorter N-terminal domain that may keep the active site more accessible for both starch and pullulan, compared to the other known CDases. Moreover, RA.04 lacks the well-conserved N-terminal Trp responsible for the substrate preference typical of CDases/MAases/PNases, suggesting a new residue is implicated in the preference for cyclic maltooligosaccharides. This study has demonstrated the usefulness of a metagenomic approach to gain novel debranching enzymes, important for the bread/food industries, from microbial environments with a high rate of plant polymer turnover, exemplified by the cow rumen.
authors.affiliation: CSIC, Institute of Catalysis, Cantoblanco, Madrid, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Ferrer
authors.forename: Ana
authors.initials: A
authors.lastname: Beloqui
authors.forename: Olga V
authors.initials: OV
authors.lastname: Golyshina
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Plou
authors.forename: Alexander
authors.initials: A
authors.lastname: Neef
authors.forename: Tatyana N
authors.initials: TN
authors.lastname: Chernikova
authors.forename: Lucia
authors.initials: L
authors.lastname: Fernández-Arrojo
authors.forename: Iraj
authors.initials: I
authors.lastname: Ghazi
authors.forename: Antonio
authors.initials: A
authors.lastname: Ballesteros
authors.forename: Kieran
authors.initials: K
authors.lastname: Elborough
authors.forename: Kenneth N
authors.initials: KN
authors.lastname: Timmis
authors.forename: Peter N
authors.initials: PN
authors.lastname: Golyshin
mesh_terms: D000687:Amylopectin / Q000378:metabolism; D000688:Amylose / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008320:Maltose / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D012417:Rumen / Q000382:microbiology*; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D047391:alpha-Cyclodextrins / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000378:metabolism; D047408:gamma-Cyclodextrins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005936:Glucans; D009844:Oligosaccharides; D047391:alpha-Cyclodextrins; D047392:beta-Cyclodextrins; D047408:gamma-Cyclodextrins; C021705:maltooligosaccharides; D008320:Maltose; C009109:pullulan; D013213:Starch; D000688:Amylose; D000687:Amylopectin; D006026:Glycoside Hydrolases; C048745:cyclomaltodextrinase; C031215:betadex; C023792:gamma-cyclodextrin; C032613:alpha-cyclodextrin