title: Identification of Pyrococcus furiosus amylopullulanase catalytic residues.
abstract: Pyrococcus furiosus amylopullulanase (PfAPU) belongs to glycosyl hydrolase family 57. Using sequence alignments of the known family 57 enzymes and site-directed mutagenesis, E291, D394, and E396 were identified as PfAPU putative catalytic residues. The apparent catalytic efficiencies (k(cat)/K(m)) of PfAPU mutants E291Q and D394N on pullulan were 123.0 and 24.4 times lower, respectively, than that of PfAPU. The activity of mutant E396Q on pullulan was too low to allow reliable determination of its catalytic efficiency. The apparent specific activities of these enzymes on starch also decreased 91.0 times (E291Q), 11.7 times (D394N), and 37.2 times (E396Q). The hydrolytic patterns for pullulan and starch were the same, while the hydrolysis rates differed as reported. Based on sequence alignment and a previous report, E291 is proposed as the catalytic nucleophile.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA.
authors.forename: S
authors.initials: S
authors.lastname: Kang
authors.forename: C
authors.initials: C
authors.lastname: Vieille
authors.forename: J G
authors.initials: JG
authors.lastname: Zeikus
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D002942:Circular Dichroism; D019641:DNA, Archaeal / Q000235:genetics; D015870:Gene Expression; D019766:Genes, Archaeal; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013213:Starch / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D019641:DNA, Archaeal; D005936:Glucans; D011994:Recombinant Proteins; C009109:pullulan; D013213:Starch; D006026:Glycoside Hydrolases; C082507:amylopullulanase