Identification of Pyrococcus furiosus amylopullulanase catalytic residues

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA.
Applied Microbiology and Biotechnology (Impact Factor: 3.34). 02/2005; 66(4):408-13. DOI: 10.1007/s00253-004-1690-7
Source: PubMed


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
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.

Download full-text


Available from: Claire Vieille, Oct 13, 2015
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cellulose and starch are the most abundant polymers on Earth. They both consist of glucose monomer units which are, however, differently bound to form polymer chains: starch contains the glucose linked up by the β-glucosidic bonds, while the glucose in cellulose is bound by the β-glucosidic linkages. Therefore these two important sources of energy for animals, plants and micro-organisms are biochemically hydrolysed by two different groups of enzymes: starch by α-glycoside hydrolases, and cellulose by β-glycoside hydrolases. Starch (amylon in Greek) consists of two distinct fractions: amylose linear α-1,4-linked glucans, and amylopectin linear α-1,4-linked glucans branched with α-1,6 linkages (Ball et al., 1996; Mouille et al., 1996), therefore the enzymes responsible for its hydrolysis are called amylolytic enzymes or simply amylases. Amylolytic enzymes form a large group of enzymes among which the most common and best known are α-amylases, β-amylases and glucoamylases. Since starch (like the structurally related glycogen) is an essential source of energy, amylolytic enzymes are produced by a great variety of living organisms (Vihinen and Mntsla, 1989). Although the different amylases mediate the same reaction they all catalyse the cleavage of the β-glucosidic bonds in the same substrate structurally and mechanistically they are quite different (MacGregor et al., 2001). Both α-amylase and β-amylase adopt the structure of a TIM-barrel fold (for a review see Pujadas and Palau, 1999), i.e. their catalytic domain consists of a (β/α)8-barrel formed by 8 parallel β-strands surrounded by 8 α-helices (Matsuura et al., 1984; Mikami et al., 1993). The barrels are, however, not similar in their details (Jespersen et al., 1991). Glucoamylase on the other hand possesses the structure of an (α/ α)6-barrel, consisting of an inner barrel composed of 6 α-helices which is surrounded by 6 more (Aleshin et al., 1992). Strands and helices of the (β/α)8-barrel domain as well as the helices of the (α/ α)6-barrel are connected by loop regions of various lengths. Based on the similarities and differences in their primary structures, amylolytic enzymes have been classified into families of glycoside hydrolases (GH) (Henrissat, 1991): (i) α-amylases family GH13; (ii) β-amylases family GH14; and (iii) glucoamylases family GH15. This classification, available online at the CAZy (Carbohydrate-Active enZymes) internet site (Coutinho and Henrissat, 1999), reflects the differences in the reaction mechanisms and catalytic machinery employed by the three types of amylase (Davies and Henrissat, 1995). Due to the enormous accumulation of new sequence data in recent years, α-amylase family GH13 has expanded so that it now contains almost 30 different enzymes and proteins (e.g. pullulanase, isoamylase, neopullulanase etc.) exhibiting sequence relatedness to α-amylases (MacGregor et al., 2001). At present all these enzymes are classified into families GH13, GH70 and GH77 which together constitute glycoside hydrolase clan GH-H (Coutinho and Henrissat, 1999). Moreover, families GH31 and GH57 contain a few amylolytic specificities with no sequence similarity to family GH13 (Henrissat and Bairoch, 1996). The present review focuses on structural characteristics of the GH families of amylases. Its main goal is to provide a brief overview of the best-known glycoside hydrolases families GH13, GH14, GH15, GH31, GH57 GH70 and GH77. Emphasis is placed on the description of their: (i) specificities with regard to the EC numbers; (ii) three-dimensional structures; and (iii) catalytic domain architecture.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Keywords: glycosyl hydrolase, alpha-glucosidase, microarrays, functional genomics, enzyme discovery, beta-amylase, hyperthermophiles. Thesis (Ph.D.)--North Carolina State University. Includes bibliographical references. Includes vita.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: cesta 21, SK-84551 Bratislava, Slovakia; phone: ++ 421 2 5930 7420, fax: ++ 421 2 5930 7416, Abstract: The amylolytic and related enzymes have been classified into the families of glycoside hydrolases (GHs). At present there are almost one hundred GH families. The main α-amylase family is the family GH-13 that forms the clan GH-H together with the families GH-70 and GH-77. β-Amylases and glucoamylases have their own families GH-14 and GH-15, respectively. Some amylolytic enzymes are grouped also in the family GH-31. The main topic of this review, the family GH-57, was established as the novel α-amylase family in 1996, based on the two sequences of amylases that were obviously dissimilar to the α-amylases of the family GH-13. The family GH-57 contains also the amylopullulanase, α-galactosidase and 4-α-glucanotransferase specificities in addition to the specificity of α-amylase. The family is remarkable by several facts: (i) it consists of only 10% of real enzymes (90% is formed by the putative proteins); (ii) the protein members originate exclusively from prokaryotic sources of extremophilic nature (many of them are archaeal hyperthermophiles); (iii) the amino acid sequences vary from less than 400 to more than 1,300 residues and are extremely diversified. There are five conserved sequence regions characteristic of the family GH-57. The catalytic domain of a GH-57 member is the (β/α)7-barrel with the Glu (nucleophile) and the Asp (proton donor) residues at the β-strands β4 and β7 of the barrel, respectively, employing the retaining mechanism. From the evolutionary point of view, the family GH-57 could be divided into several subfamilies. Importantly, the individual enzyme specificities form their own independent clusters.
Show more