Structure-guided mutational evidence and postulates explaining how a glycohydrolase from Pyrococcus furiosus functions simultaneously as an amylase and as a 4-α-glucanotransferase.

Pyrococcus furiosus exoamylase-cum-4-α-glucanotransferase (4-α-GTase; PF0272; PfuAmyGT) is reported to both (i) act upon starch, and (ii) catalyze 'disproportionation' of maltooligosaccharides (with glucose as the smallest product). PfuAmyGT shares ∼65% sequence identity with a homo-dimeric Thermococcus litoralis 4-α-GTase, for which structures are available in complex with a non-hydrolysable analog of maltotetraose (acarbose) bound to one subunit and maltose (of unknown origin) bound to the other subunit. We structurally transposed the maltose onto the acarbose-bound subunit and discovered that the two molecules lie juxtaposed in what could be perfect 'acceptor' and 'donor' substrate-binding sites, respectively. We also discovered that there is a loop between the two sites which could use an available aspartate to excise a glucose from the donor, and an available tryptophan to transfer the glucose to the non-reducing end of the acceptor glucan. We derived a structure for PfuAmyGT through homology-based modeling, identified the potential donor site, acceptor site, glucan-transferring loop, and catalytically important residues, and mutated these to alanine to examine effect(s) upon activity. Mutation D362A abolished creation of shorter, or longer, maltooligosaccharides. Mutation W365A abolished creation of longer oligosaccharides. Mutation H366A had no effect on activity. We propose that D362 facilitates glucose excision, and that W365 facilitates its transfer, either (a) directly into solution (allowing PfuAmyGT to act as an exoamylase), or (b) by glycoside bond formation with an acceptor (allowing PfuAmyGT to act as a 4-α-glucanotransferase), depending upon whether the acceptor site is vacant or occupied in a reaction cycle.

Elucidating the role of Agl in bladder carcinogenesis by generation and characterization of genetically engineered mice.

Amylo-α-1,6-glucosidase,4-α-glucanotransferase (AGL) is an enzyme primarily responsible for glycogen debranching. Germline mutations lead to glycogen storage disease type III (GSDIII). We recently found AGL to be a tumor suppressor in xenograft models of human bladder cancer (BC) and low levels of AGL expression in BC are associated with poor patient prognosis. However, the impact of low AGL expression on the susceptibility of normal bladder to carcinogenesis is unknown. We address this gap by developing a germline Agl knockout (Agl-/-) mouse that recapitulates biochemical and histological features of GSDIII. Agl-/- mice exposed to N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) had a higher BC incidence compared with wild-type mice (Agl+/+). To determine if the increased BC incidence observed was due to decreased Agl expression in the urothelium specifically, we developed a urothelium-specific conditional Agl knockout (Aglcko) mouse using a Uroplakin II-Cre allele. BBN-induced carcinogenesis experiments repeated in Aglcko mice revealed that Aglcko mice had a higher BC incidence than control (Aglfl/fl) mice. RNA sequencing revealed that tumors from Agl-/- mice had 19 differentially expressed genes compared with control mice. An 'Agl Loss' gene signature was developed and found to successfully stratify normal and tumor samples in two BC patient datasets. These results support the role of AGL loss in promoting carcinogenesis and provide a rationale for evaluating Agl expression levels, or Agl Loss gene signature scores, in normal urothelium of populations at risk of BC development such as older male smokers.

[Key enzymes in starch synthesis in plants].

Starch, the most common form of stored carbon in plants, is both the major food source for mankind and important raw material for many industries. It is composed of two types of alpha-1,4-linked glucan polymer: essentially unbranched amylose and regularly branched amylopectin, and synthesized in photosynthetic and non-photosynthetic organs. Starch is synthesized via four committed enzyme steps: ADP-Glc pyrophosphorylase, which synthesizes sugar nucleotide precursors; starch synthase, which extends the alpha-1,4-linked glucan chains using ADP-Glc; starch-branching enzymes, which introduce alpha-1,6 branch points to form amylopectin; and starch debranching enzymes, which hydrolyze alpha-1,6 branches in glucans. In this paper, recent advances in biochemical characterizations and gene engineering concerning these enzymes were reviewed, and the achievements in gene engineering involved in manipulation of starch amount and quality were also cited.

[Amylo-1,6-glucosidase--an enzyme determining the rate of glycogenolysis in teleost embryos after fertilization]. / Amilo-1,6-gliukozidaza--ferment, opredeliaiushchii skorost' glikogenoliza v zarodyshakh kostistykh ryb posle oplodotvoreniia

The activity of amylo-1,6-glucosidase (EC 3.2.1.33 dextrin: 6-glucohydrolase, "debranching enzyme") in the loach embryos is localized mainly in the blastoderm. On the basis of correlation between the increase in its activity and in the content of hexose monophosphates during embryogenesis, a suggestion is put forward to the effect that the rate of glycogenolysis is controlled by the activity of this enzyme.