Carboxyl-terminal truncations of the melibiose carrier of Escherichia coli

The melibiose carrier of Escherichia coli is predicted to possess a short NH2 terminus, 11 transmembrane segments joined by short hydrophilic regions, and a 40-residue hydrophilic carboxyl terminus of unknown function. This paper describes truncations of the carboxyl terminus at eight locations using site-specific mutagenesis to introduce stop codons. Measurement of sugar transport and cation-coupling characteristics indicate that the carboxyl tail plays no direct role in substrate recognition or energy transduction. Thirty-six amino acids could be removed from the hydrophilic carboxyl domain without the loss of sugar specificity, facilitated diffusion, uphill transport, H+-coupling or Na+-coupling characteristics. These results are consistent with the hypothesis that the sugar/cation binding site is formed by the interaction of the transmembrane helices 3, 4, 6, 9, and 10 and does not involve the carboxyl-terminal portion of the protein. When truncations were made within the hydrophobic domain of transmembrane helix 11 (truncations of 41 or more residues), the carrier was no longer found in the membrane. This suggests that the carboxyl terminus may be involved in the membrane insertion process, stabilization of the carrier within the membrane following insertion, or protection of the inserted carrier from proteolytic scavenging. A new plasmid that expresses the temperature-resistant isoform of the melibiose carrier under inducible control of a tac promoter, designated pKKMB, is also described.     

Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast.

Active loading of the phloem with sucrose in leaves is an essential part of the process of supplying non-photosynthetic tissues with carbon and energy. The transport is protein mediated and coupled to proton-symport, but so far no sucrose carrier gene has been identified. Using an engineered Saccharomyces cerevisiae strain, a cDNA from spinach encoding a sucrose carrier was identified by functional expression. Yeast strains that allow the phenotypic recognition of a sucrose carrier activity were constructed by expressing a cytoplasmic invertase from yeast, or the potato sucrose synthase gene, in a strain unable to transport or grow on sucrose due to a deletion in the SUC2 gene. A spinach cDNA expression library established from the poly(A)+ RNA from source leaves of spinach and cloned in a yeast expression vector yielded transformed yeast clones which were able to grow on media containing sucrose as the sole carbon source. This ability was strictly linked to the presence of the spinach cDNA clone pS21. Analysis of the sucrose uptake process in yeast strains transformed with this plasmid show a pH-dependent uptake of sucrose with a Km of 1.5 mM, which can be inhibited by maltose, alpha-phenylglucoside, carbonyl cyanide m-chlorophenylhydrazone and p-chloromercuribenzenesulfonic acid. These data are in accordance with measurements using both leaf discs and plasma membrane vesicles from leaves of higher plants. DNA sequence analysis of the pS21 clone reveals the presence of an open reading frame encoding a protein with a molecular mass of 55 kDa. The predicted protein contains several hydrophobic regions which could be assigned to 12 membrane-spanning regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural basis for the cyclization of the lipopeptide antibiotic surfactin by the thioesterase domain SrfTE

Many biologically active natural peptides are synthesized by nonribosomal peptide synthetases (NRPS). Product release is accomplished by dedicated thioesterase (TE) domains, some of which catalyze an intramolecular cyclization to form macrolactone or macrolactam cyclic peptides. The excised 28 kDa SrfTE domain, a member of the alpha/beta hydrolase enzyme family, exhibits a distinctive bowl-shaped hydrophobic cavity that hosts the acylpeptide substrate and tolerates its folding to form a cyclic structure. A substrate analog confirms the substrate binding site and suggests a mechanism for substrate acylation/deacylation. Docking of the peptidyl carrier protein domain immediately preceding SrfTE positions the 4'-phosphopantheinyl prosthetic group that transfers the nascent acyl-peptide chain to SrfTE. The structure provides a basis for understanding the mechanism of acyl-PCP substrate recognition and for the cyclization reaction that results in release of the macrolactone cyclic heptapeptide.     

Small-molecule glucosylation by sucrose phosphorylase: structure-activity relationships for acceptor substrates revisited

Sucrose phosphorylase catalyzes the O-glucosylation of a wide range of acceptor substrates. Acceptors presenting a suitable 1,2-diol moiety are glucosylated exclusively at the secondary hydroxyl. Production of the naturally occurring compatible solute, 2-O-α-d-glucopyranosyl-sn-glycerol, from sucrose and glycerol is a notable industrial realization of the regio- and stereoselective biotransformation promoted by sucrose phosphorylase. The acceptor substrate specificity of sucrose phosphorylase was analyzed on the basis of recent high-resolution crystal structures of the enzyme. Interactions at the acceptor binding site, observed in the crystal (d-fructosyl) and suggested by results of docking experiments (glycerol), are used to rationalize experimentally determined efficiencies and regioselectivities of enzymatic glucosyl transfer.     

Unique Substrate Recognition Mechanism of the Botulinum Neurotoxin D Light Chain

Botulinum neurotoxins are the most potent protein toxins in nature. Despite the potential to block neurotransmitter release at the neuromuscular junction and cause human botulism, they are widely used in protein therapies. Among the seven botulinum neurotoxin serotypes, mechanisms of substrate recognition and specificity are known to a certain extent in the A, B, E, and F light chains, but not in the D light chain (LC/D). In this study, we addressed the unique substrate recognition mechanism of LC/D and showed that this serotype underwent hydrophobic interactions with VAMP-2 at its V1 motif. The LC/D B3, B4, and B5 binding sites specifically recognize the hydrophobic residues in the V1 motif of VAMP-2. Interestingly, we identified a novel dual recognition mechanism employed by LC/D in recognition of VAMP-2 sites at both the active site and distal binding sites, in which one site of VAMP-2 was recognized by two independent, but functionally similar LC/D sites that were complementary to each other. The dual recognition strategy increases the tolerance of LC/D to mutations and renders it a good candidate for engineering to improve its therapeutic properties. In conclusion, in this study, we identified a unique multistep substrate recognition mechanism by LC/D and provide insights for LC/D engineering and antitoxin development.     

Human lysosomal beta-glucosidase: kinetic characterization of the catalytic, aglycon, and hydrophobic binding sites

Three binding sites on highly purified lysosomal beta-glucosidase from human placenta were identified by studies of the effects of interactions of various enzyme modifiers. The negatively charged lipids, taurocholate and phosphatidylserine, were shown to be noncompetitive, nonessential activators of 4-methylumbelliferyl-beta-D-glucoside hydrolysis. Similar results were observed using the natural substrate, glucosyl ceramide, and low concentrations of taurocholate (less than 1.8 mM) or phosphatidylserine (0.5 mM). However, higher concentrations resulted in a complex partial inhibition of glucosyl ceramide hydrolysis. Increasing concentrations of phosphatidylserine obviated the effects of taurocholate, suggesting that these compounds compete for a common binding site on the enzyme. Glucosyl sphingosine and its N-hexyl derivative were potent noncompetitive inhibitors of the enzyme activity using either substrate. Taurocholate (or phosphatidylserine) and glucosyl sphingosine were shown to be mutually exclusive, indicating competition for a common binding site. In contrast, octyl- and dodecyl-beta-glucosides were linear-mixed-type inhibitors of glucosyl ceramide or 4-methylumbelliferyl-beta-D-glucoside hydrolysis, indicating at least two binding sites on the enzyme. Inhibition by these alkyl beta-glucosides was observed only in the presence of taurocholate or phosphatidylserine. The competitive component [Ki (slope)] for the two alkyl beta-glucosides decreased with increasing alkyl chain length, and was unaffected by increasing taurocholate or phosphatidylserine concentration. The noncompetitive component [Ki (intercept)] was nearly identical for both alkyl beta-glucosides and was decreased by increasing taurocholate or phosphatidylserine concentration. These results indicated that the negatively charged lipids and alkyl beta-glucosides were not mutually exclusive, but interacted with different binding sites on the enzyme. Gluconolactone was shown to protect the enzyme from inhibition by the catalytic site-directed covalent inhibitor, conduritol B indicating an interaction at a common binding site. In the presence of substrate, taurocholate facilitated the inhibition of gluconolactone or conduritol B epoxide. These studies indicated that lysosomal beta-glucosidase had at least three binding sites: (i) a catalytic site which cleaves the beta-glucosidic moiety, (ii) an aglycon site which binds the acyl or alkyl moieties of substrates and some inhibitors, and (iii) a hydrophobic site which interacts with negatively charged lipids and facilitates enzyme catalysis.     

Trehalulose synthase native and carbohydrate complexed structures provide insights into sucrose isomerization

Various diseases related to the overconsumption of sugar make a growing need for sugar substitutes. Because sucrose is an inexpensive and readily available d-glucose donor, the industrial potential for enzymatic synthesis of the sucrose isomers trehalulose and/or isomaltulose from sucrose is large. The product specificity of sucrose isomerases that catalyze this reaction depends essentially on the possibility for tautomerization of sucrose, which is required for trehalulose formation. For optimal use of the enzyme, targeting controlled synthesis of these functional isomers, it is necessary to minimize the side reactions. This requires an extensive analysis of substrate binding modes and of the specificity-determining sites in the structure. The 1.6-2.2-A resolution three-dimensional structures of native and mutant complexes of a trehalulose synthase from Pseudomonas mesoacidophila MX-45 mimic successive states of the enzyme reaction. Combined with mutagenesis studies they give for the first time thorough insights into substrate recognition and processing and reaction specificities of these enzymes. Among the important outcomes of this study is the revelation of an aromatic clamp defined by Phe(256) and Phe(280) playing an essential role in substrate recognition and in controlling the reaction specificity, which is further supported by mutagenesis studies. Furthermore, this study highlights essential residues for binding the glucosyl and fructosyl moieties. The introduction of subtle changes informed by comparative three-dimensional structural data observed within our study can lead to fundamental modifications in the mode of action of sucrose isomerases and hence provide a template for industrial catalysts.     

Sugar Transport into Protoplasts Isolated from Developing Soybean Cotyledons : II. Sucrose Transport Kinetics, Selectivity, and Modeling Studies

The effects of metabolic inhibitors, pH, and temperature on the kinetics of sucrose uptake protoplasts isolated from developing soybean Glycine max L. cv Wye cotyledons were studied. Structural requirements for substrate recognition by the sucrose carrier were examined by observing the effects of potential alternate substrates for the saturable component on sucrose uptake.     

Sucrose phosphorylase: a powerful transglucosylation catalyst for synthesis of α-D-glucosides as industrial fine chemicals

Abstract

Sucrose phosphorylase is a bacterial transglucosidase that catalyzes conversion of sucrose and phosphate into α-D-glucose-1-phosphate and D-fructose. The enzyme utilizes a glycoside hydrolase-like double displacement mechanism that involves a catalytically competent β-glucosyl enzyme intermediate. In addition to reaction with phosphate, glucosylated sucrose phosphorylase can undergo hydrolysis to yield α-D-glucose or it can decompose via glucosyl transfer to a hydroxy group in suitable acceptor molecules, giving new α-D-glucosidic products. The glucosyl acceptor specificity of sucrose phosphorylase is reviewed, focusing on applications of the enzyme in glucoside synthesis. Polyhydroxylated compounds such as sugars and sugar alcohols are often glucosylated efficiently. Aryl alcohols and different carboxylic acids also serve as acceptors for enzymatic transglucosylation. The natural osmolyte 2-O-(α-D-glucopyranosyl)-sn-glycerol (GG) was prepared by regioselective glucosylation of glycerol from sucrose using the phosphorylase from Leuconostoc mesenteroides. An industrial process for production of GG as active ingredient of cosmetic formulations has been recently developed. General advantages of sucrose phosphorylase as a transglucosylation catalyst lie in the use of sucrose as a high-energy glucosyl donor and the usually weak hydrolase activity of the enzyme towards substrate and product.     

Crystal structure of the covalent intermediate of amylosucrase from Neisseria polysaccharea

The alpha-retaining amylosucrase from the glycoside hydrolase family 13 performs a transfer reaction of a glucosyl moiety from sucrose to an acceptor molecule. Amylosucrase has previously been shown to be able to use alpha-D-glucopyranosyl fluoride as a substrate, which suggested that it could also be used for trapping the reaction intermediate for crystallographic studies. In this paper, the crystal structure of the acid/base catalyst mutant, E328Q, with a covalently bound glucopyranosyl moiety is presented. Sucrose cocrystallized crystals were soaked with alpha-D-glucopyranosyl fluoride, which resulted in the trapping of a covalent intermediate in the active site of the enzyme. The structure is refined to a resolution of 2.2 A and showed that binding of the covalent intermediate resulted in a backbone movement of 1 A around the location of the nucleophile, Asp286. This structure reveals the first covalent intermediate of an alpha-retaining glycoside hydrolase where the glucosyl moiety is identical to the expected biologically relevant entity. Comparison to other enzymes with anticipated glucosylic covalent intermediates suggests that this structure is a representative model for such intermediates. Analysis of the active site shows how oligosaccharide binding disrupts the putative nucleophilic water binding site found in the hydrolases of the GH family 13. This reveals important parts of the structural background for the shift in function from hydrolase to transglycosidase seen in amylosucrase.     

Catalysis within the lipid bilayer-structure and mechanism of the MAPEG family of integral membrane proteins

Integral membrane enzymes of the MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) family catalyze glutathione-dependent transformations of lipophilic substrates harvested from the lipid bilayer. Recent studies of members of this family have yielded extensive insights into the structural basis for their substrate binding and catalytic activity. Most informative are the structural studies of leukotriene C4 synthase, revealing a narrow hydrophobic substrate binding pocket allowing extensive recognition of the aliphatic chain of the LTA(4) substrate. A key feature of the pocket is a tryptophan residue that pins down the omega-end of the aliphatic chain into the active site. Since MAPEG members cannot utilize a hydrophobic effect for substrate binding, this novel mode of substrate recognition appears well suited for harvesting lipophilic substrates from the membrane. The binding mode also allows for the specific alignment of the substrate in the active site, positioning the C6 of the substrate for conjugation with glutathione. The glutathione is in turn bound in a polar pocket submerged into the protein core. Structure-based sequence alignments of human MAPEG members support the notion that the glutathione binding site is highly conserved among MAPEG enzymes and that they use a similar mechanism for glutathione activation.     

Kinetic studies on dextransucrase from the cariogenic oral bacterium Streptococcus mutans

The kinetic mechanism of dextransucrase was studied using the Streptococcus mutans enzyme purified by affinity chromatography to a specific activity of 36.9 mumol/min/mg of enzyme. In addition to dextran synthesis, the enzyme catalyzed sucrose hydrolysis and isotope exchange between fructose and sucrose. The rates of sucrose hydrolysis and dextran synthesis were partitioned as a function of dextran concentration such that exclusive sucrose hydrolysis was observed in the absence of dextran and exclusive dextran synthesis at high dextran concentrations. An analogous situation was observed with fructose-dependent partitioning of sucrose hydrolysis and fructose exchange. Steady state dextran synthesis and fructose isotope exchange kinetics were simplified by assay at dextran or fructose concentrations high enough to eliminate significant contributions from sucrose hydrolysis. This limited dextran synthesis assays to dextran concentrations above apparent saturation. The limitation was diminished by establishing conditions in which the enzyme does not distinguish between dextran as a substrate and product which allowed initial discrimination among mechanisms on the basis of the presence or absence of dextran substrate inhibition. No inhibition was observed, which excluded ping-pong and all but three common sequential mechanisms. Patterns of initial velocity fructose production inhibition and fructose isotope exchange at equilibrium were consistent with dextran synthesis proceeding by a rapid equilibrium random mechanism. A nonsequential segment was apparent in the exchange reaction between fructose and sucrose assayed in the absence of dextran. However, the absence of detectable glucosyl exchange between dextrans and the lack of steady state dextran substrate inhibition indicate that glucosyl transfer to dextran must occur almost exclusively through the sequential route. A review of the kinetic constants from steady state dextran synthesis, fructose product inhibition, and fructose isotope exchange showed a consistency in constants derived from each reaction and revealed that dextran binding increases the affinity of sucrose and fructose for dextransucrase.     

The structure and specificity of Escherichia coli maltose acetyltransferase give new insight into the LacA family of acyltransferases

The crystallographic three-dimensional structure of the Escherichia coli maa gene product, previously identified as a maltose O-acetyltransferase (MAT) [Brand, B., and Boos, W. (1991) J. Biol. Chem. 266, 14113-14118] has been determined to 2.15 A resolution by the single anomalous dispersion method using data from a crystal cocrystallized with trimethyllead acetate. It is shown here that MAT acetylates glucose exclusively at the C6 position and maltose at the C6 position of the nonreducing end glucosyl moiety. Furthermore, MAT shows higher affinity toward artificial substrates containing an alkyl or hydrophobic chain as well as a glucosyl unit. The presence of a long hydrophobic patch near the acceptor site provides the structural explanation for this preference. The three-dimensional structure reveals the expected trimeric left-handed parallel beta-helix structure found in all other known hexapeptide repeat enzymes. In particular, the structure shows similarities both overall and at the putative active site to the recently determined structure of galactoside acetyltransferase (GAT), the lacA gene product [Wang, X.-G., Olsen, L. R., and Roderick, S. L. (2002) Structure 10, 581-588]. The structure, together with the new biochemical data, suggests that GAT and MAT are more closely related than previously thought and might have similar cellular functions. However, while GAT is specific for acetylation of galactosyl units, MAT is specific for glucosyl units and is able to acetylate maltooligosaccharides, an important property for biotechnological applications. Structural differences at the acceptor site reflect the differences in substrate specificity.     

Amylosucrase from Neisseria polysaccharea: novel catalytic properties

Amylosucrase is a glucosyltransferase that synthesises an insoluble alpha-glucan from sucrose. The catalytic properties of the highly purified amylosucrase from Neisseria polysaccharea were characterised. Contrary to previously published results, it was demonstrated that in the presence of sucrose alone, several reactions are catalysed, in addition to polymer synthesis: sucrose hydrolysis, maltose and maltotriose synthesis by successive transfers of the glucosyl moiety of sucrose onto the released glucose, and finally turanose and trehalulose synthesis - these two sucrose isomers being obtained by glucosyl transfer onto fructose. The effect of initial sucrose concentration on initial activity demonstrated a non-Michaelian profile never previously described.     

Weak substrate binding to transport proteins studied by NMR.

The weak binding of sugar substrates fails to induce any quantifiable physical changes in the L-fucose-H+ symport protein, FucP, from Escherichia coli, and this protein lacks any strongly binding ligands for competitive binding assays. Access to substrate binding behavior is however possible using NMR methods which rely on substrate immobiliza-tion for detection. Cross-polarization from proton to carbon spins could detect the portion of 13C-labeled substrate associated with 0.2 micromol of the functional transport system overexpressed in the native membranes. The detected substrate was shown to be in the FucP binding site because its signal was diminished by the unlabeled substrates L-fucose and L-galactose but was unaffected by a three- to fivefold molar excess of the non-transportable stereoisomer D-fucose. FucP appeared to bind both anomers of its substrates equally well. An NMR method, designed to measure the rate of substrate exchange, could show that substrate exchanged slowly with the carrier center (>10(-1) s), although its dynamics are not necessarily coupled strongly to this site within the protein. Relaxation measurements support this view that fluctuations in the interaction with substrate would be confined to the binding site in this transport system.     

Force calculations in automated docking: enzyme-substrate interactions in Fusarium oxysporum Cel7B

AutoDock is a small-molecule docking program that uses an energy function to score docked ligands. Here AutoDock's grid-based method for energy evaluation was exploited to evaluate the force exerted by Fusarium oxysporum Cel7B on the atoms of docked cellooligosaccharides and a thiooligosaccharide substrate analog. Coupled with the interaction energies evaluated for each docked ligand, these forces give insight into the dynamics of the ligand in the active site, and help to elucidate the relative importance of specific enzyme-substrate interactions in stabilizing the substrate transition-state conformation. The processive force on the docked substrate in the F. oxysporum Cel7B active site is less than half of that on the docked substrate in the Hypocrea jecorina Cel7A active site. Hydrogen bonding interactions of the enzyme with the C2 hydroxyl group of the glucosyl residue in subsite -2 and with the C3 hydroxyl group of the glucosyl residue in subsite +1 are the most significant in stabilizing the distorted14B transition-state conformation of the glucosyl residue in subsite -1. The force calculations also help to elucidate the mechanism that prevents the active site from fouling.     

Structure and substrate binding properties of cobB, a Sir2 homolog protein deacetylase from Escherichia coli

Sirtuins are NAD+-dependent protein deacetylase enzymes that are broadly conserved from bacteria to human, and have been implicated to play important roles in gene regulation, metabolism and longevity. cobB is a bacterial sirtuin that deacetylates acetyl-CoA synthetase (Acs) at an active site lysine to stimulate its enzymatic activity. Here, we report the structure of cobB bound to an acetyl-lysine containing non-cognate histone H4 substrate. A comparison with the previously reported archaeal and eukaryotic sirtuin structures reveals the greatest variability in a small zinc-binding domain implicated to play a particularly important role in substrate-specific binding by the sirtuin proteins. Comparison of the cobB/histone H4 complex with other sirtuin proteins in complex with acetyl-lysine containing substrates, further suggests that contacts to the acetyl-lysine side-chain and beta-sheet interactions with residues directly C-terminal to the acetyl-lysine represent conserved features of sirtuin-substrate recognition. Isothermal titration calorimetry studies were used to compare the affinity of cobB for a variety of cognate and non-cognate acetyl-lysine-bearing peptides revealing an exothermic reaction with relatively little discrimination between substrates. In contrast, similar studies employing intact acetylated Acs protein as a substrate reveal a binding reaction that is endothermic, suggesting that cobB recognition of substrate involves a burial of hydrophobic surface and/or structural rearrangement involving substrate regions distal to the acetyl-lysine-binding site. Together, these studies suggest that substrate-specific binding by sirtuin proteins involves contributions from the zinc-binding domain of the enzyme and substrate regions distal to the acetyl-lysine-binding site.     

Molecular cloning, characterisation and ligand-bound structure of an azoreductase from Pseudomonas aeruginosa

The gene PA0785 from Pseudomonas aeruginosa strain PAO1, which is annotated as a probable acyl carrier protein phosphodiesterase (acpD), has been cloned and heterologously overexpressed in Escherichia coli. The purified recombinant enzyme exhibits activity corresponding to that of azoreductase but not acpD. Each recombinant protein molecule has an estimated molecular mass of 23,050 Da and one non-covalently bound FMN as co-factor. This enzyme, now identified as azoreductase 1 from Pseudomonas aeruginosa (paAzoR1), is a flavodoxin-like protein with an apparent molecular mass of 110 kDa as determined by gel-filtration chromatography, indicating that the protein is likely to be tetrameric in solution. The three-dimensional structure of paAzoR1, in complex with the substrate methyl red, was solved at a resolution of 2.18 A by X-ray crystallography. The protein exists as a dimer of dimers in the crystal lattice, with two spatially separated active sites per dimer, and the active site of paAzoR1 was shown to be a well-conserved hydrophobic pocket formed between two monomers. The paAzoR1 enzyme is able to reduce different classes of azo dyes and activate several azo pro-drugs used in the treatment of inflammatory bowel disease (IBD). During azo reduction, FMN serves as a redox centre in the electron-transferring system by mediating the electron transfer from NAD(P)H to the azo substrate. The spectral properties of paAzoR1 demonstrate the hydrophobic interaction between FMN and the active site in the protein. The structure of the ligand-bound protein also highlights the pi-stacking interactions between FMN and the azo substrate.     

Characterisation of a novel amylosucrase from Deinococcus radiodurans

The BLAST search for amylosucrases has yielded several gene sequences of putative amylosucrases, however, with various questionable annotations. The putative encoded proteins share 32-48% identity with Neisseria polysaccharea amylosucrase (AS) and contain several amino acid residues proposed to be involved in AS specificity. First, the B-domains of the putative proteins and AS are highly similar. In addition, they also reveal additional residues between putative beta-strand 7 and alpha-helix 7 which could correspond to the AS B'-domain, which turns the active site into a deep pocket. Finally, conserved Asp and Arg residues could form a salt bridge similar to that found in AS, which is responsible for the glucosyl unit transfer specificity. Among these found genes, locus NP_294657.1 (dras) identified in the Deinococcus radiodurans genome was initially annotated as an alpha-amylase encoding gene. The putative encoded protein (DRAS) shares 42% identity with N. polysaccharea AS. To investigate the activity of this protein, gene NP_294657.1 was cloned and expressed in Escherichia coli. When acting on sucrose, the pure recombinant enzyme was shown to catalyse insoluble amylose polymer synthesis accompanied by side-reactions (sucrose hydrolysis, sucrose isomer and soluble maltooligosaccharide formation). Kinetic analyses further showed that DRAS follows a non-Michaelian behaviour toward sucrose substrate and is activated by glycogen, as is AS. This demonstrates that gene NP_294657.1 encodes an amylosucrase.