Structure and possible catalytic residues of Taka-amylase A

A complete molecular model of Taka-amylase A consisting of 478 amino acid residues was built with the aid of amino acid sequence data. Some typical structural features of the molecule are described. A model fitting of an amylose chain in the catalytic site of the enzyme showed a possible productive binding mode between substrate and enzyme. On the basis of the difference Fourier analysis and the model fitting study, glutamic acid (Glu230) and aspartic acid (Asp297), which are located at the bottom of the cleft, were concluded to be the catalytic residues, serving as the general acid and base, respectively.     

Studies on the substrate specificity of Taka-amylase A. XII. Investigation of the active site of Taka-amylase A by examining the properties of p-phenylazobenzoyl Taka-amylase A.

1. When p-phenylazobenzoyl Taka-amylase A (PhAB-TAA) was incubated at pH 6.5 with hydroxylamine for 3 hr at 20degrees, some of the p-phenylazobenzoyl residues that had been introduced into Taka-amylase A (TAA) [1, 4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1, Aspergillus oryzae] were liberated as a hydroxamic acid, and the activity pattern of PhAB-TAA changed to that of intact TAA. This result suggested that the p-phenylazobenzoyl residues liberated had been bound to the tyrosyl residue located near the active site in the enzyme. 2. The transferase action of TAA or PhAB-TAA was studied using phenyl alpha-maltoside as a substrate and maltotritol as an acceptor. Unlike intact TAA, PhAB-TAA was not able to transfer the maltose residue to maltotritol, and this suggested that the p-phenylazobenzoyl residue was located near one of the aglycone-binding subsites, causing steric hindrance.     

Structure of a major oligosaccharide of PASII/PMP22 glycoprotein in bovine peripheral nerve myelin

The amino acid sequence of the glycopeptide obtained from bovine PASII/PMP22 protein in the PNS myelin was determined to be Gln-Asn-Cys-Ser-Thr, where the asparagine was glycosylated. To eliminate all the contaminated P(o) glycopeptides from the PASII/PMP22 glycopeptide preparation, we used a fluorescent probe, N-[2-(2-pyridylamino)ethyl]maleimide, which reacts with the cysteine of the PASII/PMP22 glycopeptides. The labeled PASII/PMP22 glycopeptides were isolated by HPLC and were digested further with glycopeptidase A. The resultant oligosaccharides were conjugated with 2-aminopyridine (PA) as a fluorescent tag. One major PA-oligosaccharide, OPPE1, was purified by HPLC. The structure of OPPE1 was elucidated by fast atom bombardment mass spectrometry and (1)H-NMR studies and comparing the derivatives of PA-OPPE1 and PA-oligosaccharides of gamma-globulin on HPLC. The structure, SO(4)-3GlcAbeta1-3Galbeta1-4GlcNAcbeta1-2Manalpha1+ ++-6(GlcNAcbeta1-4) (GlcNAcbeta1-2Manalpha1-3)Manbeta1-4GlcNAcbeta1- 4(Fucalpha1-6)GlcNAc- PA, was identical to the pyridylaminated form of the major oligosaccharide D8 of bovine P(o) previously reported.     

Studies on the substrate specificity of Taka-amylase A1. XIV. Preparation of 6-deoxy-6-halogenomaltotrioses and their hydrolysis by Taka-amylase A.

1. O-6-Deoxy-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, O-6-chloro-6-deoxy-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, O-6-bromo-6-deoxy-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, and O-6-deoxy-6-iodo-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose were prepared, taking advantage of the substrate specificities of Taka-amylase A and glucoamylase, and the action of Taka-amylase A on these substrates was investigated. 2. The Michaelis constant Km and the molecular activity ko were determined at 37 degrees C and pH 5.2 using the modified maltotrioses. The values of Km and ko decreased upon modification of maltotriose and those of ko/Km were in agreement with the comparative initial rates for the corresponding derivatives of phenyl alpha-maltoside at low substrate concentrations. This result suggested that a subsite of the enzyme may have a specific interaction with halogen atoms in the substrate. 3. All halogenomaltotrioses examined showed substrate inhibition at high substrate concentrations.     

Site-directed mutagenesis of catalytic active-site residues of Taka-amylase A.

The cDNA encoding Taka-amylase A (EC.3.2.1.1, TAA) was isolated to identify functional amino acid residues of TAA by protein engineering. The putative catalytic active-site residues and the substrate binding residue of TAA were altered by site-directed mutagenesis: aspartic acid-206, glutamic acid-230, aspartic acid-297, and lysine-209 were replaced with asparagine or glutamic acid, glutamine or aspartic acid, asparagine or glutamic acid, and phenylalanine or arginine, respectively. Saccharomyces cerevisiae strain YPH 250 was transformed with the expression plasmids containing the altered cDNA of the TAA gene. All the transformants with an expression vector containing the altered cDNA produced mutant TAAs that cross-reacted with the TAA antibody. The mutant TAA with alteration of Asp206, Glu230, or Asp297 in the putative catalytic site had no alpha-amylase activity, while that with alteration of Lys209 in the putative binding site to Arg or Phe had reduced activity.     

Studies on the substrate specificity of Taka-amylase A. XIII. Preparation of 6-deoxy-6-iodomaltooligosaccharides and their inhibitory action against Taka-amylase A1.

O-alpha-D-Glucopyranosyl-(1 leads to 4)-O-6-deoxy-6-iodo-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose (6'-MT), O-alpha-D-glucopyranosyl-(1 leads to 4)-6-deoxy-6-iodo-D-glucopyranose (6-M), and O-6-deoxy-6-iodo-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose (6'-M) were prepared and their inhibitory action against Taka-amylase A [EC 3.2.1.1, alpha-1, 4-glucan 4-glucanohydrolase, Aspergillus oryzae] was investigated. The inhibitor constants of 6'-MT and 6'-M were 10 mM and 54 mM, respectively, and both inhibitors showed mixed-type inhibition. 6-M scarcely inhibited the enzyme action.     

Structure of the oligosaccharide of human J chain.

The complete structure of the oligosaccharide moiety of J chain isolated from a Waldenstr?ms macroglobulin Wa has been established. The oligosaccharide is present in three forms differing in the amount of sialic acid and designated J-A, J-B, and J-C. The structure and proportion of each of these is: formula : (see text) : Removal of the oligosaccharide moiety with glycosidases results in an increased mobility of J chain in sodium dodecyl sulfate polyacrylamide gel electrophoresis corresponding to a shift in apparent molecular weight from 23,500 to 19,500. The preparation and utilization of iodinated glycopeptides for sequence analysis is presented.     

Structure of the core oligosaccharide of a rough-type lipopolysaccharide of Pseudomonas syringae pv. phaseolicola.

The core structure of the lipopolysaccharide (LPS) isolated from a rough strain of the phytopathogenic bacterium Pseudomonas syringae pv. phaseolicola, GSPB 711, was investigated by sugar and methylation analyses, Fourier transform ion-cyclotron resonance ESI MS, and one- and two-dimensional 1H-, 13C- and 31P-NMR spectroscopy. Strong alkaline deacylation of the LPS resulted in two core-lipid A backbone undecasaccharide pentakisphosphates in the ratio approximately 2.5 : 1, which corresponded to outer core glycoforms 1 and 2 terminated with either L-rhamnose or 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), respectively. Mild acid degradation of the LPS gave the major glycoform 1 core octasaccharide and a minor truncated glycoform 2 core heptasaccharide, which resulted from the cleavage of the terminal Kdo residues. The inner core of P. syringae is distinguished by a high degree of phosphorylation of L-glycero-D-manno-heptose residues with phosphate, diphosphate and ethanolamine diphosphate groups. The glycoform 1 core is structurally similar but not identical to one of the core glycoforms of the human pathogenic bacterium Pseudomonas aeruginosa. The outer core composition and structure may be useful as a chemotaxonomic marker for the P. syringae group of bacteria, whereas a more conserved inner core structure appears to be representative for the whole genus Pseudomonas.     

Structure of the core oligosaccharide from lipopolysaccharide of Erwinia carotovora.

The lipopolysaccharide of Erwinia carotovora was analyzed by quantitative sugar analysis, methylation analysis, and chromic oxide oxidation. This led to the following structure of the core oligosaccharide: (Formula; see text).     

Modification of subsite Lys residue induced a large increase in maltosidase activity of Taka-amylase A.

A cross-linked modification of Taka-amylase A (TAA) by o-phthalaldehyde gave two enzymes, M1- and M2-TAA, which had optimum pH lower than that of native TAA by 0.5 to 1.0 pH units. The modified enzymes showed higher maltosidase activity, and produced glucose even at the initial period of hydrolysis, in contrast to the native TAA. The modification caused more than a 500-fold decrease in the k0 value of native TAA for alpha-amylase activity, but a definite increase in k0 value of 109. 1 min-1 (native TAA) to 460.0 min-1 (M1-TAA) and 147.1 min-1 (M2-TAA) for maltotriose was evidence for improvement of maltosidase activity of modified enzymes.