Expression of the isoamylase gene of Flavobacterium odoratum KU in Escherichia coli and identification of essential residues of the enzyme by site-directed mutagenesis.
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The isoamylase gene from Flavobacterium odoratum KU was cloned into and expressed in Escherichia coli JM109. The promoter of the gene was successful in E. coli, and the enzyme produced was excreted into the culture medium, depending on the amount of the enzyme expressed. The enzyme found in the culture medium showed almost the same M(r), heat-inactivating constant, and N-terminal sequence as those of the enzyme accumulated in the periplasmic space. This result indicated that the enzyme accumulated in an active form at the periplasm was transported out of the cell. The primary sequence of the enzyme, which was deduced from its nucleotide sequence, showed that the mature enzyme consisted of 741 amino acid residues. By changing five possible residues to Ala independently, it was found that Asp-374, Glu-422, and Asp-497 were essential. The sequences around those residues were highly conserved in isoamylases of different origins and the glycogen operon protein X, GlgX. The comparison of the distance between these essential residues with those of various amylases suggested that the bacterial and plant isoamylase but not GlgX had a longer fourth loop than the other amylases. This longer fourth loop had a possible role in accommodating the long branched chains of native glycogens and starches. (+info)
Biochemical characterization of wild-type and mutant isoamylases of Chlamydomonas reinhardtii supports a function of the multimeric enzyme organization in amylopectin maturation.
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Chlamydomonas reinhardtii mutants of the STA8 gene produce reduced amounts of high amylose starch and phytoglycogen. In contrast to the previously described phytoglycogen-producing mutants of C. reinhardtii that contain no residual isoamylase activity, the sta8 mutants still contained 35% of the normal amount of enzyme activity. We have purified this residual isoamylase and compared it with the wild-type C. reinhardtii enzyme. We have found that the high-mass multimeric enzyme has reduced its average mass at least by one-half. This coincides with the disappearance of two out of the three activity bands that can be seen on zymogram gels. Wild-type and mutant enzymes are shown to be located within the plastid. In addition, they both act by cleaving off the outer branches of polysaccharides with no consistent difference in enzyme specificity. Because the mutant enzyme was demonstrated to digest phytoglycogen to completion in vitro, we propose that its inability to do so in vivo supports a function of the enzyme complex architecture in the processing of pre-amylopectin chains. (+info)
Does human pancreas contain salivary-type isoamylase?
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Amylase isoenzyme analysis was made of extracts of normal human pancreatic tissue by first conducting ion exchange chromatography of the purified material. This gave evidence of only pancreatic type (P-type) isoamylase for all purposes. However, when effluent fractions in which salivary type isoamylase would ordinarily be expected to be present were harvested, pooled, concentrated, and rechromatographed, the pancreatic extracts were found to contain some salivary type (S-type) isoamylase. The latter accounted for approximately 0-8 to 1-7% of the total recovered amylase activity. This finding of S-type isoamylase in normal human pancreas potentially has important bearing on the interpretation of isamylase analysis. (+info)
Three isoforms of isoamylase contribute different catalytic properties for the debranching of potato glucans.
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Isoamylases are debranching enzymes that hydrolyze alpha-1,6 linkages in alpha-1,4/alpha-1,6-linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated. cDNA clones encoding three distinct isoamylase isoforms (Stisa1, Stisa2, and Stisa3) have been identified from potato. The expression patterns of the genes are consistent with the possibility that they all play roles in starch synthesis. Analysis of the predicted sequences of the proteins suggested that only Stisa1 and Stisa3 are likely to have hydrolytic activity and that there probably are differences in substrate specificity between these two isoforms. This was confirmed by the expression of each isoamylase in Escherichia coli and characterization of its activity. Partial purification of isoamylase activity from potato tubers showed that Stisa1 and Stisa2 are associated as a multimeric enzyme but that Stisa3 is not associated with this enzyme complex. Our data suggest that Stisa1 and Stisa2 act together to debranch soluble glucan during starch synthesis. The catalytic specificity of Stisa3 is distinct from that of the multimeric enzyme, indicating that it may play a different role in starch metabolism. (+info)
Hyperamylasaemia after duodenoscopy and retrograde cholangiopancreatography.
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The salivary and pancreatic isoamylases of serum were determined separately in 234 cases of duodenoscopy and retrograde cholangiopancreatography. Successful pancreatic opacification was associated with pathologically high pancreatic serum amylase activities in 60% of the cases. Extensive opacification was associated with large increases of pancreatic serum isoamylases, the maximal rise recorded was 40 times the initial value. In spite of these striking chemical events only two patients developed clinical acute pancreatitis. There were some variations in pancreatic opacification and in the elevation of pancreatic serum amylase which seemed to depend upon the particular contrast material used. A rise of the salivary serum isoamylases caused pathologically high total serum amylase activities in 7% of the cases. High levels of pancreatic serum isoamylase activity before the time of examination did not result in any different pattern of hyperamylasaemia. (+info)
Structure of di-O-alpha-maltosyl cyclodextrins produced from alpha-maltosylfluoride and cyclodextrins.
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The structures of di-O-alpha-maltosyl beta-cyclodextrins ((G2)2-beta-CDs), which were produced from alpha-maltosylfluoride (alpha-G2F) and cyclodextrin (CD) by the transfer action of debranching enzymes, were examined by the enzymic method using Bacillus subtilis saccharifying alpha-amylase (BSA). (G2)2-beta-CD was converted to (G1)2-beta-CD by treatment with glucoamylase before the examination. BSA completely hydrolyzed (G1)2-beta-CD to produce glucose, 6(3)-O-alpha-glucosylmaltotriose, and 6(3),6(5)-di-O-alpha-glucosyl maltopentaose. (G2)2-beta-CD was the mixture of 6A,6C-di-O-alpha-maltosyl beta-CD and 6A,6D-di-O-alpha-maltosyl beta-CD. The ratio of A,C/A,D in (G2)2-beta-CD synthesized with Pseudomonas isoamylase and Aerobacter pullulanase were 40:60-45:55 and 30:70, respectively. The content of 6A,6C-di-O-alpha-maltosyl gamma-CD in (G2)2-gamma-CD synthesized by isoamylase was about 35%. (+info)
Action of neopullulanase. Neopullulanase catalyzes both hydrolysis and transglycosylation at alpha-(1----4)- and alpha-(1----6)-glucosidic linkages.
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The transglycosylation reaction catalyzed by neopullulanase was analyzed. Radioactive oligosaccharides were produced when the enzyme acted on maltotriose in the presence of [U-14C]glucose. Some of the radioactive oligosaccharides had only alpha-(1----4)-glucosidic linkages, but others were suggested to have alpha-(1----6)-glucosidic linkages. The existence of alpha-(1----6)-glucosidic linkages in the products from maltotriose with neopullulanase was proven by proton NMR spectroscopy and methylation analysis. We previously reported that the one active center of neopullulanase catalyzes the hydrolysis of alpha-(1----4)- and alpha-(1----6)-glucosidic linkages (Kuriki, T., Takata, H., Okada, S., and Imanaka, T. (1991) J. Bacteriol. 173,6147-6152). These facts proved that neopullulanase catalyzed all four types of reactions: hydrolysis of alpha-(1----4)-glucosidic linkage, hydrolysis of alpha-(1----6)-glucosidic linkage, transglycosylation to form alpha-(1----4)-glucosidic linkage, and transglycosylation to form alpha-(1----6)-glucosidic linkage. The four reactions are typically catalyzed by alpha-amylase, pullulanase, cyclomaltodextrin glucanotransferase, and 1,4-alpha-D-glucan branching enzyme, respectively. These four enzymes have some structural similarities to one other, but reactions catalyzed by the enzymes are considered to be distinctive: the four reactions are individually catalyzed by each of the enzymes. The experimental results obtained from the analysis of the reaction of the neopullulanase exhibited that the four reactions can be catalyzed in the same mechanism. (+info)
Starch granule initiation is controlled by a heteromultimeric isoamylase in potato tubers.
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Starch granule initiation is not understood, but recent evidence implicates a starch debranching enzyme, isoamylase, in the control of this process. Potato tubers contain isoamylase activity attributable to a heteromultimeric protein containing Stisa1 and Stisa2, the products of two of the three isoamylase genes of potato. To discover whether this enzyme is involved in starch granule initiation, activity was reduced by expression of antisense RNA for Stisa1 or Stisa2. Transgenic tubers accumulated a small amount of a soluble glucan, similar in structure to the phytoglycogen of cereal, Arabidopsis, and Chlamydomonas mutants lacking isoamylase. The major effect, however, was on the number of starch granules. Transgenic tubers accumulated large numbers of tiny granules not seen in normal tubers. These data indicate that the heteromultimeric isoamylase functions during starch synthesis to suppress the initiation of glucan molecules in the plastid stroma that would otherwise crystallize to nucleate new starch granules. (+info)