Structural Base for Enzymatic Cyclodextrin Hydrolysis

Cyclodextrins resist hydrolysis by burying all bridge oxygens at their interior. Still, the rings can be opened by a small group of specialized enzymes, the cyclomaltodextrinases. Among them, the enzyme from Flavobacterium sp. no. 92 was mutated, crystallized and soaked with cyclodextrins, giving rise to four complex structures. One of them showed an α-cyclodextrin at the outer rim of the active center pocket. In the other complexes, α-, β-and γ-cyclodextrins were bound in a competent mode in the active center. The structures suggest that Arg464 functions as a chaperone guiding the substrates from the solvent into the active center. Over the last part of this pathway, the cyclodextrins bump on Phe274, which rotates the glucosyl group at subsite (+1) by about 120° and fixes it in the new conformation. This induced fit was observed with all three major cyclodextrins. It makes the bridging oxygen between subsites (+1) and (−1) available for protonation by Glu340, which starts the hydrolysis. The mechanism resembles a spring-lock. The structural data were supplemented by activity measurements, quantifying the initial ring opening reaction for the major cyclodextrins and the transglucosylation activity for maltotetraose. Further activity data were collected for mutants splitting the tetrameric enzyme into dimers and for active center mutants.     

Turnover of starch-bound lysophosphatidylcholine in germinating barley

Free and starch-bound lysophosphatidylcholine (LPC) in germinating barley was isolated and quantified during the first 8 days of germination. During the first 4 days the starch-bound LPC remained at a relatively constant level (ca 0.4,μmol/seed) and then declined during the next 2 days to ca 0.1 μmol/seed. There appeared to be no further loss of this starch-bound lipid on further germination. The decrease in the content of starch-bound LPC is not due to the action of phospholipase C and/or D on the starch lipid because there was no corresponding accumulation of starchbound lysophosphatidic acid or monoacylglycerol. The free LPC remained relatively constant at 0.02 to 0.04 μmol/seed during the entire germination period indicating that the LPC released from the starch during days 5 and 6 is further metabolized. Amylase activity was also measured in the germinating seed and increased 20-fold between days 2 and 4 which just precedes the rapid decline in starch-bound LPC. The starch content of the seed however declined to ca one third of the original value by day 5. LPC represents 65–70 % of the starch-bound lipid phosphorus in the dry seed. Through days 5 and 6 when the loss of LPC is most rapid there is no marked change in this percentage. After 8 days, however, the LPC is only ca 30%. of the starch-bound lipid phosphorus. Ofthe two major populations of starch-bound LPC, the one bearing a linoleyl group appears to decline more rapidly during days 4–6 than does that carrying a palmitoyl group. The role of starch-bound LPC in barley development and germination is discussed.     

Separation of Functional Domains for the α-1,4 and α-1,6 Hydrolytic Activities of a Bacillus Amylopullulanase by Limited Proteolysis with Papain

An amylopullulanase (APase) from alkalophilic Bacillus sp. KSM-1378 hydrolyzes both α-1,6 linkages in pullulan and α-1,4 linkages in other polysaccharides, each being maximally active at an alkaline pH, to generate oligosaccharides. We analyzed proteolytic fragments that were produced by exposing pure APase to various proteases, to identify its catalytic domain(s). The intact, pure 210-kDa APase was partially digested with papain for a short time, yielding simultaneously two smaller non-overlapping active fragments, designated amylose-hydrolyzing fragment (AHF114,114 kDa) and pullulan-hydrolyzing fragment (PHF102, 102 kDa). The two truncated protein fragments, each containing a single catalytic domain, were purified to homogeneity. The purified AHF114 and PHF102 had similar enzymatic properties to the amylase and pullulanase activities, respectively, of intact APase. The partial amino-terminal sequences of APase and AHF114 were both Glu-Thr-Gly-Asp-Lys-Arg-Ile-Glu-Phe-Ser-Tyr-Glu-Arg-Pro and that of PHF102 was Thr-Val-Pro-Leu-Ala-Leu-Val-Ser-Gly-Glu-Val-Leu-Ser-Asp-Lys-Leu. These results were direct evidence that the α-1,6 and α-1,4 hydrolytic activities were associated with two different active sites in this novel enzyme. Our alkaline APase is obviously a “biheaded enzyme”.     

The structure of two distinct pancreatic amylase genes in mouse strain YBR

The amylase complex on mouse chromosome 3 encodes both salivary and pancreatic amylase. It appears that one active gene is present for salivary amylase, whereas pancreatic amylase in some strains is coded by at least 4, and perhaps by more than 10, genes. Strain YBR is different from other strains in that it produces twice as much salivary amylase. Pancreatic amylase in YBR is present as two different protein forms, A and B, the sum of which amounts to only one-third of that in, for instance, strain A/J. YBR chromosomal DNA was cloned in phage , followed by restriction and heteroduplex analysis of recombinant phages carrying amylase genes. Among 32 phage isolates, 5 carried parts of the salivary amylase sequence. The remaining phage isolates contained pancreatic amylase-like sequences and represented three nonoverlapping genomic regions, i.e., one of 34 kb containing a complete gene, PAN-II; another of 41 kb with a complete but different gene, PAN-I, plus a truncated gene, PAN-1; and finally, one of 23 kb with another truncated gene, PAN-2. Parts of the amino acid sequence of A and B have previously been determined, and we report here the sequencing of a 4-kb DNA fragment from Pan-II which establishes that this gene codes for B.This work was supported by the Danish Natural Science Research Council.     

Abnormalities of caerulein- and carbamylcholine-stimulated pancreatic enzyme secretion in the obese Zucker rat

The secretory function of the exocrine pancreas has been studied in dispersed pancreatic acini from obese and homozygous lean Zucker rats at 6 and 22 wk. No abnormality was found in acini from young rats. Acini from 22 wk obese and lean rats were equally responsive to secretagogues which stimulate cAMP, i.e. vasoactive intestinal peptide (VIP) and secretin. By contrast, there was a reduction in the maximum responsiveness to caerulein and carbamylcholine in acini from obese rats. These latter secretagogues act through mobilization of intracellular Ca2+. Since obese animals are insulin resistant and amylase release is modulated by insulin, the role of insulin resistance in the secretory defect was then investigated. A group of 22 wk obese rats received treatment with Ciglitazone (a drug which reduces insulin resistance in obese laboratory animals) for 4 wk before the secretion study. Despite the expected reduction in insulin resistance there was no improvement of the secretory defect seen with caerulein and carbamylcholine stimulation. Thus, the secretory abnormality in the exocrine pancreas of adult obese Zucker rats does not appear to be directly associated with insulin resistance. Furthermore, the secretory defect is linked to those secretagogues which induce Ca2+-independent phosphoinositide hydrolysis and Ca2+ mobilization in the target cell.     

Structural basis for the flexible recognition of α‐glucan substrates by Bacteroides thetaiotaomicron SusG

Bacteria that reside in the mammalian intestinal tract efficiently hydrolyze dietary carbohydrates, including starch, that escape digestion in the small intestine. Starch is an abundant dietary carbohydrate comprised of α1,4 and α1,6 linked glucose, yet mammalian intestinal glucoamylases cannot effectively hydrolyze starch that has frequent α1,6 branching as these structures hinder recognition and processing by α1,4‐specific amylases. Here we present the structure of the cell surface amylase SusG from Bacteroides thetaiotaomicron complexed with a mixed linkage amylosaccharide generated from transglycosylation during crystallization. Although SusG is specific for α1,4 glucosidic bonds, binding of this new oligosaccharide at the active site demonstrates that SusG can accommodate α1,6 branch points at subsite ?3 to ?2, and also at subsite+1 adjacent to the site of hydrolysis, explaining how this enzyme may be able to process a wide range of limit dextrins in the intestinal environment. These data suggest that B. thetaiotaomicron and related organisms may have a selective advantage for amylosaccharide scavenging in the gut.     

The raw starch-degrading alkaline amylase ofBacillus sp IMD 370

The amylase ofBacillus sp IMD 370 is the first report of an alkaline amylase with the ability to digest raw starch. The amylase could degrade raw corn and rice starches more effectively than raw potato starch. It showed no adsorb-ability to any type of raw starch at any pH value tested. The enzyme digested raw corn starch to glucose, maltose, maltotriose and maltotetraose. The maximum pH for raw starch hydrolysis was pH 8.0 compared to pH 10.0 for soluble starch hydrolysis. The metal chelator, ethylenediaminetetraacetic acid, strongly inhibited raw starch-digestion and its effect was reversed by the addition of divalent cations. Degradation of raw starch was stimulated six-fold in the presence of -cyclodextrin (17.5 mM).     

坚强芽孢杆菌三个淀粉酶基因的克隆和表达

以pUC18为载体,用鸟枪法从产淀粉水解酶的坚强芽孢杆菌725菌株中得到三个产淀粉水解酶的重组质粒,在大肠杆菌中表达。用高压液相色谱分析了三个表达的酶的淀粉水解产物,其中pBA135和pBA150表达的酶的淀粉水解产物主要是麦芽糖,具β-淀粉酶的性质。pBA140表达的酶的淀粉水解主要产物除麦芽糖外还有一糖,三糖和四糖。pBA135编码的酶有较好的热稳定性,60℃保温30min,活性保留70％以上,最适反应温度55-60℃。而在同样条件下pBA150编码的酶仅保留37％的酶活,最适反应温度50℃。     

Cloning and characterization of a third type of human α-amylase gene, AMY2B

We have previously reported concerning the existence of a third type of human α-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229–235; Tomita et al., Gene 76 (1989) 11–18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human α-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively.Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197–1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5′ region; hence the promoter lies far upstream relative to the other two AMY genes.     

Immunological relationships and post-translational modifications of human salivary amylase (Amy1) and pancreatic amylase (Amy2) isozymes

Electrophoretic phenotypes of human salivary amylase (Amy₁) and pancreatic amylase (Amy₂) consist of complex isozyme patterns which may result from post-translational modifications of the primary products of the amylase loci. Biochemical separation of the two molecular weight families of salivary amylase and development of a new electrophoretic system have allowed the identification of complete isozyme patterns corresponding to variant alleles in Amy ₁ and Amy₂ heterozygotes. Further, immunological studies show no nonidentities among salivary isozymes and among pancreatic isozymes, which is to be expected if each series is derived from a single gene product. Both results support the hypothesis that the primary products of the amylase loci undergo post-translational modifications. Salivary and pancreatic amylase appear to be immunologically identical.This investigation was supported in part by PHS Research Grant GM-19178.Supported by PHS Training Grant DE 119.Supported by PHS Training Grant GM 1056.