Some polysaccharide processing enzymes possess secondary carbohydrate binding sites situated on the surface far from the active site. In barley alpha-amylase 1 (AMY1), two such sites, SBS1 and SBS2, are found on the catalytic (beta/alpha)(8)-barrel and the noncatalytic C-terminal domain, respectively. Site-directed mutagenesis of Trp(278) and Trp(279), stacking onto adjacent ligand glucosyl residues at SBS1, and of Tyr(380) and His(395), making numerous ligand contacts at SBS2, suggested that SBS1 and SBS2 act synergistically in degradation of starch granules. While SBS1 makes the major contribution to binding and hydrolysis of starch granules, SBS2 exhibits a higher affinity for the starch mimic beta-cyclodextrin. Compared to that of wild-type AMY1, the K(d) of starch granule binding by the SBS1 W278A, W279A, and W278A/W279A mutants thus increased 15-35 times; furthermore, the k(cat)/K(m) of W278A/W279A was 2%, whereas both affinity and activity for Y380A at SBS2 were 10% of the wild-type values. Dual site double and triple SBS1/SBS2 substitutions eliminated binding to starch granules, and the k(cat)/K(m) of W278A/W279A/Y380A AMY1 was only 0.4% of the wild-type value. Surface plasmon resonance analysis of mutants showed that beta-cyclodextrin binds to SBS2 and SBS1 with K(d,1) and K(d,2) values of 0.07 and 1.40 mM, respectively. A model that accounts for the observed synergy in starch hydrolysis, where SBS1 and SBS2 bind ordered and free alpha-glucan chains, respectively, thus targeting the enzyme to single alpha-glucan chains accessible for hydrolysis, is proposed. SBS1 and SBS2 also influence the kinetics of hydrolysis for amylose and maltooligosaccharides, the degree of multiple attack on amylose, and subsite binding energies.
The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase ( galE) can still grow on d-galactose in the presence of ammonium-but not nitrate-ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme ( araA1) did not show NAD(+)-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase ( frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD(+)-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.
This study represents the first characterisation of the substrate-binding site of Bacillus licheniformis K K-amylase (BLA). It describes the first subsite map, namely, number of subsites, apparent subsite energies and the dual product specificity of BLA. The product pattern and cleavage frequencies were determined by high-performance liquid chromatography, utilising a homologous series of chromophore-substituted maltooligosaccharides of degree of polymerisation 4^10 as model substrates. The binding region of BLA is composed of five glycone, three aglycone-binding sites and a 'barrier' subsite.Comparison of the binding energies of subsites, which were calculated with a computer program, shows that BLA has similarity to the closely related Bacillus amyloliquefaciens K Kamylase. ß
The nonreducing end of the substrate-binding site of human salivary a-amylase contains two residues Trp58 and Trp59, which belong to b2-a2 loop of the catalytic (b/a) 8 barrel. While Trp59 stacks onto the substrate, the exact role of Trp58 is unknown. To investigate its role in enzyme activity the residue Trp58 was mutated to Ala, Leu or Tyr. Kinetic analysis of the wild-type and mutant enzymes was carried out with starch and oligosaccharides as substrates. All three mutants exhibited a reduction in specific activity (150-180-fold lower than the wild type) with starch as substrate. With oligosaccharides as substrates, a reduction in k cat , an increase in K m and distinct differences in the cleavage pattern were observed for the mutants W58A and W58L compared with the wild type. Glucose was the smallest product generated by these two mutants in the hydrolysis oligosaccharides; in contrast, wild-type enzyme generated maltose as the smallest product. The production of glucose by W58L was confirmed from both reducing and nonreducing ends of CNP-labeled oligosaccharide substrates. The mutant W58L exhibited lower binding affinity at subsites )2, )3 and +2 and showed an increase in transglycosylation activity compared with the wild type. The lowered affinity at subsites )2 and )3 due to the mutation was also inferred from the electron density at these subsites in the structure of W58A in complex with acarbose-derived pseudooligosaccharide. Collectively, these results suggest that the residue Trp58 plays a critical role in substrate binding and hydrolytic activity of human salivary a-amylase.Keywords: salivary a-amylase; site-directed mutagenesis; subsite engineering; oligosaccharide hydrolysis; crystal structure.a-Amylases (a-1,4-D-glucan glucanohydrolases, EC 3.2.1.1) are endoglucanases, widely distributed in all three domains of life (Bacteria, Archaea and Eucarya), and catalyze reactions such as hydrolysis and transglycosylation of polysaccharides [1,2]. These enzymes, belonging to the glycoside hydrolase family 13 [3], possess very low overall sequence similarity among the various members; nonetheless, in four small regions around the active site, the members exhibit a strong sequence similarity [4][5][6] and harbor the (b/a) 8 barrel topology [7]. This small number of conserved but critical short regions whose residues are lined up along the surface of a deep cleft carries out substrate binding and catalysis in a-amylases [2].In humans, a-amylase is present in both salivary and pancreatic secretions; the overall primary sequences of the pancreatic and salivary a-amylases are highly homologous, and exhibit a high level of structural similarity [8,9]. Human salivary a-amylase (HSAmy) is monomeric, calcium binding protein with a single polypeptide chain of 496 amino acids [9]. The structure of HSAmy consists of three domains: domain A (residues 1-99, 170-404), domain B (residues 100-169) and domain C (residues 405-496). The domain A adopts a (b/a) 8 barrel structure bearing three catalytic residues Asp197, Glu233 an...
Iron-mediated oxidation of low-density lipoprotein has been implicated in the pathogenesis of vascular disorders such as atherosclerosis. The present investigations were performed to test whether hydrophobic fungal siderophores -hexadentate trihydroxamates desferricoprogen, desferrichrome, desferrirubin, and desferrichrysin -might suppress heme-catalyzed LDL oxidation and the toxic effects of heme-treated LDL on vascular endothelium. Indeed, two of these -desferricoprogen and desferrichrome -markedly increased the resistance of LDL to heme-catalyzed oxidation. In similar doseresponse fashion, these siderophores also inhibited the generation of LDL products cytotoxic to human vascular endothelium. When iron-free fungal siderophores were added to LDL/heme oxidation reactions, the product failed to induce heme oxygenase-1, a surrogate marker for the noncytocidal effects of oxidized LDL (not in the case of desferrichrysin). Desferricoprogen also hindered the ironmediated peroxidation of lipids from human atherosclerotic soft plaques in vitro, and was taken up in the gastrointestinal tract of rat. The absorbed siderophore was accumulated in the liver and was secreted in its iron-complexed form in the feces and urine. The consumption of mold-ripened food products such as aged cheeses and the introduction of functional foods and food additives rich in fungal iron chelators in diets may lower the risk of cardiovascular diseases.
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