Studies On Relationships Between Structure And Function Of The Raw-Starch-Degrading ?-Amylase AmyP

?-Amylases?EC 3.2.1.1?are endo-hydrolases that act on ?-?1,4?-glucosidic linkages in starch and other related poly-and oligosaccharides,thus causing the release of malto-oligosaccharides and glucose in the ?-anomeric form.?-Amylases are essential for the conversion of starch into oligosaccharides and are critical for many organisms that use starch as a primary source of energy.?-Amylases also have potential applications in a number of industrial processes,such as processes in the food,fermentation,pharmaceutical and biofuel industries.In the sequence-based classification system of all carbohydrate-active enzymes,glycoside hydrolase family 13?GH13?is the main ?-amylase family that,which,together with GH70 and GH77,forms clan GH-H,in which the catalytic domain adopts a??/??8?TIM?barrel.GH13 ?-amylases use a reaction mechanism that retains anomeric configuration,and share 4-7 conserved sequence regions and catalytic machinery,including the catalytic triad of Asp-Glu-Asp and the primary substrate binding site.The observation that some GH13 members exhibit high mutual sequence similarities has led to the current division of the GH13 family into 42 subfamilies,?-Amylase are distributed among the 11 subfamilies.The GH13₃7 subfamily is the most recently established ?-amylase subfamily.It was created on the basis of the isolation and phylogenetic analysis of a novel ?-amylase,designated AmyP,from a marine metagenomic library.It was found that AmyP is a raw starch-degrading enzyme,exhibiting a unique and remarkable ability to preferentially and very rapidly digest raw rice starch,and the final product was composed of glucose,maltose and maltotriose.It has been proposed to contain a conserved catalytic domain at the N terminus and a starch binding domain at the C terminus.Currently,the GH13₃7 subfamily contains,in addition to AmyP,approximately 56 hypothetical members,most of which are found in marine bacteria.To understand this newest subfamily,we determined the crystal structure of the E221Q mutant AmyP?sBD complexed with maltotriose.Glu221 is one of the three conserved catalytic residues,and AmyP is inactivated by the E221Q mutation.Then,different mutants were designed based on the structural analysis and the corresponding biochemical and enzymatic experiments were implemented.The AmyP?SBD is composed of A,B,and C domains and shares structural features with other GH13 ?-amylases.Domain A forms of a??/??8 barrel that contains the catalytic site.Domain C consists of an antiparallel eight-stranded P-sheet.The B domain of AmyP is the smallest among those of GH13 families of ?-amylases.It forms a loop that protrudes from domain A,has a non-regular secondary structure and does not bind Ca2+ or other metal ions.Owing to this small size of domain is a much wider and more open substrate binding pocket is created,which may facilitate access to starch granule surfaces and increase the rate of hydrolysis of raw starch.The small B domain of AmyP?SBD is situated adjacent to the active center of the enzyme and has a function to reinforce the conformation of the active site and to enhance the enzyme's thermostability.In addition,four cysteine residues in domain A are involved in two disulfide bridges?Cys2-Cys243 and Cys359-Cys365?that are conserved in the GH13₃7 subfamily.In most GH13 ?-amylases,the conserved Ca2+ is present at the junction of domains A and B.In AmyP?SBD,two calcium-binding sites are found,one of which is unique and adjacent to the-3 subsite binding loop in domain A and may be responsible for the increased thermostability of the enzyme after the addition of calcium.Moreover,Tyr36 participates in both stacking and hydrogen bonding interactions with the sugar motif at subsite-3.The observation that maltotriose is more efficient produced by the WT enzyme and maltose is more efficient produced by the Y36A mutant during the early stage of hydrolysis of starch is consistent with the structural information.In sum,this work provides the first insights into the structure of ?-amylases belonging to subfamily 37 of GH13 and may contribute to the rational design of ?-amylase mutants with enhanced performance in biotechnological applications.