A QM/MM study of the catalytic mechanism of α-1,4-glucan lyase from the red seaweed Gracilariopsis lemaneiformis

Abstract

a-1,4-Glucan lyase (GLase, EC 4.2.2.13), a unique glycoside hydrolase family member, specifically cleaves the a-1,4-glucosidic linkages in glycogen, starch and malto-oligosaccharides to produce 1,5-anhydro-Dfructose from the non-reducing end. Previous studies have proved that GLase belongs to the retaining glycoside lyase, and the catalytic reaction contains both the glycosylation and deglycosylation/ elimination steps, in which a covalent glycosyl-enzyme intermediate is involved. On the basis of the newly reported crystal structure of GLase (2X2I) and the speculated mechanism, the whole catalytic cycle of GLase has been studied by using a QM/MM method. Calculation results indicate that the whole catalytic cycle contains five elementary steps. Firstly, the aspartic acid residue D665 acts as an acid to protonate the glycoside oxygen, which is concerted with the cleavage of the glycoside bond. Then, the residue D553 functions as the nucleophile to attack the anomeric carbon to form the glycosyl-enzyme intermediate. Different from the retaining a-glucosidases whose glycosylation is a typical concerted process, the glycosylation process of glycosidic lyase follows a stepwise mechanism. For the deglycosylation/elimination step, two cases with or without the maltotriose group in the active site were considered. The departure of the maltotriose can facilitate the proceeding of this process. The deprotonated aspartic acid residue D553 further acts as a catalytic base to abstract the C2-proton of the glucosyl residue. The proton abstraction in the deglycosylation/elimination step is calculated to be the rate-limiting step of the whole catalytic reaction, which corresponds to the energy barriers of 20.69 and 18.53 kcal mol À1 for both of the two cases.Scheme 1 Catalytic reaction of glucan lyase.Scheme 2 Comparison of the catalytic mechanisms of alpha-1,4-glucan lyase (A and B) and alpha-glycosidase (A and B 0 ). This journal is

A Full QM Computational Study of the Catalytic Mechanism of α‐1,4‐Glucan Lyases

“…In this region the surface is rather flat and TS1 leads directly to the glycosyl intermediate Int1 , as depicted in Figure . Actually, these results are similar to those found by Su and coworkers: the oxocarbenium ion intermediate located by these authors (only 0.47 kcal mol −1 lower than TS1 ) is probably only a computational shortcoming with no real physical meaning, whose existence strongly depends on the level of theory and accuracy. Its arguable nature simply indicates that the formation of the glycosyl‐enzyme intermediate is a very asynchronous concerted process (S N 1‐like transition state) consisting of two phases: in the first phase a proton moves from Asp 665 to the glycosyl oxygen causing a significant weakening of the glycosidic bond; in the second phase the nucleophile Asp 553 attacks the anomeric carbon without overcoming any barrier.…”

“…Thus, single‐point computations including solvent effect on the gas‐phase optimized geometries provide reliable reaction energetics. On the whole the M06‐2X and the B3LYP+D functionals provide a similar general mechanistic picture, even if the activation barriers of glycosylation (first step) and catalyst regeneration (third step) change significantly. Importantly, however, TS2 remains the rate‐determining step at the two levels of theory and its energy does not change dramatically (18.3 and 22.7 kcal mol −1 in the former and latter case, respectively). All above results suggest that the large QM region used in our computations is crucial in determining the difference with respect to previous work . An additional effect (related to the previous one) can be ascribed to the type of functional used for estimating long‐range interactions, whose importance increase with the increasing number of residues included in the QM region.…”

“…Importantly, TS2 , corresponding to deglycosylation‐elimination, is the rate determining step of the catalytic process, as pointed out by Su and coworkers . Since it is 11.0 kcal mol −1 above Int1 (7.3 kcal mol −1 above reactants) the overall barrier that must be surmounted to form Pd1 is 18.3 kcal mol −1 .…”

“…Recently Su et al . carried out a computational study on the reaction mechanism of α‐1,4‐glucan lyases up to the formation of enol‐AF.…”

Theoretical study of the hydrolysis mechanism of 2-pyrone-4,6-dicarboxylate (PDC) catalyzed by LigI

“…These questions cannot be acquired by experiments alone. Therefore, in the present work, the catalytic mechanism of LigI was further studied by using a combined quantum mechanics and molecule mechanics (QM/MM) method, which has been successfully applied in exploring the enzymatic mechanism in the past years [17][18][19][20][21][22][23][24]. Based on the results of our calculations, the energetic details of the whole reaction cycle have been given, the structures of the reactant and its involved species, and the roles of key residues have been delineated at atomistic level.…”