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Two distinct catalytic pathways for GH43 xylanolytic enzymes unveiled by X-ray and QM/MM simulations.

ABSTRACT: Xylanolytic enzymes from glycoside hydrolase family 43 (GH43) are involved in the breakdown of hemicellulose, the second most abundant carbohydrate in plants. Here, we kinetically and mechanistically describe the non-reducing-end xylose-releasing exo-oligoxylanase activity and report the crystal structure of a native GH43 Michaelis complex with its substrate prior to hydrolysis. Two distinct calcium-stabilized conformations of the active site xylosyl unit are found, suggesting two alternative catalytic routes. These results are confirmed by QM/MM simulations that unveil the complete hydrolysis mechanism and identify two possible reaction pathways, involving different transition state conformations for the cleavage of xylooligosaccharides. Such catalytic conformational promiscuity in glycosidases is related to the open architecture of the active site and thus might be extended to other exo-acting enzymes. These findings expand the current general model of catalytic mechanism of glycosidases, a main reaction in nature, and impact on our understanding about their interaction with substrates and inhibitors.

SUBMITTER: Morais MAB

PROVIDER: S-EPMC7809346 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Two distinct catalytic pathways for GH43 xylanolytic enzymes unveiled by X-ray and QM/MM simulations.

Morais Mariana A B MAB Coines Joan J Domingues Mariane N MN Pirolla Renan A S RAS Tonoli Celisa C C CCC Santos Camila R CR Correa Jessica B L JBL Gozzo Fabio C FC Rovira Carme C Murakami Mario T MT

Nature communications 20210114 1

Xylanolytic enzymes from glycoside hydrolase family 43 (GH43) are involved in the breakdown of hemicellulose, the second most abundant carbohydrate in plants. Here, we kinetically and mechanistically describe the non-reducing-end xylose-releasing exo-oligoxylanase activity and report the crystal structure of a native GH43 Michaelis complex with its substrate prior to hydrolysis. Two distinct calcium-stabilized conformations of the active site xylosyl unit are found, suggesting two alternative ca ...[more]

PMID: 33446650

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Project description:Human arginase I (HARGI) is a metalloprotein highly expressed in the liver cytosol and catalyzes the hydrolysis of l-arginine to form l-ornithine and urea. Understanding the reaction mechanism would be highly helpful to design new inhibitor molecules for HARGI as it is a target for heart- and blood-related diseases. In this study, we explored the hydrolysis reaction mechanism of HARGI with antiferromagnetic and ferromagnetic coupling between two Mn(II) ions at the catalytic site by employing molecular dynamics simulations coupled with quantum mechanics and molecular mechanics (QM/MM). The spin states, high-spin ferromagnetic couple (S Mn1 = 5/2, S Mn2 = 5/2), low-spin ferromagnetic couple (S Mn1 = 1/2, S Mn2 = 1/2), high-spin antiferromagnetic couple (S Mn1 = 5/2, S Mn2 = -5/2), and low-spin antiferromagnetic couple (S Mn1 = 1/2, S Mn2 = -1/2) are considered, and the calculated energetics for the complex of the substrate and HARGI are compared. The results show that the high-spin antiferromagnetic couple (S Mn1 = 5/2, S Mn2 = -5/2) is more stable than other spin states. The low-spin ferromagnetic and antiferromagnetic coupled states are highly unstable compared with the corresponding high-spin states. The high-spin antiferromagnetic couple (S Mn1 = 5/2, S Mn2 = -5/2) is stabilized by 0.39 kcal/mol compared with the ferromagnetic couple (S Mn1 = 5/2, S Mn2 = 5/2). The reaction mechanism is independent of spin states; however, the energetics of transition states and intermediates are more stable in the case of the high-spin antiferromagnetic couple (S Mn1 = 5/2, S Mn2 = -5/2) than the corresponding ferromagnetic state. It is evident that the calculated coupling constants are higher for antiferromagnetic states and, interestingly, superexchange coupling is found to occur between Mn(II) ions via hydroxide ions in a reactant. The hydroxide ion enhances the coupling interaction and initiates the catalytic reaction. It is also noted that the first intermediate structure where there is no superexchange coupling is similar to the known inhibitor 2(S)-amino-6-boronohexanoic acid.

Dataset Information

Two distinct catalytic pathways for GH43 xylanolytic enzymes unveiled by X-ray and QM/MM simulations.

Publications

Two distinct catalytic pathways for GH43 xylanolytic enzymes unveiled by X-ray and QM/MM simulations.

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