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QM/MM Study of the Catalytic Reaction of Myrosinase; Importance of Assigning Proper Protonation States of Active-Site Residues

Jafari, Sonia ; Ryde, Ulf LU and Irani, Mehdi LU (2021) In Journal of Chemical Theory and Computation 17(3). p.1822-1841
Abstract

Myrosinase from Sinapis alba hydrolyzes glycosidic bonds of β-d-S-glucosides. The enzyme shows an enhanced activity in the presence of l-ascorbic acid. In this work, we employed combined quantum mechanical and molecular mechanical (QM/MM) calculations and molecular dynamics simulations to study the catalytic reaction of wild-type myrosinase and its E464A, Q187A, and Q187E mutants. Test calculations show that a proper QM region to study the myrosinase reaction must contain the whole substrate, models of Gln-187, Glu-409, Gln-39, His-141, Asn-186, Tyr-330, Glu-464, Arg-259, and a water molecule. Furthermore, to make the deglycosylation step possible, Arg-259 must be charged, Glu-464 must be protonated on OE2, and His-141 must be... (More)

Myrosinase from Sinapis alba hydrolyzes glycosidic bonds of β-d-S-glucosides. The enzyme shows an enhanced activity in the presence of l-ascorbic acid. In this work, we employed combined quantum mechanical and molecular mechanical (QM/MM) calculations and molecular dynamics simulations to study the catalytic reaction of wild-type myrosinase and its E464A, Q187A, and Q187E mutants. Test calculations show that a proper QM region to study the myrosinase reaction must contain the whole substrate, models of Gln-187, Glu-409, Gln-39, His-141, Asn-186, Tyr-330, Glu-464, Arg-259, and a water molecule. Furthermore, to make the deglycosylation step possible, Arg-259 must be charged, Glu-464 must be protonated on OE2, and His-141 must be protonated on the NE2 atom. The results indicate that assigning proper protonation states of the residues is more important than the size of the model QM system. Our model reproduces the anomeric retaining characteristic of myrosinase and also reproduces the experimental fact that ascorbate increases the rate of the reaction. A water molecule in the active site, positioned by Gln-187, helps the aglycon moiety of the substrate to stabilize the buildup of negative charge during the glycosylation reaction and this in turn makes the moiety a better leaving group. The water molecule also lowers the glycosylation barrier by ∼9 kcal/mol. The results indicate that the Q187E and E464A mutants but not the Q187A mutant can perform the glycosylation step. However, the energy profiles for the deglycosylation step of the mutants are not similar to that of the wild-type enzyme. The Glu-464 residue lowers the barriers of the glycosylation and deglycosylation steps. The ascorbate ion can act as a general base in the reaction of the wild-type enzyme only if the Glu-464 and His-141 residues are properly protonated.

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publication status
published
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in
Journal of Chemical Theory and Computation
volume
17
issue
3
pages
20 pages
publisher
The American Chemical Society (ACS)
external identifiers
  • pmid:33543623
  • scopus:85101929332
ISSN
1549-9618
DOI
10.1021/acs.jctc.0c01121
language
English
LU publication?
yes
id
aeb10ae1-0527-489f-ab01-a50b4cbe0657
date added to LUP
2021-04-25 09:06:03
date last changed
2021-06-16 01:13:18
@article{aeb10ae1-0527-489f-ab01-a50b4cbe0657,
  abstract     = {<p>Myrosinase from Sinapis alba hydrolyzes glycosidic bonds of β-d-<i>S</i>-glucosides. The enzyme shows an enhanced activity in the presence of l-ascorbic acid. In this work, we employed combined quantum mechanical and molecular mechanical (QM/MM) calculations and molecular dynamics simulations to study the catalytic reaction of wild-type myrosinase and its E464A, Q187A, and Q187E mutants. Test calculations show that a proper QM region to study the myrosinase reaction must contain the whole substrate, models of Gln-187, Glu-409, Gln-39, His-141, Asn-186, Tyr-330, Glu-464, Arg-259, and a water molecule. Furthermore, to make the deglycosylation step possible, Arg-259 must be charged, Glu-464 must be protonated on OE2, and His-141 must be protonated on the NE2 atom. The results indicate that assigning proper protonation states of the residues is more important than the size of the model QM system. Our model reproduces the anomeric retaining characteristic of myrosinase and also reproduces the experimental fact that ascorbate increases the rate of the reaction. A water molecule in the active site, positioned by Gln-187, helps the aglycon moiety of the substrate to stabilize the buildup of negative charge during the glycosylation reaction and this in turn makes the moiety a better leaving group. The water molecule also lowers the glycosylation barrier by ∼9 kcal/mol. The results indicate that the Q187E and E464A mutants but not the Q187A mutant can perform the glycosylation step. However, the energy profiles for the deglycosylation step of the mutants are not similar to that of the wild-type enzyme. The Glu-464 residue lowers the barriers of the glycosylation and deglycosylation steps. The ascorbate ion can act as a general base in the reaction of the wild-type enzyme only if the Glu-464 and His-141 residues are properly protonated.</p>},
  author       = {Jafari, Sonia and Ryde, Ulf and Irani, Mehdi},
  issn         = {1549-9618},
  language     = {eng},
  month        = {02},
  number       = {3},
  pages        = {1822--1841},
  publisher    = {The American Chemical Society (ACS)},
  series       = {Journal of Chemical Theory and Computation},
  title        = {QM/MM Study of the Catalytic Reaction of Myrosinase; Importance of Assigning Proper Protonation States of Active-Site Residues},
  url          = {http://dx.doi.org/10.1021/acs.jctc.0c01121},
  doi          = {10.1021/acs.jctc.0c01121},
  volume       = {17},
  year         = {2021},
}