2005
DOI: 10.1128/jb.187.4.1511-1514.2005
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Alpha-Subunit Positions Methionine 180 and Glutamate 214 of Pseudomonas stutzeri OX1 Toluene- o -Xylene Monooxygenase Influence Catalysis

Abstract: Alpha-subunit position M180 of toluene-o-xylene monooxygenase influences the regiospecific oxidation of aromatics (e.g., from o-cresol, M180H forms 3-methylcatechol, methylhydroquinone, and 4-methylresorcinol, whereas the wild type forms only 3-methylcatechol). Position E214 influences the rate of reaction (e.g., E214G increases p-nitrophenol oxidation 15-fold) by controlling substrate entrance and product efflux as a gate residue.Toluene monooxygenases (1,5,7,15,21) are multisubunit catalysts that oxidize ben… Show more

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Cited by 24 publications
(16 citation statements)
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“…Modification of the gating residues has been shown to change the selectivity and activity of toluene- o -xylene monooxygenase, with the E214G mutation improving oxidation of p -nitrophenol by a factor of 15. 101 A similar improvement in overall activity was achieved in a lipase from Burkholderia cepacia by the mutations L17S + L287I. This double mutant also exhibited a 10-fold increase in enantioselectivity compared to the wild-type enzyme.…”
Section: Engineering Of Gatesmentioning
confidence: 56%
“…Modification of the gating residues has been shown to change the selectivity and activity of toluene- o -xylene monooxygenase, with the E214G mutation improving oxidation of p -nitrophenol by a factor of 15. 101 A similar improvement in overall activity was achieved in a lipase from Burkholderia cepacia by the mutations L17S + L287I. This double mutant also exhibited a 10-fold increase in enantioselectivity compared to the wild-type enzyme.…”
Section: Engineering Of Gatesmentioning
confidence: 56%
“…In the wild‐type enzyme, the glutamine residue at this position forms a gate approximately 25 Å from the active site. Mutating this glutamine into a series of smaller residues revealed a proportional increase in enzyme activity . It was then proposed that replacement of glutamine with smaller residues resulted in a permanently opened gate, which was thought to be responsible for the observed increase in the rate of substrate ( p ‐nitrophenol) oxidation …”
Section: The Influence Of Protein Tunnels On Substrate Specificitymentioning
confidence: 99%
“…Furthermore, both positions E214 and D285 are located at the entrance of the tunnel, leading to the active site and facing one another. It was shown that alternating these positions influences the oxidation rate but not the product distribution (5,36). Consequently, combining mutations in the active-site entrance which enable hydroxytyrosol formation and accelerate the reaction rate with mutations in the tunnel mouth lead, as shown here, to elevated activity.…”
Section: Discussionmentioning
confidence: 92%
“…This position is analogous to position TouA E214 in ToMO, which was found by Vardar et al (36) to influence the rate, but not regioselectivity, of o-and p-nitrophenol hydroxylation. It was shown that the size of the residue at position 214 is most likely to be the factor influencing the oxidation rate, leading to 15-fold improvement for p-nitrophenol oxidation by the ToMO E214G variant (36). Accordingly, the Glu at position 214 in TmoA T4MO was chosen to be replaced with Gly, which has the smallest side chain.…”
Section: Resultsmentioning
confidence: 99%