6ju1

p-Hydroxybenzoate hydroxylase Y385F mutant complexed with 3,4-dihydroxybenzoate

spinqualitylabelsall models 6ju1, resolution 1.60Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 6ju1 is a 1 chain structure with sequence from Pseudomonas aeruginosa. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.6Å Ligands: BR, DHB, FAD, GOL, P33, PG4 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function PHHY_PSEAE Publication Abstract from PubMed p-Hydroxybenzoate hydroxylase (PHBH) is a flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (p-OHB) to 3,4-dihydroxybenzoate (3,4-DOHB). PHBH can bind to other benzoate derivatives in addition to p-OHB; however, hydroxylation does not occur on 3,4-DOHB. Replacement of Tyr385 with Phe forms a mutant, which enables the production of 3,4,5-trihydroxybenzonate (gallic acid) from 3,4-DOHB, although the catalytic activity of the mutant is quite low. In this study, we report how the L199V/Y385F double mutant exhibits activity for producing gallic acid 4.3-fold higher than that of the Y385F single mutant. This improvement in catalytic activity is primarily due to the suppression of a shunt reaction that wastes reduced nicotinamide adenine dinucleotide phosphate by producing H2O2. To further elucidate the molecular mechanism underlying this higher catalytic activity, we performed molecular dynamics simulations and quantum mechanics/molecular mechanics calculations, in addition to determining the crystal structure of the Y385F.3,4-DOHB complex. The simulations showed that the Y385F mutation facilitates the deprotonation of the 4-hydroxy group of 3,4-DOHB, which is necessary for initiating hydroxylation. Moreover, the L199V mutation in addition to the Y385F mutation allows the OH moiety in the peroxide group of C-(4a)-flavin hydroperoxide to come into the proximity of the C5 atom of 3,4-DOHB. Overall, this study provides a consistent explanation for the change in the catalytic activity of PHBH caused by mutations, which will enable us to better design an enzyme with different activities. Understanding the Molecular Mechanism Underlying the High Catalytic Activity of p-Hydroxybenzoate Hydroxylase Mutants for Producing Gallic Acid.,Moriwaki Y, Yato M, Terada T, Saito S, Nukui N, Iwasaki T, Nishi T, Kawaguchi Y, Okamoto K, Arakawa T, Yamada C, Fushinobu S, Shimizu K Biochemistry. 2019 Nov 1. doi: 10.1021/acs.biochem.9b00443. PMID:31639299 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Hydroxylases 3D structures Monooxygenase 3D structures

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