Tyr217 and His213 are important for substrate binding and hydroxylation of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1.
3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that contains FAD as a redox-active cofactor. The enzyme catalyzes para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on the enzyme crystal structure, residue His213 is located close to the hydroxyl moiety, whereas Tyr217 is close to the carboxylate group of 3HB. Y217A and Y217S did not show any perturbation of flavin absorption upon addition of 3HB, whereas Y217F has a Kd value for 3HB binding of 7.5 mm, which is ~ 50-fold larger than that found for wild-type enzyme. The results clearly indicate that Tyr217 is necessary for substrate binding. All His213 variants can bind to 3HB with similar affinity as the wild-type enzyme and form C4a-hydroperoxy intermediate. H213S, H213D and H213E produce 2,5-DHB with yields of 28 ± 5%, 52 ± 7% and 92 ± 6%, respectively, whereas H213A cannot catalyze hydroxylation. The results indicate that the interaction between the hydroxyl group of 3HB and residue 213 is important for substrate hydroxylation. Interestingly, the hydroxylation rate constant of H213E (35 s(-1) ) is similar to that of wild-type enzyme (36 s(-1) ) and this variant has an efficiency of hydroxylation (92 ± 6%) similar to the wild-type enzyme (86 ± 2%). Difference spectra of enzyme-bound substrate suggest that 3HB binds to H213E in the phenolic form. The results indicate that His213 and Glu213 in H213E may act as a catalytic base to initiate the substrate deprotonation and facilitate the electrophilic aromatic substitution of 3HB.
['Sucharitakul|Jeerus|J|', 'Medhanavyn|Dheeradhach|D|', 'Pakotiprapha|Danaya|D|', 'van Berkel|Willem J H|WJ|', 'Chaiyen|Pimchai|P|']
D001426:Bacterial Proteins D002384:Catalysis D020134:Catalytic Domain D005420:Flavoproteins D005841:Gentisates D006639:Histidine D006900:Hydroxylation D007700:Kinetics D006899:Mixed Function Oxygenases D010084:Oxidation-Reduction D010100:Oxygen D011485:Protein Binding D011487:Protein Conformation D012240:Rhodococcus D013379:Substrate Specificity D013696:Temperature D013816:Thermodynamics D014443:Tyrosine
hydroxylation 3-hydroxybenzoate 6-hydroxylase monooxygenase FAD pre-steady-state kinetics