Role of Tyr201 and Tyr385 in substrate activation by p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens

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Abstract
The crystal structure of the enzyme-substrate complex of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens shows that the hydroxyl group of 4-hydroxybenzoate interacts with the side chain of Tyr201, which is in close contact with the side chain of Tyr385. The role of this hydrogen bonding network in substrate activation was studied by kinetic and spectral analysis of Tyr-->Phe mutant enzymes. The catalytic properties of the enzymes with Tyr201 or Tyr385 replaced by Phe (Tyr201-->Phe and Tyr385-->Phe) with the physiological substrate are comparable with those of the corresponding mutant proteins of p-hydroxybenzoate hydroxylase from P. aeruginosa [Entsch, B., Palfey, B. A., Ballou, D. P. & Massey, V. (1991) J. Biol. Chem. 266, 17341-17349]. Enzyme Tyr201-->Phe has a high Km for NADPH and produces only 5% of 3,4-dihydroxybenzoate/catalytic cycle. Unlike the wild-type enzyme, the Tyr201-->Phe mutant does not stabilize the phenolate form of 4-hydroxybenzoate. With enzyme Tyr385-->Phe, flavin reduction is rate-limiting and the turnover rate is only 2% of wild type. Despite rather efficient hydroxylation, and deviating from the description of the corresponding P. aeruginosa enzyme, mutant Tyr385-->Phe prefers the binding of the phenolic form of 4-hydroxybenzoate. Studies with substrate analogs show that both tyrosines are important for the fine tuning of the effector specificity. Binding of 4-fluorobenzoate differentially stimulates the stabilization of the 4 alpha-hydroperoxyflavin intermediate. Unlike wild type, both Tyr mutants produce 3,4,5-trihydroxybenzoate from 3,4-dihydroxybenzoate. The affinity of enzyme Tyr201-->Phe for the dianionic substrate 2,3,5,6-tetrafluoro-4-hydroxybenzoate is very low, probably because of repulsion of the substrate phenolate in a more nonpolar microenvironment. In contrast to data reported for p-hydroxybenzoate hydroxylase from P. aeruginosa, binding of the inhibitor 4-hydroxycinnamate to wild-type and mutant proteins is not simply described by binary complex formation. A binding model is presented, including secondary binding of the inhibitor. Enzyme Tyr201-->Phe does not stabilize the phenolate form of the inhibitor. In enzyme Tyr385-->Phe, the phenolic pKa of bound 4-hydroxycinnamate is increased with respect to wild type. It is proposed that Tyr385-->Phe is involved in substrate activation by facilitating the deprotonation of Tyr201.

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