A Single-Site Mutation at Ser146 Expands the Reactivity of the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

Abstract

The oxygenase component (C) of p-hydroxyphenylacetate (4-HPA) 3-hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of various phenolic acids. In this report, we found that substitution of a residue close to the phenolic group binding site to yield the S146A variant resulted in an enzyme that is more effective than the wild-type in catalyzing the hydroxylation of 4-aminophenylacetate (4-APA). Product yields for both wild-type and S146A enzymes are better at lower pH values. Multiple turnover reactions of the wild-type and S146A enzymes indicate that both enzymes first hydroxylate 3-APA to give 3-hydroxy-4-aminophenylacetate (3-OH-4-APA), which is further hydroxylated to give 3,5-dihydroxy-4-aminophenylacetate, similar to the reaction of C with 4-HPA. Stopped-flow experiments showed that 4-APA can only bind to the wild-type enzyme at pH 6.0 and not at pH 9.0, while it can bind to S146A under both pH conditions. Rapid-quench flow results indicate that the wild-type enzyme has low reactivity toward 4-APA hydroxylation, with a hydroxylation rate constant (k) for 4-APA of 0.028 s compared to 17 s for 4-HPA, the native substrate. In contrast, for S146A, the hydroxylation rate constants for both substrates are very similar (2.6 s for 4-HPA versus 2.5 s for 4-APA). These data indicate that Ser146 is a key catalytic residue involved in optimizing C reactivity toward a phenolic compound. Removing this hydroxyl group expands C activity toward a non-natural aniline substrate. This understanding should be helpful for future rational engineering of other two-component flavin-dependent monooxygenases that have this conserved Ser residue.

Oxidative dehalogenation and denitration by a flavin-dependent monooxygenase is controlled by substrate deprotonation

“…In another two-component flavin-dependent monooxygenase, p -hydroxyphenylacetate hydroxylase (C 2 ), His 120 was identified as a residue which potentially serves as an active site base to abstract a proton from the phenolic moiety. 85 , 86 Overall, the key catalytic features to deprotonate the phenolic substrate to facilitate the hydroxylation reaction in two-component and single-component monooxygenases are completely different.…”

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

“…Furthermore, the individual rate constants associated with each step and the mechanism of reduced flavin transfer between the two components have been reported (24 -30). This knowledge has been useful for applying HPAH for the synthesis of bioactive phenolic acids and in the hydroxylation of aniline derivatives (31,32).…”

Unique Biochemical and Sequence Features Enable BluB To Destroy Flavin and Distinguish BluB from the Flavin Monooxygenase Superfamily

“…For example, cyclohexanone monooxygenase (and other Baeyer-Villiger flavoprotein catalysts) requires a deprotonated C4a-peroxyflavin (C4a-OO − ) intermediate that is favored at high pH, rather than a hydroperoxyflavin (C4a-OOH),42 and in the flavin-containing monooxygenase enzyme SidA, the C4a-OOH hydroxylates the substrate more efficiently at a higher pH 43. In contrast, the hydroxylation efficiency of a modified oxygenase component of p -hydroxyphenylacetate-3-hydroxylase is more efficient at a lower pH 44. To examine whether any steps in the BluB reaction are influenced by pH, we conducted stopped-flow analysis on reactions initiated by mixing FMNH 2 -bound BluB using buffers ranging from 6.0 to 9.5.…”