Biocatalytic Potential of p‐Hydroxybenzoate Hydroxylase from Rhodococcus rhodnii 135 and Rhodococcus opacus 557

Abstract

AbstractThe biocatalytic potential of the NADH‐dependent p‐hydroxybenzoate hydroxylases (PHBH) from Rhodococcus rhodnii 135 and Rhodococcus opacus 557 was investigated. Monofluorinated 4‐hydroxybenzoates were efficiently hydroxylated, albeit at different rates. 2‐Fluoro‐4‐hydroxybenzoate was a true substrate for PHBH from R. rhodnii 135 but a substrate inhibitor for PHBH from R. opacus 557. Monochlorinated 4‐hydroxybenzoates also acted as PHBH substrates, but with these compounds strong uncoupling of hydroxylation (formation of hydrogen peroxide) occurred. PHBH from R. rhodnii 135 preferred the 5′‐hydroxylation of 2‐chloro‐4‐hydroxybenzoate but the enzyme from R. opacus 557 favored the formation of 2‐chloro‐3,4‐dihydroxybenzoate. Conversely, PHBH from R. rhodnii 135 regioselectively hydroxylated 2‐fluoro‐4‐hydroxybenzoate to 2‐fluoro‐3,4‐dihydroxybenzoate whereas the enzyme from R. opacus 557 also produced significant amounts of 2‐fluoro‐4,5‐dihydroxybenzoate. At high NADH/substrate ratio, both 2‐fluorodihydroxybenzoate products were further converted to 2‐fluoro‐3,4,5‐trihydroxybenzoate. PHBH from R. rhodnii 135 and R. opacus 557 preferred the 5′‐hydroxylation of 3‐chloro‐4‐hydroxybenzoate. However, conversion of 3‐fluoro‐4‐hydroxybenzoate involved considerable dehalogenation affording nearly equal amounts of 3,4‐dihydroxybenzoate and 5‐fluoro‐3,4‐dihydroxybenzoate. At high NADH/substrate ratio, the latter compound was further converted to 3,4,5‐trihydroxybenzoate. The results are discussed in relation to the properties of the NADPH‐specific PHBH from Pseudomonas fluorescens.