Substitution of Arg214 at the substrate‐binding site of p, ‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens

Abstract

The gene encoding p-hydroxybenzoate hydroxylase from Pseudomonas,fluorescens was cloned in Escherichia coli to provide DNA for mutagenesis studies on thc protein product. A plasmid containing a 1.65-kbp insert of P. fluorescens chromosomal DNA was obtained and its nucleotide sequence determined. The DNA-derived amino acid sequence agrees completely with the chemically determined amino acid sequence of the isolated protein. The enzyme is strongly expressed under influence of the vector-encoded lac promotor and is purified to homogeneity in a simple three-step procedure.The relation between substrate binding, the effector role of substrate and hydroxylation efficiency was studied by use of site-directed rnutagenesis. Arg214, in ion-pair interaction with the carboxy moiety of p-hydroxybenzoate, was replaced with Lys, Gln and Ala, respectively. The affinity of the free enzymes for NADPH is unchanged, whereas the affinity for the aromatic substrate is strongly decreased. For enzymes Arg214jAla and Arg214+Gln, the effector role of substrate is lost. For enzyme Arg214+Lys, binding of p-hydroxybenzoate highly stimulates the rate of flavin reduction.In the presence of substrate or substrate analogues, the reduced enzyme Arg214jLys fails to stabilize the 4a-hydroperoxyflavin intermediate, essential for efficient hydroxylation. Like the wild-type, enzyme Arg214+Lys is susceptible to substrate inhibition. From spectral and kinetic results it is suggested that secondary binding of the substrate occurs at the re side of the flavin, where the nicotinamide moiety of NADPH is supposed to bind.p-Hydroxybenzoate hydroxylase is a member of the class of flavin-dependent monooxygenases [I]. The enzyme catalyzes the conversion of 4-hydroxybenzoate to 3,4-dihydroxybenzoate, an intermediate step in the degradation of aromatic compounds in soil bacteria [2]. Like the majority of the flavoprotein aromatic hydroxylases, p-hydroxybenzoate hydroxylase shows a narrow substrate specificity [l, 31. The substrate also acts as an effector, strongly stimulating the reduction of protein-bound FAD by NADPH [3,4].p-H ydroxybenzoate hydroxylase from Pseudomonas Juorescens is one of the best studied flavoproteins [l]. Catalytic and biophysical properties have been investigated in detail [5 -81 and high-resolution crystal structures of the enzyme in complex with substrate or product are available [9 -121. The structure of the free enzyme is less well resolved [13], and the binding mode of NADPH remains to be elucidated [14].

Crystal structure of p‐hydroxybenzoate hydroxylase reconstituted with the modified fad present in alcohol oxidase from methylotrophic yeasts: Evidence for an arabinoflavin

“…Partial uncoupling of substrate hydroxylation results in a relatively high oxidase activity (production of hydrogen peroxide). Such an impaired hydroxylation capacity has also been observed with various mutant enzymes (Entsch et al, 1991van Berkel et al, 1992van Berkel et al, , 1994Eschrich et al, 1993) and with native enzyme reconstituted with artificial flavins (Entsch et al, 1980, Fig. 5.…”

“…The dissociation constant for NADPH as derived from this plot is in the same range as found for the native enzyme-substrate complex (Table 3). The maximal rate of reduction in the presence of 4-hydroxybenzoate is higher than found for native enzyme (Howell et al, 1972;van Berkel et al, 1992; Table 3). Interestingly, an enhanced rate of reduction with respect to the native enzyme-substrate complex has so far only been observed with p-hydroxybenzoate hydroxylase-containing 2-thio-FAD (Claiborne & Massey, 1983).…”

Hydroxylation of Indole by Laboratory-evolved 2-Hydroxybiphenyl 3-Monooxygenase

“…Determination of Dissociation Constants-Dissociation constants between the enzymes and substrates were determined by monitoring the absorption changes of the enzyme-bound FAD upon binding of substrate (27). For this, 12 M purified wild-type HbpA and HbpA ind were titrated with known concentrations of 2-hydroxybiphenyl or indole, and the resulting spectra were recorded using a Varian Cary E1 UV-visible spectrophotometer.…”

4‐Hydroxybenzoate Hydroxylase from Pseudomonas Sp. CBS3

“…Up to now, amino acid sequences of 4-hydroxybenzoate hydroxylase have been obtained from the pobA gene of six related bacterial strains (Entsch et al, 1988;van Berkel et al, 1992;DiMarco et al, 1993;Shuman and Dix, 1993;Wong et al, 1994). Based on the three-dimensional structure of 4-hydroxybenzoate hydroxylase from f!…”

“…No information is available about the biochemical properties and genetic background of the enzyme converting 4-hydroxybenzoate. To date, the DNA sequences of the pobA genes encoding 4-hydroxybenzoate hydroxylase from Pseudomonus aeruginosu (Entsch et al, 1988), PseudomonasJluorescens (van Berkel et al, 1992;Shuman and Dix, 1993), Acinetobacter calcoaceticus (DiMarco et al, 1993), and Rhizobium legumiriosmwn (Wong el al., 1994) have been reported. However, none of these FAD-dependent hydroxylases are involved in the biodegradation of a halogenated aromatic compound.…”