Cloning, nucleotide sequence, and expression of a p-hydroxybenzoate hydroxylase isozyme gene from Pseudomonas fluorescens

B Shuman,T.A Dix

doi:10.1016/s0021-9258(19)85301-2

Abstract

A gene encoding for a putative isozyme of p-hydroxy-benzoate hydroxylase (PHBH) has been isolated from Pseudomonas fluorescens (ATCC 13525). A comparison of the translated amino acid sequence with that of the known PHBH from P. fluorescens revealed that the new enzyme contains 3 additional amino acids and has 73% absolute homology to the previously known enzyme; conservation of secondary and active-site structures implied that the isozyme and known enzyme share the same general tertiary structure. Subsequent expression of the isozyme in Escherichia coli produced an enzyme with a specific activity about half that of the previously characterized PHBHs from P. fluorescens and Pseudomonas aeruginosa; in addition, somewhat weaker binding affinities for both NADPH and p-hydroxybenzoate were observed. Speculations are made on the reason for the existence of the isozyme, which does not appear to be expressed routinely in P. fluorescens.

Highlights

A gene encoding for a putative isozymofep-hydroxy- zymes is relatively untapped
Pseudomonas fluorescens (ATCC 13525).Acomparisonof (Schreuder et al, 1988, 1989); the enzyme is an ideal the translatedaminoacidsequencewiththat of the candidatfeosrite-directedmutagenesistructure-function known PHBH from €? fluorescens revealed that the new studies with the developmentof general oxygentransfer cataenzyme contains 3 additional aminoacids and has 73% lysts as a desirable, and feasible, goal (Ulmer,1983)
Kinetic Characterization of Purified Protein-The dissociation constants for the enzyme-p-hydroxybenzoate andenzyme-NADPH complexes were determinedat 4 ”C in potassium phosphate(50mw), pH 6.6, containing10 mM EDTA, Experiments were conducted in a manner similar t o that described by Husain and Massey (1979) Hanodwell et (11. (1972).To determine the binding of p-hydroxybenzoate t o the oxidized enzyme, 33 VM PHBH was titrated with increasing amounts of p-hydroxybenzoate (0.025-0.065 mM),and the decrease in absorbancaet 497 nm was recorded

Summary

Isolation and Expression of PHBH Isozyme Gene

Model W-185-F sonicator (Heat Systems-Ultrasonics, h e . , Plainview, NY) equipped with a microtip was used for cell lysis. The cells (from radiolabeled with Ia-:'2PldATPas described by Sambrook et al (1989), liters) preparedas described above were centrifuged and taken up in 40 except that SequenaseO Version 2.0 DNA polymerase was usedin place ml of potassium phosphate (50mM), pH 7.25, containing0.3 mM EDTA of the Klenow fragment of E. coli DNA polymerase I. The ends potassium phosphate(7.0 mM), pH 7.0, and dialyzed against two 2-liter were thenflushedwith Klenow fragment,and EcoRI linkerswere changes of the same buffer This solution was loaded onto a hyadded. Kinetic Characterization of Purified Protein-The dissociation constants for the enzyme-p-hydroxybenzoate andenzyme-NADPH complexes were determinedat 4 ”C in potassium phosphate(50mw), pH 6.6, containing mM EDTA, Experiments were conducted in a manner similar t o that described by Husain and Massey (1979) Hanodwell et The dissociation constant was calculated from a double reciprocal plot of the rate of reduction L’ersus NADPH concentration by the method of Benesi and Hildebrand (1949)

RESULTS

Kdb NADPH KJ"''

Findings

DISCUSSION