PRIMARY AND TERTIARY STRUCTURE STUDIES OF PARA-HYDROXYBENZOATE HYDROXYLASE FROM PSEUDOMONAS-FLUORESCENS - ISOLATION AND ALIGNMENT OF THE CNBR PEPTIDES - INTERACTIONS OF THE PROTEIN WITH FLAVIN ADENINE-DINUCLEOTIDE

Abstract

P-Hydroxybenzoate hydroxylase from P. fluorescens contains 6 methionine residues, one of which is N-terminal. After CNBr cleavage, 5 peptides, ranging from 13-158 residues in length, and free homoserine were isolated and purified by repeated gel filtration. The alignment of the CNBr fragments was deduced from a 0.25 nm electron density map and sequence data. The isolated fragments account for the entire polypeptide chain. The amino acid sequence of the N-terminal quarter of the polypeptide chain was determined. The X-ray results together with the sequence data yielded details of the binding of FAD. The AMP moiety was bound to a .beta..alpha..beta. unit resembling that found in the dehydrogenases. Hydrogen bonds were present between the protein and the ribityl residue and the isoalloxazine ring. A homology was found between the N-terminal amino acid sequence of p-hydroxybenzoate hydroxylase and another enzyme containing FAD, D-amino acid oxidase. The presence of a mononucleotide binding fold is suggested at the N terminus of the latter.