Open AccessPrimary and Tertiary Structure Studies of p ‐Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens
Author(s) -
HOFSTEENGE Jan,
VEREIJKEN Johan M.,
WEIJER Wicher J.,
BEINTEMA Jaap J.,
WIERENGA Rik K.,
DRENTH Jan
Publication year - 1980
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1980.tb06148.x
Subject(s) - moiety , pseudomonas fluorescens , stereochemistry , residue (chemistry) , chemistry , protein primary structure , biochemistry , peptide sequence , methionine , enzyme , amino acid , biology , bacteria , genetics , gene
p ‐Hydroxybenzoate hydroxylase from Pseudomonas fluorescens contains six methionine residues, one of which is N‐terminal. After CNBr cleavage five peptides, ranging from 13 to 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 βαβ unit resembling that found in the dehydrogenases. Hydrogen bonds were present between the protein and the ribityl residue and the isoalloxazine ring. Furthermore, a homology was found between the N‐terminal amino acid sequence of p ‐hydroxybenzoate hydroxylase and another enzyme containing FAD, viz. d ‐amino acid oxidase. This finding suggests the presence of a mononucleotide binding fold at the N terminus of the latter.