A hydrophobic sequence motif common to N-hydroxylating enzymes.

The first committed step in the biosynthesis of various bacterial and fur,gal siderophores (low-molecular-weight iron chelators that are produc~ in response '~o iron deficiency) of the hydroxamate type, such as aerobactin, alcaligin an6, ferrichrome, involves N-hydroxylatie a of a primary amino group. This reaction is catalyzed at the expense of NADPH by a family o[ FAD-dependent enzym ~s. Some of the siderophores act as virulence factors i. A similar reaction is carried out by a family of mammalian flavin-containing dimethylaniline monooxygenases. In the latter case, the subs~rates are tertiary and secondary diet-derived alkylamines and, as such, these enzymes play a role in the degradation of xenobiotics. Deficiency in this enzyme activi:y was recognized as the cause of the inheritable 'fish-odor syndrome' (trime:hylaminuria) which is characterized [,y an increased excretion of malodorous teimethylamine'. A BLAST sear~ch 3 in the NCBI non-redundant database (as of 5 August 1997) and seqt~ence alignment with CLUSTALX (ReL 4)and MACAW (Ref. 5) of four siderophore biosynthetic enzymes from EschericMa coil (aerA; iucD) 6,7, Pseudomonu:~ aeruginosa (pvdA) ", Bordetella b:'onchiseptica (alcA) 9 and Ustilago macdis (sid 1) 1° and about 30 sequences of flavin-containing mammalian monooxygenases (nine representati-,e sequences are shown in Fig. l) revealed three dominant areas of similarity (Fig, la, b and c). As expected, all proteins contained two nucleotide-binding folds; the N-terminal fold was assigned as the FAD and the one towards the centre as the NADP binding site (Fig. la and b, respectively)! u2. The FAD-binding site of the mammalian monooxygenases has the typical fingerprint sequence GXGXXG, whereas the siderophore biosynthetic enzymes (alcA, iucD, pvdA and sidl, see Fig. 1) exhibit an exchange of the last glycine to proline. This quite unusual replacement is unique among FAD-dependent enzymes and it was assumed to be the cause of the weak binding of FAD to lysine N~-hydroxylase (EC 1.14.13...)L~. Similarly, alcA and pvdA possess an alanine and sidl a serine instead o[ the last glycine in the putative NADP-binding site. The third and new sequence similarity was discovered in the C-terminal part of the proteins (Fig. lc). The similarity starts with a highly conserved aspartate and is followed by eight hydrophobic amino acids. The core region consists of the sequence L/FATGY and ends with a proline after four variable amino acids. An exception was found in the two ornithine NS-hydroxylases