A hydrophobic sequence• n motif commo to N.hydroxylating enzymesThe 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

-hydroxylase (EC 1.14.13.99) exhibits an unusual proline in a position where a highly conserved glycine is found in other FAD dependent hydroxylases. We have studied the role of this proline by mutating it to glycine in [P14G]aerA, which was expressed in Escherichia coli M15-2 and purified to homogeneity. The mutation has marked effects on the affinities of the cofactors FAD and NADPH as well as the substrate, lysine. Compared to the wild-type enzyme, the activity vs. pH profile of the mutant protein indicates a shift of the apparent pK' a s (7.8 and 8.7 for wild-type and 6.8 and 7.7 for the P14G-mutant enzyme) and of the activity maximum (pH 8 for wild-type and pH 7 for the P14G-mutant enzyme). While the activity of the mutant enzyme is much lower under conditions found to be optimal for the wild-type enzyme, adjustment of substrate and cofactor concentrations and pH leads to comparable activities for the mutant enzyme. These results suggest that the proline fulfils an important structural role in the proposed FAD binding site.

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