A hydrophobic sequence motif common to N-hydroxylating enzymes

Abstract

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

Pseudobactin Biogenesis in the Plant Growth-Promoting Rhizobacterium Pseudomonas Strain B10: Identification and Functional Analysis of the l -Ornithine N 5 -Oxygenase ( psbA ) Gene

“…The conserved N-terminal region (residues 2 to 34 relative to PsbA) is prevalently hydrophobic and contains the putative flavin adenine dinucleotide (FAD)-binding domain. The type 1 signature of the FAD-dependent oxidoreductases (motif GXGXXG; residues 16 to 21 of PsbA) shows a typical replacement of the last glycine with proline; this is a unique feature of hydroxylases involved in siderophore biosynthesis (34). Although such deviation is predicted to distort the ␣-helical structure within the secondary ␤␣␤ fingerprint of flavin-binding proteins (45), recent studies suggest that the deviated sequence is still compatible with the binding of FAD and association with the cytoplasmic membrane (35,36).…”

Mammalian flavin-containing monooxygenases: structure/function, genetic polymorphisms and role in drug metabolism

“…The ATG fingerprint, D(X) 4 ATG, retains only the D and G residues in many of its flavoenzyme relatives. However, N-hydroxylating siderphore enzymes from bacteria and fungi have retained the closest reiteration of the FATGY motif (Stehr et al, 1998) which can be written as D(X) 3 (L/F)ATGY(X) 4 P, where L can be substituted for F among siderphore enzymes. Like other FMO relatives these enzymes also bind FAD (via a modified GXGXXP motif) and NADPH.…”

Studies with Lysine N6-Hydroxylase. Effect of a Mutation in the Assumed FAD Binding Site on Coenzyme Affinities and on Lysine Hydroxylating Activity

“…However, close inspection of the 3D structure of para-hydroxybenzoate hydroxylase and phenol hydroxylase, two monooxygenases belonging to the same family, reveals that the FAD-cofactor is bound in an elongated form with various hydrogen bond interactions between amino acids and the isoalloxazine, ribityl and adenine moieties (Wierenga, 1979;Enroth et al, 1998). Although the N-termini in these two proteins are involved in FAD-binding, as proposed also for lysine N 6 -hydroxylase (Stehr et al, 1998), the majority of interactions occurs with central and even C-terminal parts of the protein. Since the relative contributions of these interactions for FAD binding are not yet known, it is conceivable that deletion of the N-terminus in lysine N 6 -hydroxylase does not lead to a complete loss of FAD binding and thus activity might be retained by the truncated protein.…”

“…The putative FAD binding site of lysine N 6 -hydroxylase and other hydroxylases involved in siderophore biosynthesis consists of a GxGxGP motif in the N-terminal part of the protein (Stehr et al, 1998). The unusual proline in this sequence prompted us to investigate the role of this residue in determining the affinity for FAD and its possible influence on catalysis.…”

“…Most of these enzymes have a conserved amino acid motif for FAD binding consisting of the sequence Gly-X-Gly-X-X-Gly located near the Nterminus of an ␣-helix (Wierenga et al, 1985). We have recently emphasized that all known N-hydroxylating enzymes involved in siderophore biosynthesis carry a proline instead of the last glycine in this nucleotide fingerprint motif (Stehr et al, 1998). This observation raises the question whether this unusual replacement has any effect on FAD binding to lysine N 6 -hydroxylase (LH).…”