Identification of a novel conserved sequence motif in flavoprotein hydroxylases with a putative dual function in FAD/NAD(P)H binding

Abstract

Abstract:A novel conserved sequence motif has been located among the flavoprotein hydroxylases. Based on the crystal structure and site-directed mutagenesis studies of p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas jluorescens, this amino acid fingerprint sequence is proposed to play a dual function in both FAD and NAD(P)H binding. In PHBH, the novel sequence motif (residues 153-166) includes strand A4 and the N-terminal part of helix H7. The conserved amino acids Asp 159, Gly 160, and Arg 166 are necessary for maintaining the structure. The backbone oxygen of Cys 158 and backbone nitrogens of Gly 160 and Phe 161 interact indirectly with the pyrophosphate moiety of FAD, whereas it is known from mutagenesis studies that the side chain of the moderately conserved His 162 is involved in NADPH binding.Keywords: fingerprint; flavoprotein family; NADPH-binding; p-hydroxybenzoate hydroxylase; sequence alignment Flavoprotein hydroxylases are monooxygenases that catalyze the insertion of one atom of molecular oxygen into the substrate, using pyridine nucleotides as external electron donor (van Berkel & Muller, 1991). These enzymes play an important role in the biodegradation of lignin-derived aromatic compounds as well as environmental pollutants, and in the biosynthesis of sterols, antibiotics, and plant hormones. They lack a known fingerprint sequence for NAD(P)H binding, but possess two fingerprint motifs for the FAD binding. The first FAD motif identifies the dinucleotide binding pap-fold, which binds the ADP moiety of FAD (Wierenga et al., 1986), whereas the second motif represents residues that are in contact with the riboflavin moiety of FAD (Eggink et al., 1990).PHBH (EC 1.14.13.2) is the prototype of FAD-dependent hydroxylases, and the only enzyme in this class of flavoproteins for which a three-dimensional structure is known in atomic detail .In the past few years, the number of flavoprotein hydroxylase cloned genes has increased tremendously, and about 50 amino acid sequences are known currently. Therefore, and in view of the unknown binding mode of NADPH in this class of flavoenzymes, it was of interest to search for the presence of conserved sequence motifs. This report describes the identification of a novel sequence motif in flavoprotein hydroxylases, which appears to be important for the binding of both FAD and NAD(P)H. Discussion:Sequence alignments have classified a number of gene products to flavoprotein hydroxylases (Kahn et al., 1992; Kukor & Olsen, 1992;Nakahigashi et al., 1992;Blanco et al., 1993;Filippini et al., 1995; Haigler et al., 1996;Marin et al., 1996; Seibold et al., 1996;Tsuji et al., 1996;Yang et al., 1996). These sequence data, together with that of PHBH from Pseudomonas jluorescens (van Berkel, 1992), were the starting points for a thorough screening of different databases. This search was performed with BEAUTY, which is an BLAST-enhanced alignment utility that integrates multiple biological information resources (Worley et al., 1995). From the 50 collected sequences, small ...

Crystal Structure of 3-Hydroxybenzoate 6-Hydroxylase Uncovers Lipid-assisted Flavoprotein Strategy for Regioselective Aromatic Hydroxylation

“…3 and 4A), resulting in a tight protein-cofactor interaction. One-third of the protein-FAD contacts are strictly conserved among flavoprotein hydroxylases and cluster around the three fingerprint sequences (29).…”

Characterization of a Novel Nicotine Hydroxylase from Pseudomonas sp. ZZ-5 That Catalyzes the Conversion of 6-Hydroxy-3-Succinoylpyridine into 2,5-Dihydroxypyridine

“…WP_031302470.1, 60% identity), respectively. Multiple sequence alignment analyses revealed that HSPHZZ contained the conserved motif Gly-Ala-Glu-GlyAla (amino acids 166-170), which is associated with FAD-and NAD(P)H-binding [25] (Supplementary Figure S2). At the amino acid sequence level, HSPHZZ also showed high identity to p-nitrophenol monooxygenases from Acidovorax sp.…”

Is Renalase a Novel Player in Catecholaminergic Signaling? The Mystery of the Catalytic Activity of an Intriguing New Flavoenzyme

“…Notably, in renalase the isoalloxazine N5 position is solvent exposed [20]. In addition to the Rossman fold GXGXXG signature, flavoprotein hydroxylases display two highly conserved consensus sequences: the 'GD' and the 'DG' motifs [54]. Whereas, the former polypeptide region participates in FAD binding and is conserved in renalase, the latter motif, critical for the interaction with the nicotinamide nucleotide, is absent.…”

“…Whereas, the former polypeptide region participates in FAD binding and is conserved in renalase, the latter motif, critical for the interaction with the nicotinamide nucleotide, is absent. In particular, Gly160 and His162 of PHBH, which are very important for NADPH binding [54], in renalase are both replaced by Pro residues (Pro162 and Pro164), excluding the presence of a functional NAD(P)-binding site in this protein. These considerations, combined with the observation that the FAD structural environment is incompatible with the required chemistry (confirmed by the production of superoxide upon reaction with O 2 [33] instead of hydrogen peroxide as typical in hydroxylases [52]), led to the logical conclusion that renalase cannot be a monooxygenase.…”