The coenzyme analog adenosine 5-diphosphoribose displaces FAD in the active site of p-hydroxybenzoate hydroxylase. An x-ray crystallographic investigation

Laan, J.M. van der; Schreuder, Herman; Swarte, M.B.A.; Wierenga, R.K.; Kalk, K.H.; Hol, W.G.J.; Drenth, Jan

doi:10.1021/bi00444a011

Cited by 24 publications

(9 citation statements)

References 21 publications

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“…Often, the ADP moiety strongly contributes to the interaction between FAD and the apoprotein24585960. However, in TtProDH, the adenosine moiety of FAD does not show any interaction with the enzyme32 (Fig.…”

Section: Discussionmentioning

confidence: 99%

Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor

Huijbers

Martínez‐Júlvez

Westphal

et al. 2017

Sci Rep

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Flavoenzymes are versatile biocatalysts containing either FAD or FMN as cofactor. FAD often binds to a Rossmann fold, while FMN prefers a TIM-barrel or flavodoxin-like fold. Proline dehydrogenase is denoted as an exception: it possesses a TIM barrel-like fold while binding FAD. Using a riboflavin auxotrophic Escherichia coli strain and maltose-binding protein as solubility tag, we produced the apoprotein of Thermus thermophilus ProDH (MBP-TtProDH). Remarkably, reconstitution with FAD or FMN revealed that MBP-TtProDH has no preference for either of the two prosthetic groups. Kinetic parameters of both holo forms are similar, as are the dissociation constants for FAD and FMN release. Furthermore, we show that the holo form of MBP-TtProDH, as produced in E. coli TOP10 cells, contains about three times more FMN than FAD. In line with this flavin content, the crystal structure of TtProDH variant ΔABC, which lacks helices αA, αB and αC, shows no electron density for an AMP moiety of the cofactor. To the best of our knowledge, this is the first example of a flavoenzyme that does not discriminate between FAD and FMN as cofactor. Therefore, classification of TtProDH as an FAD-binding enzyme should be reconsidered.

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Section: Discussionmentioning

confidence: 99%

Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor

Huijbers

Martínez‐Júlvez

Westphal

et al. 2017

Sci Rep

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“…[24][25][26][27] Unlike many other pyridine nucleotide-dependent enzymes, PHBH lacks a Rossmann fold for binding NADPH. Based on sitedirected mutagenesis studies, 26,28,29 an interdomain binding for NADPH was proposed 26 and a switch in coenzyme specificity was achieved.…”

Section: 12mentioning

confidence: 99%

Time-resolved fluorescence analysis of the mobile flavin cofactor in p-hydroxybenzoate hydroxylase

Berg

Grever

Hoek³

et al. 2007

J Chem Sci

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Abstract. Conformational heterogeneity of the FAD cofactor in p-hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations; the 'in' conformation with the isoalloxazine ring located in the active site, and the 'out' conformation with the isoalloxazine ring disposed towards the protein surface. Fluorescence-lifetime analysis of these complexes revealed similar lifetime distributions for the 'in' and 'out' conformations. The reason for this is twofold. First, the active site of PHBH contains various potential fluorescence-quenching sites close to the flavin. Fluorescence analysis of uncomplexed PHBH Y222V and Y222A showed that Tyr222 is responsible for picosecond fluorescence quenching free enzyme. In addition, other potential quenching sites, including a tryptophan and two tyrosines involved in substrate binding, are located nearby. Since the shortest distance between these quenching sites and the isoalloxazine ring differs only little on average, these aromatic residues are likely to contribute to fluorescence quenching. Second, the effect of flavin conformation on the fluorescence lifetime distribution is blurred by binding of the aromatic substrates: saturation with aromatic substrates induces highly efficient fluorescence quenching. The flavin conformation is therefore only reflected in the small relative contributions of the longer lifetimes.

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“…The structure of PHBH is unusual because there is no NADPH-binding domain. So far, crystallographic analysis did not reveal a structure of the enzyme complexed with NADPH, and soaking experiments with the coenzyme analogue ADPR resulted in displacement of FAD by ADPR (van der Laan et al, 1989). Site-directed mutagenesis studies have pointed to the involvement of Arg 44 (Eppink et al, 1995) and His 162 in NADPH binding.…”

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confidence: 99%

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

Eppink

Schreuder²,

Berkel³

1997

Protein Science

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135

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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 ...

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The coenzyme analog adenosine 5-diphosphoribose displaces FAD in the active site of p-hydroxybenzoate hydroxylase. An x-ray crystallographic investigation

Cited by 24 publications

References 21 publications

Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor

Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor

Time-resolved fluorescence analysis of the mobile flavin cofactor in p-hydroxybenzoate hydroxylase

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

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