On the Structure of Flavin-Oxygen Intermediates Involved in Enzymatic Reactions

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66396/1/j.1432-1033.1977.tb11579.x.pd

Purification, properties, and oxygen reactivity of p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa

The monooxygenase, p-hydroxybenzoate hydroxylase (4-hydroxybenzoate, NADPH: oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) has been isolated and purified from Pseudomonas aeruginosa. The reaction catalysed is linked to the pathways for degradation of aromatic compounds by microorganisms. The enzyme has been quantitatively characterizd in this paper for use in the mechanistic analysis of the protein by site-directed mutagenesis. This can be achieved when the results presented are used in combination with the information on the sequence and structure of the gene for this protein and the high-resolution crystallographic data for the protein from P. fluorescens. The protein is a dimer of identical sub-units in solution, and has one FAD per polypeptide with a monomeric molecular weight of 45 000. A full steady-state kinetic analysis was carried out at the optimum pH (8.0). A Vmax of 3750 min-1 at 25[deg]C was calculated, and the enzyme has a concerted-substitution mechanism, involving the substrates, NADPH, oxygen, and p-hydrobenzoate. Extensive analyses of the reactions of reduced enzyme with oxygen were carried out. The quality of the data obtained confirmed the mechanisms of these reactions as proposed earlier by the authors for the enzyme from P. fluorescens. It was found that the amino acid residue differences between enzyme from P. fluorescence and aeruginosa do marginally change some observed transient state kinetic parameters, even though the structure of the enzyme shows they have no direct role in catalysis. Thus, transient state kinetic analysis is an excellent tool to examine the role of amino acid residues in catalysis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27633/1/0000009.pd

The cyanobiont in an Azolla fern is neither Anabaena nor Nostoc

The cyanobacterial symbionts in the fern Azolla have generally been ascribed to either the Anabaena or Nostoc genera. By using comparisons of the sequences of the phycocyanin intergenic spacer and a fragment of the 16S rRNA, we found that the cyanobiont from an Azolla belongs to neither of these genera.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75153/1/S0378-1097_03_00784-5.pd

Flavin-Mediated Hydroxylation Reactions

Flavins react with oxygen and can form stable flavin peroxides in an aprotic solvent or buried in a protein. It is this hydroperoxide or peroxide that is the oxygenating agent in flavoproteins. This property is used in nature to carry out aromatic hydroxylations, halogenations, Baeyer-Villiger oxygenations, hydroxylation of xenobiotics and some metabolites, as well as light emission from luciferase. Several groups of enzymes seem to have evolved hydroxylating properties independently of each other. One group consists of the two-component flavin-dependent hydroxylases that sue many of the same principles as the single component hydroxylases, although they also have some special requirements. After a brief introduction to the reactivity of flavins with oxygen, we examine p-hydroxybenzoate hydroxylase as the paradigm for the chemistry and protein functions exhibited by these enzymes. We then discuss unique features of each group of enzymes and the exciting prospects for future research

The reaction mechanisms of Groups A and B flavoprotein monooxygenases

Flavoprotein monooxygenases, found in species ranging from microorganisms to mammals, transfer one oxygen atom derived from O₂ to a substrate, oxidizing it. In this chapter, we review the enzymes in Groups A and B, which accomplish all their chemistry with just one protein. The catalytic cycles of both groups are roughly similar. NAD (P)H reduces the enzyme-bound flavin, which then reacts with oxygen to form a flavin C4a-(hydro)peroxide - the key oxygenating intermediate. The terminal oxygen of the (hydro)peroxide is transferred to the substrate, leaving the hydroxyflavin, which eliminates water to form oxidized enzyme. Catalysis in both groups is strictly regulated, but in very different ways, to limit NAD(P)H oxidase activity. Group A enzymes only allow the fast reaction of NAD(P)H when the substrate to be oxygenated is bound. In contrast, Group B monooxygenases do not require substrate to be present for rapid flavin reduction, but after the flavin is reduced, NAD(P) remains bound, stabilizing the flavin (hydro)peroxide until it encounters the substrate to be oxygenated. The enzymes in Group A are aromatic hydroxylases; they add oxygen to an activated aromatic ring by electrophilic substitution. The most studied flavoprotein monooxygenase, p-hydroxybenzoate hydroxylase, belongs to this group and will be discussed in detail. The enzymes in Group B catalyze nucleophilic and electrophilic oxygenations. Their substrates include aldehydes, ketones, amines, thiols, boronates, selenides, and thioethers. Conformational changes are important for controlling catalysis in both Group A and Group B monooxygenases

Characterization of the Tryptamine Pathway of Auxin Biosynthesis in Developing Rice Grains

The importance of flavin monooxygenases (OsYUCCA), tryptophan decarboxylases (OsTDC), nitrilases (OsNIT), and aldehyde oxidases (OsAO) for auxin production in developing rice grains was investigated. Indole-3-acetic acid (IAA) levels in grains increased from approximately 20ng/g FW to 2 μg/g FW during 14 days after flowering (DAF), with the largest increase in IAA (4 and 7 days DAF) correlating with the major gain in grain fresh weight. The rice genome was found to contain 14 OsYUCCA, 7 OsTDC, 4 OsAO, and 2 OsNIT genes. Phylogenetic analysis showed that OsTDC1 has orthologues across the plant kingdom. OsTDC1 was expressed in developing grains at 1, 7, and 21 DAF, however quantitative RT-PCR analysis did not show a clear correlation between OsTDC1 expression and IAA synthesis. Phylogenetic analysis of OsYUCCAs classified OsYUCCAs 9-14, in the same clade as AtYUCCA10 and AtYUCCA11, which are reported to be involved in seed development. A strong correlation between expression of OsYUCCAs 9 & 11 and IAA content suggested these genes are crucial for IAA synthesis in rice grains. Phylogenetic analysis of AOs suggested that major plant groups inherited one AO sequence with isoforms being products of recent gene duplication. This was surprising as AOs have proposed involvement in both abscisic acid and IAA synthesis. Expression analysis and sequencing showed that of four rice AOs, two (OsAO1 and OsAO2) were expressed in grains at 1, 7, and 21 DAF. OsNIT1 and OsNIT2 are part of a conserved clade with members from a diverse group of plants. RT-PCR results as well as on-line microarray data showed expression of OsNITs in grains at 1, 7, and 21 DAF. Despite evidence of expression there was no clear correlation observed between AO or NIT transcripts and IAA content. Data thus provided strong evidence for the involvement of YUCCA in auxin synthesis in developing grains, but only weak evidence for involvement of TDC, AO and NIT. The observed correlation between expression of tryptophan amino transferase (OsTAA1), and OsYUCCA 9 & 11 however suggested that an alternative pathway which involves both YUCCA and TAA should be considered. This would require a different catalytic activity for YUCCA than that previously proposed

The cyanobiont in an 'Azolla' fern is neither 'Anabaena' nor 'Nostoc'

The cyanobacterial symbionts in the fern 'Azolla' have generally been ascribed to either the 'Anabaena' or 'Nostoc' genera. By using comparisons of the sequences of the phycocyanin intergenic spacer and a fragment of the 16S rRNA, we found that the cyanobiont from an 'Azolla' belongs to neither of these genera

Protein dynamics and electrostatics in the function of 'p'-hydroxybenzoate hydroxylase

'para'-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor, FAD, by NADPH in response to binding 'p'-hydroxybenzoate to the enzyme, then oxidation of reduced FAD by oxygen to form a hydroperoxide, which oxygenates 'p'-hydroxybenzoate to form 3,4-dihydroxybenzoate. These diverse reactions all occur within a single polypeptide and are achieved through conformational rearrangements of the isoalloxazine ring and protein residues within the protein structure. In this review, we examine the complex dynamic behavior of the protein that enables regulated fast and specific catalysis to occur. Original research papers (principally from the past 15 years) provide the information that is used to develop a comprehensive overview of the catalytic process. Much of this information has come from detailed analysis of many specific mutants of the enzyme using rapid reaction technology, biophysical measurements, and high-resolution structures obtained by X-ray crystallography. We describe how three conformations of the enzyme provide a foundation for the catalytic cycle. One conformation has a closed active site for the conduct of the oxygen reactions, which must occur in the absence of solvent. The second conformation has a partly open active site for exchange of substrate and product, and the third conformation has a closed protein structure with the isoalloxazine ring rotated out to the surface for reaction with NADPH, which binds in a surface cleft. A fundamental feature of the enzyme is a H-bond network that connects the phenolic group of the substrate in the buried active site to the surface of the protein. This network serves to protonate and deprotonate the substrate and product in the active site to promote catalysis and regulate the coordination of conformational states for efficient catalysis

Analysis of the pobA and pobR genes controlling expression of p-hydroxybenzoate hydroxylase in 'Azotobacter chroococcum'

We report the cloning and analysis of a gene and its cognate regulatory element from a member of the 'Azotobacteriaceae' which are involved in the breakdown of an aromatic compound. The genes from 'Azotobacter chroococcum' encoding p-hydroxybenzoate hydroxylase (pobA) and its regulatory protein (pobR) were cloned from a genomic library and sequenced. Sequence analysis of pobA revealed homology with other bacterial p-hydroxybenzoate hydroxylase enzymes. Residues essential to the structure and function of the enzyme have been conserved. The pobR gene encodes a DNA binding regulatory protein with similarity to proteins from the AraC/XylS family of transcriptional activators. A fragment containing both pobA and pobR was cloned into pUC19 and p-hydroxybenzoate hydroxylase activity was induced in 'Escherichia coli' by the addition of p-hydroxybenzoate. A frame-shift mutation introduced into the pobR gene prevented expression of p-hydroxybenzoate hydroxylase, indicating that PobR is the protein required for transcription of pobA. Interestingly, 'A. chroococcum' PobR has no homology to the PobR protein that is the transcriptional activator of pobA in 'Acinetobacter' strain ADP1, a protein that is homologous to the IclR family of transcriptional regulators. However, PobR from 'A. chroococcum' is homologous to several other proteins, suggesting that these proteins will also function as transcriptional activators of pobA