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W S McIntire

Publications and source records attributed to W S McIntire.

At least 19 recordsLinked to original sources
PubMed2001

Effects of noncovalent and covalent FAD binding on the redox and catalytic properties of p-cresol methylhydroxylase.

Each flavoprotein subunit (alpha or PchF) of the alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida contains FAD covalently attached to Tyr384. PCMH oxidizes p-cresol to 4-hydroxybenzyl alcohol, which is oxidized subsequently by PCMH to 4-hydroxybenzaldehyde. The Y384F mutant form of PchF (apo-PchF[Y384F]) displayed stoichiometric noncovalent FAD binding. PchF[Y384F]FAD associated with the cytochrome subunit (beta or PchC) (producing PCMH[Y384F]), although not as avidly as with wild-type PchF containing covalently bound FAD (PchF(C)). Dramatic increases in the two-electron E(m,7) (NHE) values for FAD were observed when it bound noncovalently to either apo-PchF or apo-PchF[Y384F], and the two-electron E(m,7) value for FAD was increased further by about 75 mV upon covalent binding to PchF, i.e., PchF(C). The E(m,7) values increased by approximately 20 and 45 mV, respectively, when PchF(C) and PchF[Y384F]FAD associated with PchC. The two-electron E(m,7) for covalently bound FAD in PCMH is 84 mV, the highest measured for a flavoprotein. The values for the one-electron redox potentials (E(m,7), NHE) for FAD were measured also for various forms of PchF. Under anaerobiosis, the reduction of PchF[Y384F]FAD by substrates was similar to that observed previously for PchF containing noncovalently bound FAD. Stopped-flow kinetic studies indicated a rapid substrate reduction of the FAD and heme in PCMH[Y384F] which produced PchF[Y384F]FAD(rad) x PchC, the mutant enzyme containing the flavin radical and reduced heme. These experiments also revealed a slow reduction of unassociated PchC(ox) by PchF[Y384F]FAD(rad) x PchC. Steady-state kinetic studies of the reaction of PCMH[Y384F] with p-cresol indicated that the K(m) for this substrate was unchanged relative to that of PCMH, but that the k(cat) was diminished by an order of magnitude. The data indicate that the covalent attachment of FAD to PchF assists catalysis by raising the E(m,7) of the flavin. Contributions to this effect likely result from conformational changes.

Amino Acid Substitution↗
PubMed2000

Structures of the flavocytochrome p-cresol methylhydroxylase and its enzyme-substrate complex: gated substrate entry and proton relays support the proposed catalytic mechanism.

The degradation of the toxic phenol p-cresol by Pseudomonas bacteria occurs by way of the protocatechuate metabolic pathway. The first enzyme in this pathway, p-cresol methylhydroxylase (PCMH), is a flavocytochrome c. The enzyme first catalyzes the oxidation of p-cresol to p-hydroxybenzyl alcohol, utilizing one atom of oxygen derived from water, and yielding one molecule of reduced FAD. The reducing electron equivalents are then passed one at a time from the flavin cofactor to the heme cofactor by intramolecular electron transfer, and subsequently to cytochrome oxidase within the periplasmic membrane via one or more soluble electron carrier proteins. The product, p-hydroxybenzyl alcohol, can also be oxidized by PCMH to yield p-hydroxybenzaldehyde. The fully refined X-ray crystal structure of PCMH in the native state has been obtained at 2. 5 A resolution on the basis of the gene sequence. The structure of the enzyme-substrate complex has also been refined, at 2.75 A resolution, and reveals significant conformational changes in the active site upon substrate binding. The active site for substrate oxidation is deeply buried in the interior of the PCMH molecule. A route for substrate access to the site has been identified and is shown to be governed by a swinging-gate mechanism. Two possible proton transfer pathways, that may assist in activating the substrate for nucleophilic attack and in removal of protons generated during the reaction, have been revealed. Hydrogen bonding interactions between the flavoprotein and cytochrome subunits that stabilize the intramolecular complex and may contribute to the electron transfer process have been identified.

Catalysis↗
PubMed2000

Heterologous expression in Pseudomonas aeruginosa and purification of the 9.2-kDa c-type cytochrome subunit of p-cresol methylhydroxylase.

The 9.2-kDa c-type cytochrome subunit (PchC) of the flavocytochrome p-cresol methylhydroxylase from Pseudomonas putida NCIMB 9869 has been overexpressed in recombinant form in Pseudomonas aeruginosa PAO1-LAC, using the recently developed pUCP-Nde vector. Efforts to produce the cytochrome in Escherichia coli using a pET vector, with or without its signal peptide, were generally unsuccessful, yielding relatively low levels of the protein. In contrast, the mature form of PchC accumulated in the periplasmic space of P. aeruginosa PAO1-LAC to about 1 mg/g wet cell paste. A periplasmic fraction enriched to about 12% (w/w) of total protein with recombinant PchC was isolated from the remainder of the cells by a washing procedure using ethylenediaminetetraacetate in the presence of sucrose. The cytochrome was purified to homogeneity from the periplasmic extract by anion-exchange chromatography on DEAE-Sepharose CL-6B followed by chromatofocusing on PolyBuffer Exchanger 94. Purified PchC was obtained in a yield of about 50% and was shown to be identical to that resolved from the native flavocytochrome isolated from P. putida. This system may prove to be of general use for the production of recombinant c-type cytochromes.

Catalytic Domain↗
PubMed1999

Properties of p-cresol methylhydroxylase flavoprotein overproduced by Escherichia coli.

The alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida is composed of a flavoprotein homodimer (alpha(2) or PchF(2); M(r) = 119 kDa) with a cytochrome monomer (beta, PchC; M(r) = 9.3 kDa) bound to each PchF subunit. Escherichia coli BL21(DE3) has been transformed with a vector for expression of the pchF gene, and PchF is overproduced by this strain as the homodimer. During purification, it was recognized that some PchF had FAD bound, while the remainder was FAD-free. However, unlike PchF obtained from PCMH purified from P. putida, FAD was bound noncovalently. The FAD was conveniently removed from purified E. coli-expressed PchF by hydroxyapatite chromatography. Fluorescence quenching titration indicated that the affinity of apo-PchF for FAD was sufficiently high to prevent the determination of the dissociation constant. It was found that p-cresol was virtually incapable of reducing PchF with noncovalently bound FAD (PchF(NC)), whereas 4-hydroxybenzyl alcohol, the intermediate product of p-cresol oxidation by PCMH, reduced PchF(NC) fairly quickly. In contrast, p-cresol rapidly reduced PchF with covalently bound FAD (PchF(C)), but, unlike intact PCMH, which consumed 4 electron equiv/mol when titrated with p-cresol (2 electrons from p-cresol and 2 from 4-hydroxybenzyl alcohol), PchF(C) accepted only 2 electron equiv/mol. This is explained by extremely slow release of 4-hydroxybenzyl alcohol from reduced PchF(C). 4-Hydroxybenzyl alcohol rapidly reduced PchF(C), producing 4-hydroxybenzaldehyde. It was demonstrated that p-cresol has a charge-transfer interaction with FAD when bound to oxidized PchF(NC), whereas 4-bromophenol (a substrate analogue) and 4-hydroxybenzaldehyde have charge-transfer interactions with FAD when bound to either PchF(C) or PchF(NC). This is the first example of a "wild-type" flavoprotein, which normally has covalently bound flavin, to bind flavin noncovalently in a stable, redox-active manner.

Bacterial Proteins↗
PubMed1999

Organization and sequences of p-hydroxybenzaldehyde dehydrogenase and other plasmid-encoded genes for early enzymes of the p-cresol degradative pathway in Pseudomonas putida NCIMB 9866 and 9869.

The gene (designated pchA) encoding the aldehyde dehydrogenase that is required to metabolise the p-hydroxybenzaldehyde produced by the degradation of p-cresol in Pseudomonas putida NCIMB 9866 and 9869 has been identified on plasmids pRA4000 and pRA500, respectively. The gene lies immediately upstream of the pchC and pchF genes encoding the subunits of p-cresol methylhydroxylase (PCMH), the preceeding enzyme in the p-cresol degradative pathway. In pRA500 the latter genes are followed by the genes encoding the alpha (pcaG) and beta (pcaH) subunits of protocatechuate-3,4-dioxygenase, whereas in pRA4000 the genes encoding PCMH are followed by an open reading frame encoding a protein that is similar to the maturase-related protein of P. alcaligenes. A gene, designated pchX, that encodes a protein of unknown function was identified between the pchC and pchF genes in both plasmids.

Aldehyde Oxidoreductases↗
PubMed1998

cDNA cloning of two splice variants of a human copper-containing monoamine oxidase pseudogene containing a dimeric Alu repeat sequence.

Two alternatively spliced transcripts, psiHLAO1 and psiHLAO2, of a copper-containing monoamine oxidase pseudogene have been isolated from a human-liver cDNA library. The larger psiHLAO1 cDNA (2073bp) contains a 5'-flanking segment of 134bp, followed by an apparent open reading frame (ORF) of 1725bp. The deduced amino acid sequence of this ORF (574 residues) shares 81.0% similarity with the 763-residue monoamine oxidase from human placenta (HPAO) (the N-terminal 533 residues of psiHLAO1 share 86.7% similarity with HPAO). The psiHLAO1 ORF is interrupted by an in-frame stop codon corresponding to amino acid 225 and terminates within a type S(a) dimeric Alu repeat sequence. psiHLAO2 appears to be an alternatively spliced variant of psiHLAO1 that has 413 bases of psiHLAO1 excised according to the 'GT-AG' rule. The slightly longer 3' end of the psiHLAO2 transcript shows that the Alu repeat is followed by an 11-bp poly(A) tract that, in turn, is followed by an AT-rich (81%) sequence of 105bp. A reverse transcriptase-polymerase chain reaction (RT-PCR) protocol was used to confirm that both psiHLAO1 and psiHLAO2 are transcribed in human liver and placenta. A search of the expressed sequence tag (EST) database indicates that, like HPAO, psiHLAO derives also from the region 17q21 of the human genome.

Alternative Splicing↗
PubMed1998

Crystallographic and spectroscopic studies of native, aminoquinol, and monovalent cation-bound forms of methylamine dehydrogenase from Methylobacterium extorquens AM1.

Various monovalent cations influence the enzymatic activity and the spectroscopic properties of methylamine dehydrogenase (MADH). Here, we report the structure determination of this tryptophan tryptophylquinone-containing enzyme from Methylobacterium extorquens AM1 by high resolution x-ray crystallography (1.75 A). This first MADH crystal structure at low ionic strength is compared with the high resolution structure of the related MADH from Paracoccus denitrificans recently reported. We also describe the first structures (at 1.95 to 2.15 A resolution) of an MADH in the substrate-reduced form and in the presence of trimethylamine and of cesium, two competitive inhibitors. Polarized absorption microspectrophotometry was performed on single crystals under various redox, pH, and salt conditions. The results show that the enzyme is catalytically active in the crystal and that the cations cause the same spectral perturbations as are observed in solution. These studies lead us to propose a model for the entrance and binding of the substrate in the active site.

Bacterial Proteins↗
PubMed1998

Proposed steady-state kinetic mechanism for Corynebacterium ammoniagenes FAD synthetase produced by Escherichia coli.

The bifunctional enzyme, FAD synthetase (FS), from Corynebacterium ammoniagenes was overproduced in Escherichia coli and purified, and its steady-state kinetic properties were investigated. Although FMN is an intermediate product in the conversion of riboflavin to FAD, FMN must be released after formation, and then rebind for adenylylation. It was shown that adenylylation of FMN is reversible; FAD and pyrophosphate can be converted to FMN and ATP by the enzyme. In contrast, under the conditions studied, phosphorylation of riboflavin is irreversible. A method is described for analysis of two catalytic cycles, occurring on one enzyme, which have a substrate and/or product in common. The binding order for the phosphorylation cycle of FS was established as riboflavin(in), ATP(in), ADP(out), and FMN(out). The order for the adenylylation cycle was ATP(in), FMN(in), pyrophosphate(out), and FAD(out). A set of steady-state constants was determined, and without additional optimization, these constants were sufficient to describe experimental progress curves for conversion of riboflavin to FAD. In independent studies, it was demonstrated that FMN binds to apo-FS with a dissociation constant of 6-7 microM, which is 2 orders of magnitude higher than the KD value for riboflavin. For the steady-state kinetic analysis, this represents reversible binding of FMN(out) in the phosphorylation cycle (cycle I), which effectively inhibits catalysis in the adenylylation cycle (cycle II).

Adenosine Triphosphate↗
PubMed1998

Crystallographic study of azurin from Pseudomonas putida.

Azurin from Pseudomonas putida is a blue copper protein which functions as an electron carrier. Two crystal forms of azurin were grown, one in the presence and the other in the absence of zinc acetate; each belongs to space group P21 and contains two molecules per asymmetric unit. The zinc-free crystals have cell dimensions a = 43.25, b = 50.65, c = 54.60 A, beta = 107.79 degrees, while the crystals grown from zinc-containing solution have cell dimensions a = 40.76, b = 51.22, c = 54.96 A, beta = 103.12 degrees. The latter crystals were found to have four zinc ions incorporated into the crystal lattice. Both crystal structures were solved by the molecular-replacement method using the program MERLOT. The search model was the structure of azurin from Alcaligenes denitrificans. The crystallographic R factor for native azurin is 0.169 (Rfree = 0. 257) from 8 to 1.92 A resolution, while that for zinc azurin is 0. 181 (Rfree = 0.248) from 10 to 1.6 A resolution; for each structure the root-mean-square deviation in bond lengths from ideal values is 0.007 A. In both crystal structures the Cu atom forms three strong bonds in the equatorial plane, two with Ndelta1 from His46 and His117, and one with the thiolate S atom of Cys112. Two longer axial approaches are made by the Sgamma from Met121 and the carbonyl O atom from Gly45. This results in a distorted trigonal bipyramidal co-ordination around the Cu atom. It further confirms the presence of a weak fifth bond to the copper in P. putida azurin, as with other azurin structures described at high resolution. The Ndelta1 atom of His35 is protonated, as it is in the low-pH form of azurin from Pseudomonas aeruginosa but unlike the low-pH form of the azurins from Alcaligenes denitrificans or Alcaligenes xylosoxidans. In each crystal form the two molecules of azurin in the asymmetric unit are related by a local twofold axis and form a dimer stabilized by the interaction of a pair of hydrophobic patches surrounding the partially exposed His117 side chain. In the other known azurin crystal structures, analogous dimer formation is observed, but with different relative orientations of the molecules. The four zinc ions introduced during crystallization of zinc azurin are bound to the protein and participate in five- and sixfold ligand coordination with no affect on the copper binding site. The zinc ligands are Ndelta from His, carboxylate O atoms from Asp and Glu, Ogamma from Ser and water molecules. One of the zinc ions, located on a non-crystallographic twofold axis, links the dimers of the asymmetric unit into continuous chains parallel to the crystallographic (-101) direction and is primarily responsible for the altered unit-cell parameters. Two of the other zinc ions bind to His83, one in each molecule.

Amino Acid Sequence↗
PubMed1998

Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: the current state of affairs.

The first identified covalent flavoprotein, a component of mammalian succinate dehydrogenase, was reported 42 years ago. Since that time, more than 20 covalent flavoenzymes have been described, each possessing one of five modes of FAD or FMN linkage to protein. Despite the early identification of covalent flavoproteins, the mechanisms of covalent bond formation and the roles of the covalent links are only recently being appreciated. The main focus of this review is, therefore, one of mechanism and function, in addition to surveying the types of linkage observed and the methods employed for their identification. Case studies are presented for a variety of covalent flavoenzymes, from which general findings are beginning to emerge.

Amino Acid Sequence↗
PubMed1998

Newly discovered redox cofactors: possible nutritional, medical, and pharmacological relevance to higher animals.

Research spurred by the discovery of pyrroloquinoline quinone (PPQ) in 1979 led to the discovery of four additional oxidation-reduction (redox) cofactors, all of which result from transmogrification of amino acyl side chains in respective enzymes. These cofactors are (a) topa quinone in copper-containing amine oxidases, enzymes found in nearly all forms of life, including human; (b) lysyl topa quinone of the copper protein lysyl oxidase, an enzyme required for proper cross-linking of collagen and elastin; (c) tryptophan tryptophylquinone of alkylamine dehydrogenases from gram-negative soil bacteria; and (d) the copper-complexed cysteinyltyrosyl radical of fungal galactose oxidase. Originally, PQQ was thought to be a covalently bound cofactor in numerous enzymes from eukaryotes and prokaryotes. Today, PQQ is only found as a noncovalent cofactor in bacterial enzymes. The ubiquity of PQQ in the environment and its steady accessibility in the human diet has raised questions concerning its role as a vitamin, or an essential or helpful nutrient. The relevance to nutrition, medicine, and pharmacology of PQQ, topa quinone, lysyl topa quinone, tryptophan trytophylquinone, the galactose oxidase cofactor, and the enzymes harboring these cofactors are discussed in this review.

Amino Acid Oxidoreductases↗
PubMed1997

Crystal structures of the copper-containing amine oxidase from Arthrobacter globiformis in the holo and apo forms: implications for the biogenesis of topaquinone.

The crystal structures of the copper enzyme phenylethylamine oxidase from the Gram-positive bacterium Arthrobacter globiformis (AGAO) have been determined and refined for three forms of the enzyme: the holoenzyme in its active form (at 2.2 A resolution), the holoenzyme in an inactive form (at 2.8 A resolution), and the apoenzyme (at 2.2 A resolution). The holoenzyme has a topaquinone (TPQ) cofactor formed from the apoenzyme by the post-translational modification of a tyrosine residue in the presence of Cu2+. Significant differences between the three forms of AGAO are limited to the active site. The polypeptide fold is closely similar to those of the amine oxidases from Escherichia coli [Parsons, M. R., et al. (1995) Structure 3, 1171-1184] and pea seedlings [Kumar, V., et al. (1996) Structure 4, 943-955]. In the active form of holo-AGAO, the active-site Cu atom is coordinated by three His residues and two water molecules in an approximately square-pyramidal arrangement. In the inactive form, the Cu atom is coordinated by the same three His residues and by the phenolic oxygen of the TPQ, the geometry being quasi-trigonal-pyramidal. There is evidence of disorder in the crystals of both forms of holo-AGAO. As a result, only the position of the aromatic group of the TPQ cofactor, but not its orientation about the Cbeta-Cgamma bond, is determined unequivocally. In apo-AGAO, electron density consistent with an unmodified Tyr occurs at a position close to that of the TPQ in the inactive holo-AGAO. This observation has implications for the biogenesis of TPQ. Two features which have not been described previously in amine oxidase structures are a channel from the molecular surface to the active site and a solvent-filled cavity at the major interface between the two subunits of the dimer.

Amine Oxidase (Copper-Containing)↗
PubMed1996

Cloning and sequencing of a copper-containing, topa quinone-containing monoamine oxidase from human placenta.

A 4040-bp cDNA was cloned from a human placenta library by screening with a polymerase chain reaction-amplified fragment. The fragment was generated from the library using primers corresponding to conserved sequences encompassing the topa quinone (TPQ) cofactor sites of the copper-containing proteins, bovine serum amine oxidase (BSAO) and human kidney diamine oxidase (DAO). The cloned cDNA contains a coding sequence from positions 161 to 2449. Between bases 2901 and 2974, in a very long 1591-bp 3'-untranslated region, there is a G/A-rich region in the minus strand, which contains a (AGG)5 tandem repeat. The human placenta cDNA sequence and its translated amino acid sequence are 84% and 81% identical to the corresponding BSAO sequences, while the identities for the placenta sequences and those for human kidney DAO are 60% and 41%, respectively. The TPQ consensus nucleotide and protein sequences are identical for the placenta enzyme and BSAO, but the corresponding sequences for human kidney DAO are nonidentical. Three His residues that have been identified as Cu(II) ligands in other amine oxidases are conserved in the human placenta amine oxidase protein sequence. It was concluded that the placenta cDNA open-reading frame codes for a copper-containing, TPQ-containing monoamine oxidase. A putative 19-amino acid signal peptide was identified for human placenta amine oxidase. The resulting mature protein would be composed of 744 amino acids, and would have a Mr of 82,525. Comparison of the human placenta amine oxidase with DNA sequences found in GenBank suggests that the gene for this enzyme is located in the q21 region of human chromosome 17, near the BRCA1 gene.

Amine Oxidase (Copper-Containing)↗
PubMed1996

Purification, crystallization and preliminary X-ray crystal structure analysis of copper amine oxidase from Arthrobacter globoformis.

beta-Phenylethylamine oxidase from the Gram-positive bacterium Arthrobacter globoformis has been crystallized as three crystal forms. Two belong to space group C2 and one to space group P2(1)2(1)2(1), respectively. The unit-cell volumes are consistent with one infunit of 70 644 Da per asymmetric unit for the two monoclinic forms, and with two infunits per asymmetric unit for the orthorhombic crystals. Three-dimensional intensity data have been recorded to 2.8A resolution for one of the monoclinic crystal forms and to 3A, resolution for the orthorhombic crystal form.

Journal Article↗
PubMed1995

The cytochrome subunit is necessary for covalent FAD attachment to the flavoprotein subunit of p-cresol methylhydroxylase.

When p-cresol methylhydroxylase (PCMH) is expressed in its natural host Pseudomonas putida, or when the genes of the alpha and beta subunits of the enzyme are expressed together in the heterologous host Escherichia coli, flavin-adenine dinucleotide (FAD) is covalently attached to Tyr384 of the alpha subunit and the correct alpha 2 beta 2 form of the enzyme is assembled. The apoflavoprotein has been expressed in E. coli in the absence of the beta cytochrome c subunit and purified. While noncovalent FAD binding to apoflavoprotein in the absence of the cytochrome subunit could not be directly demonstrated, circumstantial evidence suggests that this indeed occurs. Covalent flavinylation requires one molecule each of FAD and cytochrome for each flavoprotein subunit. The flavinylation process leads to the 2-electron-reduced form of covalently bound FAD, and the resulting alpha 2 beta 2 enzyme is identical to wild-type PCMH. This work presents clear evidence that covalent flavinylation occurs by a self-catalytic mechanism; an external enzyme or chaperon is not required, nor is prior chemical activation of FAD or of the protein. This work is the first to define the basic chemistry of covalent flavinylation of an enzyme to produce the normal, active species, and confirms a long standing, postulated chemical mechanism of this process. It also demonstrates, for the first time, the absolute requirement for a partner subunit in the post-translational modification of a protein. It is proposed that the covalent FAD bond to Tyr384 and the phenolic portion of this Tyr are part of the essential electron transfer path from FAD to heme.

Amino Acid Sequence↗