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W S McIntire

Publications and source records attributed to W S McIntire.

At least 37 records · Page 2Linked to original sources
PubMed1995

cDNA sequences of variant forms of human placenta diamine oxidase.

Genes for two forms of human placenta diamine oxidase (dao) were cloned from a cDNA library and sequenced. One gene, pdao1, is identical in length to human kidney dao but differs from it by two bases in the coding region and differs slightly in the 3'- and 5'-noncoding regions. The second gene, pdao2, is nearly identical to these genes in the coding region, except that it has an extra 57-nucleotide coding segment near the 3' end of this region. This segment corresponds to the contiguous sequence of the 3' end of intron 3 of human kidney dao.pdao2 also differs significantly from pdao1 and human kidney dao in a 13-base sequence in the 5'-noncoding region. It is proposed that pdao1 and human kidney dao are polymorphic forms of the same allele. Whether pdao2 is a polymorph of these two is not certain, because of the significant differences in the coding and noncoding regions. Pdao2 may represent a different allele.

Amine Oxidase (Copper-Containing)↗
PubMed1994

Influence of monovalent cations on the ultraviolet-visible spectrum of tryptophan tryptophylquinone-containing methylamine dehydrogenase from bacterium W3A1.

The influence of the monovalent cations on the UV-visible spectra of the methylamine dehydrogenase (MADH) from bacterium W3A1 was investigated. The spectra for the oxidized and 1- and 2-electron-reduced forms, unperturbed by bound cations, were obtained for the enzyme, and the extinction coefficients for these forms were determined. The binding of the following cations was investigated: Li+, Na+, K+, Rb+, Cs+, NH4+, (CH3)3NH+, and (CH3)4N+. It was shown that each cation produced unique spectral changes, some of which were pH-dependent. Except for NH4+, all spectral changes produced by binding of the monovalent cations can be explained by assuming two different binding sites in MADH (type I and type II sites). Na+ and K+ displayed monophasic binding to the type II site, (CH3)3NH+ and (CH3)4N+ displayed monophasic binding to the type I site, and Li+, Rb+, and Cs+ displayed either monophasic or biphasic binding to one or both sites depending on pH. The pH dependence for binding to the two sites is different, i.e. plots of log(Kd) versus pH have negative slopes approximately 1 for the type II site, whereas the negative slope is significantly less than 1 (0.6-0.8) for the type I site. This difference leads to pH-dependent changes in spectral features produced by binding of Li+, Rb+, and Cs+. The spectral changes seen during titrations with NH+4 were unlike those seen for any other cation. The binding of NH+4 was biphasic, and the spectra produced in each phase were unaffected by pH. It is assumed that this cation binds to the tryptophan tryptophylquinone cofactor to produce the iminoquinone in the first phase and then binds to the type I monovalent cation binding site in the second phase. It is suggested that binding of NH+4 (and CH3NH+3) to the type I site is a prelude to binding to the cofactor.

Bacteria↗
PubMed1994

Quinoproteins.

As used today, the word quinoprotein defines three distinct groups of enzymes. Before 1979, the structures of the essential, quinonoid oxidation-reduction cofactors were a mystery for all these enzymes. The first proteins proven to harbor this type prosthetic group are those with noncovalently bound pyrroloquinoline quinone (PQQ). PQQ-containing enzymes can be described as alcohol dehydrogenases, with the exception of a single protein, which is an amine dehydrogenase. More recently, it was discovered that copper-containing amine oxidases contain 6-hydroxydopa quinone, also known as topa quinone (TQ), whereas certain bacterial amine dehydrogenases require 2',4-bitryptophan-6,7-dione (tryptophan tryptophylquinone, TTQ) for activity. These latter two quinones are formed, by unknown processes, from a specific tyrosyl residue for the amine oxidases, and from two widely separate tryptophyl residues in the polypeptide of the amine dehydrogenases.

Coenzymes↗
PubMed1994

Genetic organization of the mau gene cluster in Methylobacterium extorquens AM1: complete nucleotide sequence and generation and characteristics of mau mutants.

The nucleotide sequence of the methylamine utilization (mau) gene region from Methylobacterium extorquens AM1 was determined. Open reading frames for 11 genes (mauFBEDACJGLMN) were found, all transcribed in the same orientation. The mauB, mauA, and mauC genes encode the periplasmic methylamine dehydrogenase (MADH) large and small subunit polypeptides and amicyanin, respectively. The products of mauD, mauG, mauL, and mauM were also predicted to be periplasmic. The products of mauF, mauE, and mauN were predicted to be membrane associated. The mauJ product is the only polypeptide encoded by the mau gene cluster which is predicted to be cytoplasmic. Computer analysis showed that the MauG polypeptide contains two putative heme binding sites and that the MauM and MauN polypeptides have four and two FeS cluster signatures, respectively. Mutants generated by insertions in mauF, mauB, mauE, mauD, mauA, mauG, and mauL were not able to grow on methylamine or any other primary amine as carbon sources, while a mutant generated from an insertion in mauC was not able to utilize methylamine as a source of carbon but utilized C2 to C4 n-alkylamines as carbon sources. Insertion mutations in mauJ, mauM, and mauN did not impair the ability of the mutants to utilize primary n-alkylamines as carbon sources. All mau mutants were able to utilize methylamine as a nitrogen source, implying the existence of an alternative (methyl)amine oxidation system, and a low activity of N-methylglutamate dehydrogenase was detected. The mauD, mauE, and mauF mutants were found to lack the MADH small subunit polypeptide and have a decreased amount of the MADH large subunit polypeptide. In the mauG and mauL mutants, the MADH large and small subunit polypeptides were present at wild-type levels, although the MADHs in these strains were not functional. In addition, MauG has sequence similarity to cytochrome c peroxidase from Pseudomonas sp. The mauA, mauD, and mauE genes from Paracoccus denitrificans and the mauD and mauG genes from Methylophilus methylotrophus W3A1 were able to complement corresponding mutants of M. extorquens AM1, confirming their functional equivalence. Comparison of amino acid sequences of polypeptides encoded by mau genes from M. extorquens AM1, P. denitrificans, and Thiobacillus versutus shows that they have considerable similarity.

Amines↗
PubMed1994

Organization of the methylamine utilization (mau) genes in Methylophilus methylotrophus W3A1-NS.

The organization of genes involved in utilization of methylamine (mau genes) was studied in Methylophilus methylotrophus W3A1. The strain used was a nonmucoid variant termed NS (nonslimy). The original mucoid strain was shown to be identical to the NS strains on the basis of chromosomal digest and hybridization patterns. An 8-kb PstI fragment of the chromosome from M. methylotrophus W3A1-NS encoding the mau genes was cloned and a 6,533-bp region was sequenced. Eight open reading frames were found inside the sequenced area. On the basis of a high level of sequence identity with the Mau polypeptides from Methylobacterium extorquens AM1, the eight open reading frames were identified as mauFBEDAGLM. The mau gene cluster from M. methylotrophus W3A1 is missing two genes, mauC (amicyanin) and mauJ (whose function is unknown), which have been found between mauA and mauG in all studied mau gene clusters. Mau polypeptides sequenced so far from five different bacteria show considerable identity. A mauA mutant of M. methylotrophus W3A1-NS that was constructed lost the ability to grow on all amines as sources of nitrogen but still retained the ability to grow on trimethylamine as a source of carbon. Thus, unlike M. extorquens AM1 and Methylobacillus flagellatum KT, M. methylotrophus W3A1-NS does not have an additional methylamine dehydrogenase system for amine oxidation. Using a promoter-probe vector, we identified a promoter upstream of mauF and used it to construct a potential expression vector, pAYC229.

Amino Acid Sequence↗
PubMed1994

Cloning, sequencing, and expression of the structural genes for the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase from two strains of Pseudomonas putida.

The structural genes for the flavoprotein subunit and cytochrome c subunit of p-cresol (4-methylphenol) methylhydroxylase (PCMH) from Pseudomonas putida NCIMB 9869 (National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland) and P. putida NCIMB 9866 were cloned and sequenced. The genes from P.putida NCIMB 9869 were for the plasmid-encoded A form of PCMH, and the genes from P.putida NCIMB 9866 were also plasmid encoded. The nucleotide sequences of the two flavoprotein genes from P.putida NCIMB 9869 and P.putida NCIMB 9866 (pchF69A and pchF66, respectively) were the same except for 5 bases out of 1,584, and the translated amino acid sequences were identical. The nucleotide sequences of the genes for the cytochrome subunits of PCMH from the two bacteria (pchC69A and pchC66) varied by a single nucleotide in their 303-base sequences, and the translated amino acid sequences differed by a single residue at position 41 (Asp in PchC69A and Ala in PchC66). Both cytochromes had 21-residue signal sequences, as expected for periplasmic proteins, and these sequences were identical. On the other hand, no signal sequences were found for the flavoproteins.pchF69A and pchC69A were expressed, separately or together, in Escherichia coli JM109 and P.putida RA4007, with active PCMH produced in both bacteria. The E. coli-expressed flavocytochrome was purified. Our studies indicated that the E.coli-expressed subunits were identical to the subunits expressed in P.putida NCIMB 9869: molecular weights, isoelectric points, UV-visible spectra, and steady-state kinetic parameters were the same for the two sets of proteins. The subunits readily associated upon mixing two crude extracts of E.coli, one extract containing PchC69A and the other containing PchF69A. The courses of association of PchC69A and PchF69A were essentially identical for pure E. coli-expressed subunits and pure P. putida 9869-expressed subunits. E. coli-expressed PchC69A and PchF69A contained covalently bound heme and covalently bound flavin adenine dinucleotide, respectively, as the proteins expressed in nature.

Amino Acid Sequence↗
PubMed1993

The amino acid sequence of Pseudomonas putida azurin.

The low molecular weight "blue" copper protein, azurin, has been purified from Pseudomonas putida (NCIB 9869) to homogeneity using various chromatographic techniques including reverse-phase HPLC. The amino acid sequence of the N-terminus of the reduced and carboxymethylated protein yielded a single sequence corresponding to AECKV. The complete sequence, comprising 128 amino acid residues with a C-terminal sequence corresponding to TVTLK, was determined from the peptides obtained from a Staphylococcus aureus V8 digest of the protein and confirmed using peptides obtained following cyanogen bromide and endoprotease Asp-N digests. The amino acid sequence contained three cysteine residues at positions 3, 26, and 112, was devoid of tryptophan, and showed closest similarity (90% identical residues) to the previously determined sequence of azurin isolated from Pseudomonas fluorescens biotype B [Ambler, R.P. (1971) in Developpements Recents Dans L'Etude Chimique De La Structures Des proteins (Preverio, A., Pechere, J.-F., and Coletti-Preverio, M.-A., Eds.), pp. 289-305, INSERM, Paris]. Examination of the complete sequence indicated P. putida azurin contained unique Asp and Ala residues at positions 19 and 21, respectively, that have not been found in any other azurin sequence.

Amino Acid Sequence↗
PubMed1993

Cloning, sequencing, expression, and regulation of the structural gene for the copper/topa quinone-containing methylamine oxidase from Arthrobacter strain P1, a gram-positive facultative methylotroph.

Deoxyoligonucleotides corresponding to amino acid sequences of methylamine oxidase and polyclonal anti-methylamine oxidase antibodies were used to probe Arthrobacter strain P1 plasmid and chromosomal DNA libraries. Two open reading frames, maoxI and maoxII, which are greater than 99% homologous, were cloned from the chromosomal library. The deduced amino acid sequences of the coding regions are identical except for two residues near the C termini. On the other hand, the 5'- and 3'-flanking regions of maoxI and maoxII are quite different. While either gene could code for methylamine oxidase, the dissimilarity in the 5'-flanking regions indicates that the genes are differently regulated. It was determined that maoxII alone encodes methylamine oxidase. The tyrosyl residue which is converted to topa quinone in the mature enzyme was located by comparison with amino acid sequences at the cofactor sites in other copper/topa quinone-containing amine oxidase. Transcriptional start sites and possible regulatory elements were identified in the 5' region of maoxI and maoxII, and stem-loop structures were found in the 3'-flanking regions. High levels of methylamine oxidase are produced when Arthrobacter strain P1 is grown on methylamine alone or on glucose plus methylamine, but growth on LB medium plus methylamine resulted in very low production of the enzyme. Expression of maoxII from its own promoter in Escherichia coli grown on glucose or LB medium with or without methylamine gave the same level of production of methylamine oxidase.

Amino Acid Sequence↗
PubMed1992

Wither PQQ.

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Amine Oxidase (Copper-Containing)↗
PubMed1991

Subunit interactions change the heme active-site geometry in p-cresol methylhydroxylase.

The enzyme p-cresol methylhydroxylase [4-cresol: (acceptor) oxidoreductase (methyl-hydroxylating), EC 1.17.99.1] contains two subunits: a cytochrome c (electron transfer) subunit (cytochrome cpc) and a flavin (catalytic) subunit. When these subunits are separated by isoelectric focusing, a stable cytochrome subunit is obtained. Significant differences are observed between the one-dimensional NMR spectra of oxidized cytochrome cpc and of oxidized p-cresol methylhydroxylase. Analysis of the two-dimensional nuclear Overhauser enhancement and exchange spectroscopy (NOESY) spectrum of reduced cytochrome cpc suggests that the axial ligand, Met-50, of the stable subunit reorients by a rotation about the C gamma-S delta bond when cytochrome cpc binds to the flavin subunit. This reorientation must result in a change in bonding at the heme, which is reflected both in the para-magnetically shifted resonances and in the redox potential. p-Cresol methylhydroxylase thereby provides an interesting example of the coupling of subunit interactions to active-site structure and reactivity.

Allosteric Regulation↗
PubMed1991

A new cofactor in a prokaryotic enzyme: tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase.

Methylamine dehydrogenase (MADH), an alpha 2 beta 2 enzyme from numerous methylotrophic soil bacteria, contains a novel quinonoid redox prosthetic group that is covalently bound to its small beta subunit through two amino acyl residues. A comparison of the amino acid sequence deduced from the gene sequence of the small subunit for the enzyme from Methylobacterium extorquens AM1 with the published amino acid sequence obtained by the Edman degradation method, allowed the identification of the amino acyl constituents of the cofactor as two tryptophyl residues. This information was crucial for interpreting 1H and 13C nuclear magnetic resonance, and mass spectral data collected for the semicarbazide- and carboxymethyl-derivatized bis(tripeptidyl)-cofactor of MADH from bacterium W3A1. The cofactor is composed of two cross-linked tryptophyl residues. Although there are many possible isomers, only one is consistent with all the data: The first tryptophyl residue in the peptide sequence exists as an indole-6,7-dione, and is attached at its 4 position to the 2 position of the second, otherwise unmodified, indole side group. Contrary to earlier reports, the cofactor of MADH is not 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), a derivative thereof, or pro-PQQ. This appears to be the only example of two cross-linked, modified amino acyl residues having a functional role in the active site of an enzyme, in the absence of other cofactors or metal ions.

Amino Acid Sequence↗
PubMed1991

Laser flash photolysis study of intermolecular and intramolecular electron transfer in trimethylamine dehydrogenase.

Laser flash photolysis has been used to investigate the kinetics of reduction of trimethylamine dehydrogenase by substoichiometric amounts of 5-deazariboflavin semiquinone, and the subsequent intramolecular electron transfer from the FMN cofactor to the Fe4S4 center. The initial reduction event followed second-order kinetics (k = 1.0 x 10(8) M-1 s-1 at pH 7.0 and 6.4 x 10(7) M-1 s-1 at pH 8.5) and resulted in the formation of the neutral FMN semiquinone and the reduced iron-sulfur cluster (in a ratio of approximately 1:3). Following this, a slower, protein concentration independent (and thus intramolecular) electron transfer was observed corresponding to FMN semiquinone oxidation and iron-sulfur cluster reduction (k = 62 s-1 at pH 7.0 and 30 s-1 at pH 8.5). The addition of the inhibitor tetramethylammonium chloride to the reaction mixture had no effect on these kinetic properties, suggesting that this compound exerts its effect on the reduced form of the enzyme. Treatment of the enzyme with phenylhydrazine, which introduces a phenyl group at the 4a-position of the FMN cofactor, decreased both the rate constant for reduction of the protein and the extent of FMN semiquinone production, while increasing the amount of iron-sulfur center reduction, consistent with the results obtained with the native enzyme. Experiments in which the kinetics of reduction of the enzyme were determined during various stages of partial reduction were also consistent with these results, and further indicated that the FMN semiquinone form of the enzyme is more reactive toward the deazariboflavin reductant than is the oxidized FMN.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteria↗
PubMed1991

A Cu(I)-semiquinone state in substrate-reduced amine oxidases.

The role of copper in copper-containing amine oxidases has long been a source of debate and uncertainty. Numerous electron paramagnetic resonance (EPR) experiments, including rapid freeze-quench studies, have failed to detect changes in the copper oxidation state in the presence of substrate amines. One suggestion that copper reduction might occur, has never been confirmed. Copper amine oxidases contain another cofactor, recently identified as 6-hydroxydopa quinone (topa quinone), which is reduced by substrates. Copper has been implicated in the reoxidation of the substrate-reduced enzyme, but the failure to detect any copper redox change has led to proposals that Cu(II) acts as a Lewis acid, that it has an indirect role in catalysis, or that it serves a structural role. We present evidence for the generation of a Cu(I)-semiquinone state by substrate reduction of several amine oxidases under anaerobic conditions, and suggest that the Cu(I)-semiquinone may be the catalytic intermediate that reacts directly with oxygen.

Amine Oxidase (Copper-Containing)↗
PubMed1991

Resonance Raman spectroscopy of methylamine dehydrogenase from bacterium W3A1.

Resonance Raman spectroscopy has been used to probe the structure of the covalently bound quinone cofactor in methylamine dehydrogenase from the bacterium W3A1. Spectra were obtained on the phenylhydrazine and 2-pyridylhydrazine derivatives of the native enzyme, on the quinone-containing subunit labeled with phenylhydrazine, and on an active-site peptide also labeled with phenylhydrazine. Comparisons of these spectra to the corresponding spectra of copper-containing amine oxidase derivatives indicate that the quinones in these two classes of quinoproteins are not identical. The resonance Raman spectra of the native enzyme and small subunit have also been measured. 16O/18O exchange permitted the carbonyl modes of the quinone to be identified in the resonance Raman spectrum of oxidized methylamine dehydrogenase: a band at 1614 cm-1, together with a shoulder at 1630 cm-1, are assigned as modes containing substantial C = O stretching character. D2O/H2O exchange has pronounced effects on the resonance Raman spectrum of the oxidized enzyme, suggesting that the quinone may have numerous hydrogen bonds to the protein or that it is unusually sensitive to the local environment. Resonance Raman spectra of the isolated small subunit, and its phenylhydrazine derivative, are considerably different from the corresponding spectra of the intact protein. An attractive explanation for these observations is that the quinone cofactor in methylamine dehydrogenase from W3A1 is located at the interface between the large and small subunits, as found for the enzyme from Thiobacillus versutus [Vellieux, F. M. D., Huitema, F., Groendijk, H., Kalk, K. H., Frank, J. Jzn., Jongejan, J. A., & Duine, J. A. (1989) EMBO J. 8, 2171-2178].(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteria↗
PubMed1991

Three-dimensional structure of p-cresol methylhydroxylase (flavocytochrome c) from Pseudomonas putida at 3.0-A resolution.

p-Cresol methylhydroxylase (PCMH) isolated from Pseudomonas putida is an alpha 2 beta 2 tetramer of approximate subunit Mr 49,000 and 9,000. It is a flavocytochrome c containing covalently bound FAD in the larger subunit and covalently bound heme in the smaller. Crystals in space group P2(1)2(1)2(1) with unit-cell parameters a = 140.3 A, b = 130.6 A, and c = 74.1 A contain one full molecule per asymmetric unit and diffract anisotropically to about 2.8-A resolution in two directions and to about 3.3-A resolution in the third. An electron density map has been computed at a nominal resolution of 3.0 A by use of area detector data from native crystals and from two derivatives. The phases were improved with the B.C. Wang solvent leveling procedure, and the map was averaged about the noncrystallographic 2-fold axis. The cytochrome subunit, whose amino acid sequence is known, has been fitted to the electron density on a graphics system. The course of the polypeptide chain of the flavoprotein subunit, whose sequence is mostly unknown, has been traced in a minimap and a model of polyalanine fitted to the electron density on the graphics system. The flavoprotein subunit consists of three domains in close contact. The N-terminal domain consists largely of beta-structure and contains most of the FAD binding site. The second domain contains a seven-stranded antiparallel beta-sheet of unusual topology connected by antiparallel alpha-helices on one side. The flavin ring lies at the juncture of the first two domains. The third domain lies against the first domain and helps cover the rest of the FAD chain. The cytochrome subunit resembles other small cytochromes such as c-551 and c5 and fits into a depression on the surface of the large flavoprotein subunit. The flavin and heme planes are nearly perpendicular, the normals to the planes being approximately 65 degrees apart. The two groups are separated by about 8 A, the distance from one of the vinyl methylene carbon atoms of the heme to the 8 alpha-methyl group of the flavin ring.

Amino Acid Sequence↗
PubMed1990

Methylamine oxidase from Arthrobacter P1 as a prototype of eukaryotic plasma amine oxidase and diamine oxidase.

Methylamine oxidase (MAOx) from Gram-positive soil bacterium Arthrobacter P1 catalyzes the oxidation of CH3NH2 to H2C = O and NH4+ via reduction of O2 to H2O2. Past work indicates that MAOx is similar to mammalian plasma amine oxidase (PAO) and diamine oxidase (DAO), plant DAO, and yeast peroxisomal amine oxidase (YAO). All have Mr congruent to 170,000 and are composed of 2 identical subunits, each of which contains 1 atom of Cu(II) and one molecule of quinonoid cofactor. Herein, we report further evidence as to the striking similarity of these enzymes, and describe properties of MAOx which offer insights into understanding the eukaryotic oxidases. It is our belief that the structure of the quinone cofactor, and the Cu(II) site in MAOx are identical to these sites in PAO and DAO.

Amine Oxidase (Copper-Containing)↗