α/β Barrel evolution and the modular assembly of enzymes: Emerging trends in the flavin oxidase/dehydrogenase family

Scrutton, Nigel S.

doi:10.1002/bies.950160208

Cited by 40 publications

(49 citation statements)

References 30 publications

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“…A barrel domain related to OYE forms part of trimethylamine dehydrogenase (Lim et al, 1986), and is used as a module in several other multidomain proteins (Scrutton, 1994), such as E. coli 2,4-dienoyl-CoA-reductase (He et al, 1997) and enoate reductases of clostridia (Rohdich et al, 2001). Whilst some of the putative active site residues differ in these distant relatives of OYE, there is a strong sequence conservation in a core region of around 40 amino acids.…”

Section: An Old Yellow Enzyme Family Of Flavoproteinsmentioning

confidence: 99%

‘New uses for an Old Enzyme’ – the Old Yellow Enzyme family of flavoenzymes

2002

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Section: An Old Yellow Enzyme Family Of Flavoproteinsmentioning

confidence: 99%

‘New uses for an Old Enzyme’ – the Old Yellow Enzyme family of flavoenzymes

2002

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“…Trimethylamine dehydrogenase (TMADH) 1 from the soil pseudomonad Methylophilus methylotrophus (formerly known by the isolate designation W 3 A 1 ) is a flavin-and iron-sulfurcontaining enzyme that catalyzes the oxidative demethylation of trimethylamine to dimethylamine and formaldehyde (1). The physiological oxidant for TMADH is the electron-transferring flavoprotein (ETF) of the organism, which becomes reduced only to the level of the semiquinone under physiological conditions (2), giving the following overall reaction stoichiometry: N(CH 3 ) 3 ϩ H 2 O ϩ 2ETF ox ¡ NH(CH 3 ) 2 ϩ H 2 CϭO ϩ 2ETF sq (Eq.…”

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confidence: 99%

Radiolytic Studies of Trimethylamine Dehydrogenase

Anderson¹,

Jang²,

Hille³

2000

Journal of Biological Chemistry

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Trimethylamine dehydrogenase from the pseudomonad Methylophilus methylotrophus has been examined using the technique of pulse radiolysis to rapidly introduce a single reducing equivalent into the enzyme. Using enzyme that has had its iron-sulfur center rendered redox-inert by prior reaction with ferricenium hexafluorophosphate, we determined the spectral change associated with formation of both the anionic and neutral forms that were generated at high and low pH, respectively, of the unique 6-cysteinyl-FMN of the enzyme. With native enzyme, electron transfer was observed within the radiolytically generated one-electron reduced enzyme but only at low pH (6.0). The kinetics and thermodynamics of this electron transfer in oneelectron reduced enzyme may be compared with that studied previously in the two-electron reduced enzyme. In contrast to previous studies with two-electron reduced enzyme in which a pK a of ϳ8 was determined for the flavin semiquinone, in the one-electron reduced enzyme the semiquinone was not substantially protonated even at pH 6.0. These results indicate that reduction of the iron-sulfur center of the enzyme significantly decreases the pK a of the flavin semiquinone of the active site. This provides further evidence, in conjunction with the strong magnetic interaction known to exist between the centers in the two-electron reduced enzyme, that the two redox-active centers in trimethylamine dehydrogenase are in intimate contact with one another in the active site of the enzyme. Trimethylamine dehydrogenase (TMADH)1 from the soil pseudomonad Methylophilus methylotrophus (formerly known by the isolate designation W 3 A 1 ) is a flavin-and iron-sulfurcontaining enzyme that catalyzes the oxidative demethylation of trimethylamine to dimethylamine and formaldehyde (1). The physiological oxidant for TMADH is the electron-transferring flavoprotein (ETF) of the organism, which becomes reduced only to the level of the semiquinone under physiological conditions (2), giving the following overall reaction stoichiometry:The reaction is thought to proceed via an initial imine product, which is then nonenzymatically hydrolyzed to the secondary amine and formaldehyde. TMADH is a member of a small but important group of amine-oxidizing flavoproteins that includes the monoamine oxidases A and B, sarcosine oxidase, and methyltryptophan oxidase. In each of these enzymes the flavin cofactor is covalently linked to the polypeptide in the active sites. In the case of TMADH this covalent linkage is to Cys-30 of the polypeptide at position 6 of the isoalloxazine ring of the FMN cofactor (4, 5). Loss of this linkage by mutation of Cys-30 to alanine results in a 6-fold decrease in the initial reduction of the enzyme-bound flavin, although this step remains at least an order of magnitude faster than catalysis, which is principally rate limited by product dissociation. More significantly, loss of the covalent linkage predisposes the flavin to nucleophilic attack by hydroxide to yield 6-hydroxyl-FMN, which is catalytically in...

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“…The deduced amino acid sequence of PETN reductase was compared to sequences in protein and nucleic acid databases using the BLAST program of the GCG package (6). The most similar proteins found were members of the old yellow enzyme family of ␣/␤-barrel flavoprotein oxidoreductases (17). These include old yellow enzyme of Saccharomyces carlsbergensis and Saccharomyces cerevisiae (11,14,18), homologs from the yeast Kluyveromyces lactis (10) and the protozoan Trypanosoma cruzii (OWL:U31282), a steroid-binding protein from the yeast Candida albicans (9), and morphinone reductase from the bacterium Pseudomonas putida M10 (4,5).…”

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Sequence and properties of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2

1996

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Pentaerythritol tetranitrate reductase, which reductively liberates nitrite from nitrate esters, is related to old yellow enzyme. Pentaerythritol tetranitrate reductase follows a ping-pong mechanism with competitive substrate inhibition by NADPH, is strongly inhibited by steroids, and is capable of reducing the unsaturated bond of 2-cyclohexen-1-one.We previously reported the isolation of Enterobacter cloacae PB2 on the basis of its ability to use nitrate esters such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate (GTN) as nitrogen sources. E. cloacae PB2 possesses a soluble PETN reductase capable of reductively liberating nitrite from nitrate esters with oxidation of NADPH (1) (Fig. 1). PETN reductase is a monomeric flavoprotein of M r 40 000. Recently, White et al. (20) reported the isolation of a strain of Agrobacterium radiobacter capable of growth with GTN as the sole nitrogen source and showed that cell extracts from this organism liberated nitrite from GTN and PETN with oxidation of NADH, suggesting the activity of a similar enzyme.Nitrate esters, though produced in large amounts for use as explosives and vasodilators (13), are rare in nature (7, 12), and multiply substituted nitrate esters are not known to occur naturally. The origin of enzymes apparently specialized for their breakdown is therefore of interest. To investigate this question, the structural gene encoding PETN reductase, designated onr (for organic nitrate reductase), was cloned using degenerate oligonucleotide probes.Cloning and sequence analysis of onr. The N-terminal sequence of PETN reductase purified from E. cloacae PB2 as previously described (1) was found to be SAEKLFTPLKV GAVTAPNRVFMAPLT. On the basis of E. cloacae typical codon usage, the following oligonucleotide probes were designed, based on residues 2 to 11 and 18 to 26, respectively:Southern blots using standard procedures (15) showed that both probes bound to the same region of PB2 genomic DNA. A 1,525-bp NcoI-ClaI genomic DNA fragment was cloned in pBluescript SKϩ (Stratagene) to give pONR1. Sequencing indicated the presence of an open reading frame beginning with codons matching the known N-terminal sequence of PETN reductase. The sequence of onr and deduced amino acid sequence of PETN reductase are shown in Fig. 2. The sequence predicts a protein of 364 residues with an M r of 39,358 excluding the N-terminal methionine, consistent with the M r of approximately 40,000 estimated for PETN reductase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (1). A putative ribosome-binding site and transcription termination sequence were detected. No obvious 70 -like promoter sequence was present, but three regions between a putative upstream termination sequence and the ribosome-binding site have significant homology to Escherichia coli S -dependent promoters (21). The deduced amino acid sequence of PETN reductase was compared to sequences in protein and nucleic acid databases using the BLAST program of the GCG package (6). The most similar prote...

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α/β Barrel evolution and the modular assembly of enzymes: Emerging trends in the flavin oxidase/dehydrogenase family

Cited by 40 publications

References 30 publications

‘New uses for an Old Enzyme’ – the Old Yellow Enzyme family of flavoenzymes

‘New uses for an Old Enzyme’ – the Old Yellow Enzyme family of flavoenzymes

Radiolytic Studies of Trimethylamine Dehydrogenase

Sequence and properties of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2

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