Mechanistic studies on cyclohexanone oxygenase

Ryerson, Carol C.; Ballou, David P.; Walsh, Christopher T.

doi:10.1021/bi00540a011

Cited by 215 publications

(128 citation statements)

References 49 publications

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“…S2B in the supplemental material). The latter finding indicated that the product is not a 7-member ring compound in which oxygen is inserted between N-3 and C-4 (analogous to the Baeyer-Villiger rearrangement observed with cyclohexanone [40]), as did its mass (Fig. 4A).…”

Section: Discussionmentioning

confidence: 79%

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The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems

Kim

Pelton

Inwood³

et al. 2010

J Bacteriol

103

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The Rut pathway is composed of seven proteins, all of which are required by Escherichia coli K-12 to grow on uracil as the sole nitrogen source. The RutA and RutB proteins are central: no spontaneous suppressors arise in strains lacking them. RutA works in conjunction with a flavin reductase (RutF or a substitute) to catalyze a novel reaction. It directly cleaves the uracil ring between N-3 and C-4 to yield ureidoacrylate, as established by both nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Although ureidoacrylate appears to arise by hydrolysis, the requirements for the reaction and the incorporation of 18 O at C-4 from molecular oxygen indicate otherwise. Mass spectrometry revealed the presence of a small amount of product with the mass of ureidoacrylate peracid in reaction mixtures, and we infer that this is the direct product of RutA. In vitro RutB cleaves ureidoacrylate hydrolytically to release 2 mol of ammonium, malonic semialdehyde, and carbon dioxide. Presumably the direct products are aminoacrylate and carbamate, both of which hydrolyze spontaneously. Together with bioinformatic predictions and published crystal structures, genetic and physiological studies allow us to predict functions for RutC, -D, and -E. In vivo we postulate that RutB hydrolyzes the peracid of ureidoacrylate to yield the peracid of aminoacrylate. We speculate that RutC reduces aminoacrylate peracid to aminoacrylate and RutD increases the rate of spontaneous hydrolysis of aminoacrylate. The function of RutE appears to be the same as that of YdfG, which reduces malonic semialdehyde to 3-hydroxypropionic acid. RutG appears to be a uracil transporter.The rut (pyrimidine utilization) operon of Escherichia coli K-12 contains seven genes (rutA to -G) (31,38). A divergently transcribed gene (rutR) codes for a regulator. The RutR regulator is now known to control not only pyrimidine degradation but also pyrimidine biosynthesis and perhaps a number of other things (44,45). In the presence of uracil, RutR repression of the rut operon is relieved.Superimposed on specific regulation of the rut operon by RutR is general control by nitrogen regulatory protein C (NtrC), indicating that the function of the Rut pathway is to release nitrogen (31, 59). The rut operon was discovered in E. coli K-12 as one of the most highly expressed operons under NtrC control. In vivo it yields 2 mol of utilizable nitrogen per mol of uracil or thymine and 1 mol of 3-hydroxypropionic acid or 2-methyl 3-hydroxypropionic acid, respectively, as a waste product (Fig. 1). Waste products are excreted into the medium. (Lactic acid is 2-hydroxypropionic acid.) Wild-type E. coli K-12 can use uridine as the sole nitrogen source at temperatures up to 22°C but not higher. It is chemotactic to pyrimidine bases by means of the methyl-accepting chemoreceptor TAP (taxis toward dipeptides), but this response is not temperature dependent (30).In the known reductive and oxidative pathways for degradation of the pyrimidine ring (22, 48, 52), the C-5-C-6 double bond ...

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Section: Discussionmentioning

confidence: 79%

“…We infer that RutA catalyzes synthesis of ureidoacrylate peracid (see text). Although our work did not address the specific role of FMN, it is plausible that flavin hydroperoxide, a well-known intermediate in related reactions (40), would participate (37). We postulate that ureidoacrylate peracid is the primary substrate for RutB (see text).…”

Section: Discussionmentioning

confidence: 95%

The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems

Kim

Pelton

Inwood³

et al. 2010

J Bacteriol

103

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“…The 1 -deazaflavin form of cyclohexanone mono-oxygenase is fully competent in carrying out the normal mono-oxygenation reaction of this enzyme [65], and reduced l-deaza-FMN is competent in the bioluminescence reaction of bacterial luciferase [66]. In contrast, when 1-deazaflavin is introduced into all aromatic hydroxylases so far studied, i.e.…”

Section: Mechanism Probesmentioning

confidence: 99%

New flavins for old: artificial flavins as active site probes of flavoproteins

Ghisla¹,

Massey²

1986

Biochemical Journal

195

143

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“…The ketone-to-lactone conversion is the biological equivalent of Baeyer-Villiger oxidation. All monooxygenases known to date that effect Baeyer-Villigertype conversions are flavoproteins, and of these, the best characterized is the cyclohexanone monooxygenase from acinetobacteria (4,6,16,19; J. A. Latham, Jr., Ph.D. thesis, Massachusetts Institute of Technology, Cambridge, 1985).…”

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confidence: 99%

Acinetobacter cyclohexanone monooxygenase: gene cloning and sequence determination

1988

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The gene coding for cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871 was isolated by immunological screening methods. We located and determined the nucleotide sequence of the gene. The structural gene is 1,626 nucleotides long and codes for a polypeptide of 542 amino acids; 389 nucleotides 5' and 108 nucleotides 3' of the-coding region are also reported. The complete amino acid sequence of the enzyme was derived by translation of the nucleotide sequence. From a comparison of the amino acid sequence with consensus sequences of nucleotide-binding folds, we identified a potential flavin-binding site at the NH2 terminus of the enzyme (residues 6 to 18) and a potential nicotinamide-binding site extending from residue 176 to residue 208 of the protein. An overproduction system for the gene to facilitate genetic manipulations was also constructed by using the tac promoter vector pKK223-3 in Escherichia coli. Bacteria can degrade a large variety of organic compounds by oxygenating metabolic routes. The degradation of monocyclic aromatic, polycyclic aromatic, and complex heteroaromatic structures such as lignin by both monooxygenases and dioxygenases has been intensively studied. Microbes can also decompose alicyclic compounds by oxygenating routes, with the breakdown of the bicyclic ketone camphor and the monocyclic ketone cyclohexanone by soil bacteria being prototypic cases. In these instances, a difficult step for dissimilatory metabolism is the ring fragmentation to yield readily metabolizable acyclic fragments. In both camphor and cyclohexanone degradation as well as in steroidal ketone utilization, the fragmentation strategy is executed in two steps: (i) oxygenating ring expansion of cyclic ketone to lactone followed by (ii) hydrolytic decomposition of lactone to acyclic hydroxy acid. The ketone-to-lactone conversion is the biological equivalent of Baeyer-Villiger oxidation. All monooxygenases known to date that effect Baeyer-Villigertype conversions are flavoproteins, and of these, the best characterized is the cyclohexanone monooxygenase from acinetobacteria (4, 6, 16, 19; J. A. Latham, Jr., Ph.D. thesis, Massachusetts Institute of Technology, Cambridge, 1985).We have previously shown that the purified cyclohexanone monooxygenase (Mr, 59,000) is in fact a remarkably versatile oxygenation catalyst that uses the bound flavin adenine dinucleotide (FAD)-4a-OOH oxygenating intermediate to initiate oxygen transfer to both electrophilic substrate sites, such as the carbonyl of ketones and aldehydes, and nucleophilic substrate sites, such as sulfides and selenides, to yield the corresponding sulfoxide and selenoxide products. Further, this oxygenase oxygenates at nitrogen, trivalent phosphorus, and boron sites in boronic acids, making it the most broad-based flavoprotein oxygenase known. To date, none of the Baeyer-Villiger-type flavoprotein monooxygenases have been the subject of significant structural analysis. To that end, we report the cloning of the gene for the Acinetobacter sp. strain NCIB 987...

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Mechanistic studies on cyclohexanone oxygenase

Cited by 215 publications

References 49 publications

The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems

The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems

New flavins for old: artificial flavins as active site probes of flavoproteins

Acinetobacter cyclohexanone monooxygenase: gene cloning and sequence determination

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