The purification and characterization of 4-ethylphenol methylenehydroxylase, a flavocytochrome from <i>Pseudomonas putida</i> JD1

Reeve, Christopher D.; Carver, Mark A.; Hopper, David J.

doi:10.1042/bj2630431

Cited by 52 publications

(32 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This pathway contrasts with the degradation pathways for p-cresol by P. putida NCIMB 9869 and NCIMB 9866 (Kim et al, 1994) and 4-ethylphenol by Pseudomonas sp. JD1 (Reeve et al, 1989), which start degradation from the side chain, but is compatible with the p-cresol degradation pathways present in P. putida U (Bayly et al, 1966), Alcaligenes eutrophus 345 (Hughes et al, 1984) and Pseudomonas sp. CF600 (Shingler, 1996), and part of the degradation pathway for alkylbenzoates in P. putida containing the TOL plasmid (Ramos et al, 1987).…”

Section: Discussionmentioning

confidence: 96%

3- and 4-alkylphenol degradation pathway in Pseudomonas sp. strain KL28: genetic organization of the lap gene cluster and substrate specificities of phenol hydroxylase and catechol 2,3-dioxygenase

Jeong

Kim

et al. 2003

Microbiology

View full text Add to dashboard Cite

The enzymes and genes responsible for the catabolism of higher alkylphenols have not been characterized in aerobic bacteria. Pseudomonas sp. strain KL28 can utilize a wide range of alkylphenols, which include the 4-n-alkylphenols (C 1 -C 5 ). The genes, designated as lap (for long-chain alkylphenols), encoding enzymes for the catabolic pathway were cloned from chromosomal DNA and sequenced. The lap genes are located in a 13?2 kb region with 14 ORFs in the order lapRBKLMNOPCEHIFG and with the same transcriptional orientation. The lapR gene is transcribed independently and encodes a member of the XylR/DmpR positive transcriptional regulators. lapB, the first gene in the lap operon, encodes catechol 2,3-dioxygenase (C23O). The lapKLMNOP and lapCEHIFG genes encode a multicomponent phenol hydroxylase (mPH) and enzymes that degrade derivatives of 2-hydroxymuconic semialdehyde (HMS) to TCA cycle intermediates, respectively. The P lapB promoter contains motifs at positions "24(GG) and "12(GC) which are typically found in s 54 -dependent promoters. A promoter assay using a P lapB : : gfp transcriptional fusion plasmid showed that lapB promoter activity is inducible and that it responds to a wide range of (alkyl)phenols. The structural genes encoding enzymes required for this catabolism are similar (42-69 %) to those encoded on a catabolic pVI150 plasmid from an archetypal phenol degrader, Pseudomonas sp. CF600. However, the lap locus does not include genes encoding HMS hydrolase and ferredoxin. The latter is known to be functionally associated with C23O for use of 4-alkylcatechols as substrates. The arrangement of the lap catabolic genes is not commonly found in other meta-cleavage operons. Substrate specificity studies show that mPH preferentially oxidizes 3-and 4-alkylphenols to 4-alkylcatechols. C23O preferentially oxidizes 4-alkylcatechols via proximal (2,3) cleavage. This indicates that these two key enzymes have unique substrate preferences and lead to the establishment of the initial steps of the lap pathway in strain KL28. INTRODUCTIONPhenol is produced naturally by the breakdown of plant materials during microbial degradation. In contrast, phenols with bulky alkyl group(s) that are found in the environment originate predominantly from anthropogenic sources. For instance, nonyl-and octylphenol are pollutants that are commonly found in aquatic environments. They are recalcitrant intermediates that are formed during the microbial breakdown of alkylphenol ethoxylates (Ferguson et al., 2001;Giger et al., 1984). Alkylphenol 3Present address: KOMED Co., Yatap 151, Bundang, Sungnam, Kyunggi, Korea.The GenBank accession numbers for the sequences reported in this study are AY324319 (the partial sequence of 16S rDNA) and AY324644 (orf1lapRBKLMNOPCEHIFG).Abbreviations: C12O, catechol 1,2-dioxygenase; C23O, catechol 2,3-dioxygenase; GFP, green fluorescent protein; HMS, 2-hydroxymuconic semialdehyde; IHF, integration host factor; lap, long-chain alkylphenol; mPH, multicomponent phenol hydroxylase. ethoxylates are highly effec...

show abstract

Section: Discussionmentioning

confidence: 96%

3- and 4-alkylphenol degradation pathway in Pseudomonas sp. strain KL28: genetic organization of the lap gene cluster and substrate specificities of phenol hydroxylase and catechol 2,3-dioxygenase

Jeong

Kim

et al. 2003

Microbiology

View full text Add to dashboard Cite

show abstract

“…However, unlike for some of the PCMHs, these could not be separated by isoelectric focusing. This presumably reflects a similarity in the pIs of the two subunits but is not unique within this class of enzyme, as it is also found for one of the PCMHs from P. putida NCIB 9869 and also the flavocytochrome 4-ethylphenol methylenehydroxylase (19), neither of which can be resolved by isoelectric focusing. The retention of the flavin through the various treatments and the hypsochromic shift of the 375-nm peak of flavin adenine dinucleotide to 354 nm (Fig.…”

Section: Discussionmentioning

confidence: 96%

p-cresol methylhydroxylase from a denitrifying bacterium involved in anaerobic degradation of p-cresol

1991

Self Cite

View full text Add to dashboard Cite

A bacterium, strain PC-07, previously isolated as part of a coculture capable of growing on p-cresol under anaerobic conditions with nitrate as the acceptor was identified as an Achromobacter sp. The first enzyme of the pathway, p-cresol methylhydroxylase, which converts its substrate into p-hydroxybenzyl alcohol, was purified.The enzyme had an Mr of 130,000 and the spectrum of a flavocytochrome. It was composed of flavoprotein subunits of Mr 54,000 and cytochrome subunits of Mr 12,500. The midpoint redox potential of the cytochrome was 232 mV. The-Km and kcat for p-cresol were 21 ,uM and 112 s-1 respectively, and the Km for phenazine methosulfate, the artificial acceptor used ig the assays, was determined to be 1.7 mM. These properties place the enzyme in the same class as the p-cresol methylhydroxylases from aerobically isolated Pseudomonas spp.

show abstract

“…The enzyme comprises two C-type cytochrome subunits and two flavoprotein subunits, each possessing a single FAD cofactor covalently associated at its 8a-carbon via an ether linkage with the phenolic oxygen of Tyr 384 (McIntire et al, 1981). The same link is found in the related enzyme 4-ethylphenol methylenehydroxylase (Reeve et al, 1989). The genes for the two component subunits of PCMH have been expressed in the heterologous host E. coli, singly and together (Kim et al, 1994).…”

mentioning

confidence: 77%

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

1998

View full text Add to dashboard Cite

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.Keywords: covalent flavoproteins; flavin adenine dinucleotide; flavin mononucleotide; flavinylation; redox cofactors Cofactors are recruited by protein molecules to extend the chemistry available within the active sites of enzymes. The chemistries displayed are variable and the range of protein-specific cofactors available bears testimony to the types of biological reaction achieved by many enzyme molecules. Cofactors may be present in the freely dissociable form, but others, such as lipoic acid and biotin, are permanently linked to the host protein. Other cofactors (e.g., heme and pyridoxyl phosphate) fall into both categories in that they can be attached covalently to the protein or operate in the freely dissociable form. The covalent addition of specific cofactors to unique residues within a polypeptide chain is a fundamental problem in studies of biological molecular recognition. In many cases, this specificity is purchased at the expense of recruiting modification enzymes that direct the covalent addition of a cofactor to the host protein. For example, the additions of biotin and lipoic acid to the €-amino groups of specific Lys residues in carboxyltransferases and the 2-oxoacid dehydrogenases are directed by a biotin holoenzyme synthase (Sweetman et al., 1985;Takai et al., 1987) and Reprint requests to: N.S. Scmtton, Department of Biochemistry, University of Leicester, Adrian Building, University Road, Leicester LE1 7RH UK; e-mail: nss4@le.ac.uk; or W.S. McIntire, Molecular Biology Division, Department of Veterans Affairs Medical Center, San Francisco, California 94121; e-mail: wsm@itsa.ucsf.edu.lipoate-protein ligases (Brookfield et al., 1991; Moms et al., 1994), respectively. In a similar fashion, a heme cytochrome c lyase (Steiner et al., 1996) is responsible for the covalent addition of heme to two Cys residues via thioether linkages. In some enzymes, an existing side chain is modified to yield what is in effect a covalently attached cofactor, although no pre-existing cofactor molecule is incorporated into the enzyme. Several examples of this type of modification are provided in the next paragraph.For histidine decarboxylase of Lactobacillus 30A (Recsei & Snell, 1984), a pyruvoyl group is generated from an existing residue, Ser 82. The enzyme is synthesized as a proenzym...

show abstract

The purification and characterization of 4-ethylphenol methylenehydroxylase, a flavocytochrome from Pseudomonas putida JD1

Cited by 52 publications

References 26 publications

3- and 4-alkylphenol degradation pathway in Pseudomonas sp. strain KL28: genetic organization of the lap gene cluster and substrate specificities of phenol hydroxylase and catechol 2,3-dioxygenase

3- and 4-alkylphenol degradation pathway in Pseudomonas sp. strain KL28: genetic organization of the lap gene cluster and substrate specificities of phenol hydroxylase and catechol 2,3-dioxygenase

p-cresol methylhydroxylase from a denitrifying bacterium involved in anaerobic degradation of p-cresol

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

Contact Info

Product

Resources

About