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

Abstract

The structural genes for the flavoprotein subunit and cytochrome c subunit of p-cresol (4-methylphenol) methyihydroxylase (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 PCMH has been isolated from several Pseudomonas species and other microorganisms (24,25,28,33,41,51,57). A similar enzyme, 4-ethylphenol methylenehydroxylase from Pseudomonas putida JD1, has also been purified and studied (55).The best-characterized enzyme from this group is PCMH69A, the plasmid-encoded A form of PCMH from P. putida NCIMB 9869 (National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, Scotland). Its 3-A (1 A = 0.1 nm) three-dimensional structure is known (42) stereochemistry of its oxidation ofp-cresol, 4-ethylphenol, and 1-(4'-hydroxyphenyl)ethanol (45,46). The flavoprotein and cytochrome subunits have been separated, characterized, and reconstituted into native enzyme (35,36). Further, the amino acid sequences of the entire cytochrome subunit and the N terminus and flavin attachment site of the flavoprotein of PCMH69A have been determined (44,48).In the absence of the complete amino acid sequence, several important features have been gleaned from the three-dimensional structure of PCMH69A. For example, the cytochrome subunit structure is similar to those of other c-type cytochromes, but the structure of the flavoprotein subunit is unlike that of any other known protein structure. It was found that the planes of the isoalloxazine and heme rings form a 650 angle, and a heme vinyl methylene carbon is -9 A from the 8ot-methyl group of the flavin (42). To obtain further detailed structural information from the crystallographic data, knowledge of the complete amino acid sequence of the flavoprotein is essential. These data are also important in our quest to better understand the mechanism of action of PCMH; in this regard, investigation of site-specifically mutated forms of PCMH will be extremely useful.P. putida NCIMB 9866 (hereafter referred to as P. putida 9866) produces a single form of PCMH (PCMH66), while P. putida NCIMB 9869 (hereafter referred to as P. putida 9869) manufactures an A and a B form of the enzyme. The genes for 6349 on May 9, 2018 by guest

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

“…This motif is also found in enzymes with noncovalently bound flavin (van Driessche et al, 1996); however, L-gulono-y-lactone oxidase, p-cresol methylhydroxylase, and 6-hydroxy-~-nicotine oxidase are covalent flavoproteins that do not have this so-called "fingerprint" (Brandsch, 1993;Kim et al, 1994). Additionally, the regions very near the flavin attachment sites in numerous covalent flavoproteins (see Table 1) display either -Gly-Gly-(succinate dehydrogenase, fumarate reductase, 6-hydroxy-~-nicotine oxidase, monoamine oxidase, berberin bridge enzyme), -Gly-Gly-Gly-(Lgulono-y-lactone oxidase), -Gly-Gly-Gly-Gly-(p-cresol methylhydroxylase), or -Gly-Ala-Gly-(dimethyl-and trimethylamine dehydrogenases) motifs.…”

“…In the future, we will likely encounter and marvel at other unusual structures. Other examples of aminoacyl groups recruited as cofactors can be found elsewhere (Rucker & Wold, 1988;Rucker & McGee, 1993;McIntire, 1994).The covalent attachment of flavins to proteins has been recognized for about 40 years following the work of Singer and coworkers (Keamey & Singer, 1955a, 1955bSinger et al, 1956;Keamey et al, 1971) on mammalian succinate dehydrogenase. Since then, many covalent flavoproteins have been identified.…”

Group II intron from Pseudomonas alcaligenes NCIB 9867 (P25X): entrapment in plasmid RP4 and sequence analysis

“…In searching for nucleotide sequence homology with the databases, we found that the region of Xln6 which could fold into the domain VI RNA secondary structure (nucleotides showed sequence identity, with only three point differences, with a noncoding region immediately downstream of the p-cresol hydroxylase gene (pchF) from both Pseudomonas putida NCIB 9869 (strain P35X) and P. putida NCIB 9866 (strain P24X) (Kim et al, 1994) (Fig. 5b).…”

Induction of aromatic catabolic activity in Sphingomonas aromaticivorans strain F199

“…This is not surprising in light of the fact that p-cresol methylhydroxylase is localized in the periplasm in some Gram-negative bacteria such as P. putida [33]. The cytochrome c component of the methylhydroxylase (PchC) is predicted to occur in the membrane in both S. aromaticivorans F199 ( Figure 1) and P. putida.…”

“…Homologs of the methylhydroxylase flavin component, PchFa and PchFb (pNL1), PchF (P. putida), and VaoA (Penicillium simplicissimum), do not possess a typical N-terminal leader sequence. Analysis of C-terminal residues in PchF suggests that it is translocated across the membrane by a sec-independent pathway [33]. Similarly, C-terminal residues in VaoA suggest that this protein is translocated to the peroxisome [15].…”