The ferroprotein component of a methylene hydroxylase

Cushman, David; Tsai, R. L.; Gunsalus, I. C.

doi:10.1016/0006-291x(67)90104-0

Cited by 110 publications

(38 citation statements)

References 9 publications

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“…In heterologous reconstitution assays, however, only limited enzymatic activity of P-4501in is found in coupling the NADH via the camphor reductase and camphor redoxin, nor is P-450cam fully active with linalool reductase and linalool redoxin (12,65). A similar selectivity for the iron-sulfurcontaining redoxins camphor redoxin and adrenodoxin was reported previously for P-450cam and the three-component P-450 steroid hydroxylase system of adrenal mitochondria (4,9,13,26).…”

supporting

confidence: 85%

Cloning and expression of a member of a new cytochrome P-450 family: cytochrome P-450lin (CYP111) from Pseudomonas incognita

1993

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Cytochrome P-4501in catalyzes the 8-methyl hydroxylation of linalool as the first committed step of its utilization by Pseudomonas incognita as the sole carbon source. By using a polymerase chain reaction-based cloning strategy, a 2.1-kb DNA fragment containing the cytochrome P-4501in gene (linC) was isolated. An open reading frame of 406 amino acids has been identified as that of P-4501in on the basis of amino acid sequence data from peptides of the native protein. Heterologous expression of functional holoprotein is exhibited by Escherichia coli transformed with pUC18 containing the subcloned linC gene under constitutive transcriptional control of the lac promoter. The G+C content oflinC was found to be 55% overall and 58% in the third codon position. An optimized amino acid sequence alignment of P-4501in with cytochrome P-450cam shows that the two enzymes have only 25% identity. P-4501in was found to exhibit the expected conservation in the axial cysteine heme ligand-containing peptide and the threonine region postulated to form an 02-binding pocket (T. L. Poulos, B. C. Finzel, and A. J. Howard, J. Mol. Biol. 195:687-700, 1987). The low amino acid sequence identity between P-4501in and all other P-450 sequences has shown that P45O1in is the first member of the CYPIll P-450 gene family.Members of the genus Pseudomonas play a major role in the metabolism of a variety of complex organic molecules, including hydrocarbons, utilizing oxygen as a cosubstrate. Therefore, it is not surprising that members of the equally diverse family of cytochrome P-450 monooxygenases are recognized to participate in chemical transformations leading to their use as biological energy and carbon sources (53, 67). P-450s have been shown to be involved in xenobiotic transformation as well as biosynthesis of secondary metabolites in other prokaryotes as well (39, 53-55, 64, 69). In eukaryotes, oxygen insertion at unactivated carbon centers by cytochrome P-450 is key to numerous essential biosynthetic pathways, including those for steroid hormones and prostoglandins, as well as in detoxification of foreign substances (8,21,68).Cytochrome P-450cam (cam indicates that cytochrome P-450 is specific for camphor), the first Pseudomonas P-450 to be cloned and sequenced (27,66), has long been the prototype for not only the prokaryotic P-450s but also the entire P-450 superfamily, with extensive physical and biochemical characterization dating back to the early 1970s (5,11,57,(71)(72)(73). The availability of a high-resolution tertiary structure for P-450cam, including substrate-free (41), substrate-bound (42), and several inhibitor-bound (43, 44) structures, has paved the way for the use of rational site-directed mutagenesis to further probe the structure-function relationships in P-450cam (for reviews, see references 32, 56, and 61).Pseudomonas which catalyzes the 8-methyl hydroxylation of linalool by utilizing reducing equivalents ultimately derived from NADH (45, 46). The monooxygenase, P-4501in, as well as its redox partner proteins, lin-redox...

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confidence: 85%

Cloning and expression of a member of a new cytochrome P-450 family: cytochrome P-450lin (CYP111) from Pseudomonas incognita

1993

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“…Although its cluster resembles that of plant-type ferredoxins, in that it gives a rhombic EPR signal, XylT absorbance in the visible region of the spectrum is closer to that of adrenodoxin (31) and putidaredoxin (32), which instead yield a nearly axial EPR signal (33). The EPR analysis revealed that XylT has intermediate properties, which might reflect greater flexibility of the protein around the cluster.…”

Section: Discussionmentioning

confidence: 93%

A Novel [2Fe-2S] Ferredoxin from Pseudomonas putidamt2 Promotes the Reductive Reactivation of Catechol 2,3-Dioxygenase

Hugo¹,

Armengaud²,

Gaillard³

et al. 1998

Journal of Biological Chemistry

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Catechol 2,3-dioxygenase (XylE) is a component of the TOL plasmid-encoded pathway for the degradation of toluene and xylenes and catalyzes the dioxygenolytic cleavage of the aromatic ring. Purified XylE is oxygensensitive and unstable in vitro, particularly in the presence of substituted catechol substrates, but it is stabilized in vivo by another protein, XylT, encoded by the xylT gene located just upstream of xylE. In this study, we have purified to homogeneity the XylT product from a recombinant Escherichia coli strain containing a hyperexpressible xylT gene and characterized it as a novel [2Fe-2S] ferredoxin. It is the first example of a soluble ferredoxin with a net positive charge at neutral pH. The EPR signal of the iron sulfur cluster has rhombic symmetry as is the case for plant-type ferredoxins, but the XylT absorbance spectrum resembles more closely that of adrenodoxin. The midpoint redox potential was determined to be ؊373 ؎ 6 mV, at pH 8.5. XylT was unusually unstable for a [2Fe-2S] ferredoxin, with half-lives of 69 min at 25°C in air and 70 min at 37°C in argon. With photochemically reduced 5-deazaflavin for the controlled generation of reductant, it was demonstrated that XylT mediates the rapid reactivation of purified inactive catechol 2,3-dioxygenase in vitro. Inactivation of XylE by 4-methylcatechol resulted in oxidation of the active site iron to a high spin ferric state that was detectable by EPR. Spectroscopic evidence presented here demonstrates that XylT reactivates XylE through reduction of the iron atom in the active site of the enzyme. It is the first instance of a ferredoxin-mediated reactivation of an enzyme. The level of expression of XylT in Pseudomonas putida mt2 cells is low and the calculated XylT/XylE molar ratio is consistent with the proposal that XylE reactivation involves catalytic nonstoichiometric amounts of XylT.Pseudomonas putida mt2 carries the TOL plasmid pWW0, which encodes a set of enzymes responsible for the transformation of toluene and xylene to central pathway intermediates (1).The catabolic (xyl) genes are organized in two operons, the so-called upper and meta-operons. The enzymes encoded by the upper operon catalyze the sequential oxidation of toluene to benzoate, whereas the enzymes of the meta-operon convert benzoate to Krebs cycle intermediates (2). In the meta-pathway, the enzyme catechol 2,3-dioxygenase encoded by xylE catalyzes the extradiol cleavage of the aromatic ring. This reaction has been studied in detail, and a general mechanism for the oxidative cleavage of catechol by extradiol dioxygenases has been proposed (3). The two atoms of the oxygen molecule are incorporated in the catechol substrate on two adjacent carbon atoms of the aromatic ring, one of which already carries a hydroxyl substituent of the diol and the other of which is unsubstituted. At the active site, the enzyme possesses a single iron atom that binds the substrate and oxygen and participates in the catalytic cycle.

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“…Class IIA RO is a three-component oxygenase in which the electron-transfer components comprise a simple flavoprotein and a putidaredoxin-type ferredoxin containing a putidaredoxin-type [2Fe-2S] cluster ([2Fe-2S] Pu ). 44,45) Almost all ROs are classified into class IIB or III. Only a few examples of class IIA ROs are known, including pyrazon dioxygenase from an unidentified bacterium, 46) dioxin dioxygenase from Sphingomonas wittichii RW1, 47) and dicamba O-demethylase from P. maltophilia DI-6.…”

Section: Structural Basis Of the Electron Transfer Interaction Ammentioning

confidence: 99%

Structural and Molecular Genetic Analyses of the Bacterial Carbazole Degradation System

Nojiri

2012

Bioscience, Biotechnology, and Biochemistry

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The ferroprotein component of a methylene hydroxylase

Cited by 110 publications

References 9 publications

Cloning and expression of a member of a new cytochrome P-450 family: cytochrome P-450lin (CYP111) from Pseudomonas incognita

Cloning and expression of a member of a new cytochrome P-450 family: cytochrome P-450lin (CYP111) from Pseudomonas incognita

A Novel [2Fe-2S] Ferredoxin from Pseudomonas putidamt2 Promotes the Reductive Reactivation of Catechol 2,3-Dioxygenase

Structural and Molecular Genetic Analyses of the Bacterial Carbazole Degradation System

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