Pseudomonas putida hydroxylase

The oxidation by strains of Pseudomonas putida of the methyl group in arenes containing a hydroxyl group in the para position is, however, carried out by a different mechanism. The initial step is dehydrogenation to a quinone methide followed by hydration (hydroxylation) to the benzyl alcohol (Hopper 1976) (Figure 3.7). The reaction with 4-ethylphenol is partially stereospecific (Mclntire et al. 1984), and the enzymes that catalyze the first two steps are flavocytochromes (Mclntire et al. 1985). The role of formal hydroxylation in the degradation of azaarenes is discussed in the section on oxidoreductases (hydroxylases). 

The alkane hydroxylase belongs to a family of nonheme iron oxygenases. There is some structural similarity between the nucleotide sequence of the integral-membrane alkane hydroxylase and the subunits of the monooxygenase encoded by xylA and xylM in the TOL plasmid that are involved in hydrox-ylation of the methyl groups in toluene and xylene in Pseudomonas putida PaWl (Suzuki et al. 1991). 

The degradation of 4-ethylphenol and related componnds is initiated not by oxygenation bnt by dehydrogenation to a qninone methide followed by hydroxylation (Hopper and Cottrell 2003) (Figure 8.39), and the flavocy tochrome 4-ethylphenol methylene hydroxylase in Pseudomonas putida stain JDl has been characterized (Reeve et al. 1989). 

Reeve CD, MA Carver, DJ Hopper (1989) The purification and characterization of 4-ethylphenol methylene hydroxylase, a flavocytochrome from Pseudomonas putida JDl. Biochem J 263 431-437. 

Kitcher JO, PW Trudgill, JS Rees (1972) Purification and properties of 2-furoyl-coenzyme A hydroxylase from Pseudomonas putida F2. Biochem J 130 121-132. 

Smith CA, MR Flyman (2004) Oxidation of methyl tcrt-butyl ether by alkane hydroxylase in dicyclopro-pyUcetone-induced and n-octane-grown Pseudomonas putida Gpol. Appl Environ Microbiol 70 4544-4550. 

Blase, M. Bruntner, C. Tshisuaka, B., et al., Cloning, Expression, and Sequence Analysis of the Three Genes Encoding Quinoline 2-Oxidoreductase, a Molybdenum-Containing Hydroxylase From Pseudomonas Putida 86. J, Biol Chem,. 1996. 271(38) pp. 23068-23079. 

A first application using ferroceneboronic acid as mediator [45] was described for the transformation of p-hydroxy toluene to p-hydroxy benzaldehyde which is catalyzed by the enzyme p-cresolmethyl hydroxylase (PCMH) from Pseudomonas putida. This enzyme is a flavocytochrome containing two FAD and two cytochrome c prosthetic groups. To develop a continuous process using ultrafiltration membranes to retain the enzyme and the mediator, water soluble polymer-bound ferrocenes [50] such as compounds 3-7 have been applied as redox catalysts for the application in batch electrolyses (Fig. 12) or in combination with an electrochemical enzyme membrane reactor (Fig. 13) [46, 50] with excellent results. 

The enzyme p-ethylphenol methylene hydroxylase (EPMH), which is very similar to PCMH, can also be obtained from a special Pseudomonas putida strain. This enzyme catalyzes the oxidation of p-alkylphenols with alkyl chains from C2 to C8 to the optically active p-hydroxybenzylic alcohols. We used this enzyme in the same way as PCMH for continuous electroenzymatie oxidation of p-ethylphenol in the electrochemical enzyme membrane reactor with PEG-ferrocene 3 (MW 20 000) as high molecular weight water soluble mediator. During a five day experiment using a 16 mM concentration of p-ethylphenol, we obtained a turnover of the starting material of more than 90% to yield the (f )-l-(4 -hydroxyphenyl)ethanol with 93% optical purity and 99% enantiomeric excess (glc at a j -CD-phase) (Figure 14). The (S)-enantiomer was obtained by electroenzymatie oxidation using PCMH as production enzyme. 

Putidaredoxin. Cushman et al. (36) isolated a low molecular iron-sulfur protein from camphor-grown Pseudomonas putida. This protein, putidaredoxin, is similar to the plant type ferredoxins with two irons attached to two acid-labile sulfur atoms (37). It has a molecular weight of 12,000 and shows absorption maxima at 327, 425 and 455 nm. Putidaredoxin functions as an electron transfer component of a methylene hydroxylase system involved in camphor hydroxylation by P. putida. This enzyme system consists of putidaredoxin, flavoprotein and cytochrome P.cQ (38). The electron transport from flavoprotein to cytochrome P.cq is Smilar to that of the mammalian mixed-function oxidase, but requires NADH as a primary electron donor as shown in Fig. 4. In this bacterial mixed-function oxidase system, reduced putidaredoxin donates an electron to substrate-bound cytochrome P. g, and the reduced cytochrome P. g binds to molecular oxygen. One oxygen atom is then used for substrate oxidation, and the other one is reduced to water (39, 40). 

Fe2S2] clusters are part of the molybdenum containing hydroxylases. Typically, apart from molybdenum and two EPR-distinct iron-sulfur centres there can be FAD as additional cofactor. In Chlostridium purinolyticum a selenium-dependent purine hydroxylase has been characterized as molybdenum hydroxylase. The EPR of the respective desulfo molybdenum (V) signal indicated that the Mo-ligands should differ from those of the well known mammalian corollary xanthine oxidase.197 For the bacterial molybdenum hydroxylase quinoline oxidoreductase from Pseudomonas putida an expression system was developed in order to be able to construct protein mutants for detailed analysis. EPR was used to control the correct insertion of the cofactors, specifically of the two [Fe2S2] clusters.198... 

NADH NADH-putidaredoxin reductase (fp), putidaredoxin (Fe-S) P-450 Pseudomonas putida D-camphor hydroxylase 40-42)... 

The ability of Pseudomonas putida to grow onoctanol is due to two alkane-inducible enzymes - a hydroxylase (this strain also contains a chromosomal gene coding for a hydroxylase) and a dehydrogenase, located on a CAM plasmid (Chakrabarty et al., 1973). 

Nieder, M. Shapiro, J. (1975). Physiological function of Pseudomonas putida PpG6 (Pseudomonas oleovarans) alkane hydroxylase monoterminal oxidation of alkanes and fatty acids. Journal of Bacteriology, 122, 93-8. 

A metabolic engineering approach has now provided direct evidence for the role of salicylic acid in systemic acquired resistance. Transgenic tobacco plants were produced expressing the "nah G" gene from Pseudomonas putida, which encodes a salicylate hydroxylase that converts salicylic acid... 

The P450 cam camphor hydroxylase from Pseudomonas putida is one of the best characterised of all enzymes. It catalyses the 5-exo hydroxyla-tion of camphor, the first step in the breakdown of the eompound as an energy source (Sligar and Gunsalus, 1976). The atomie strueture of the P450 has been solved in a variety of different forms (substrate-free, eamphor-bound, inhibitor-bound, mutant forms) and was the first P450 for which a... 

Models have been developed to accommodate the results of the hydroxyla-tion of substrates with different structures. The cytochrome P450CAM camphor hydroxylase from the bacterium Pseudomonas putida has been studied by X-ray crystallography. The importance of hydrophilic interactions with a valine (VAL-247) and a polar interaction mediated by hydrogen bonding to a tyrosine residue (TYR-96) has been noted. A model based on the hydroxylation of numerous cyclic amides by Beauveria sulfurescens (originally named Sporotrichum sulfurescens) showed that hydroxylation occurred preferentially at a methylene group about 5.5 A from an electron-rich substituent on the substrate. 

You, I.-S., R.I. Murray, D. Jollie, and I.C. Gunsalus. 1990. Purification and characterization of salicylate hydroxylase from Pseudomonas putida PpG7. Biochem. Biophys. Res. Commun. 169 1049-1054. 

Figure 3.5 Plots of the -values of Mo(v) species against g -anisotropy for members of the XO enzymes family. In red, slow-type signals from Dg AOR slow-type aquo from milk XO (2), with %poxanthine from Clostridium purinolyticum Purine Hydroxylase (3). In black, rapid-type signal from milk XO (4), rapid q from Pseudomonas putida Quinoline 2-Oxidore-ductase (5), rapid type 1 with 1-methybcanthine from milk XO (6), Thauera aromatica 4-Hydroxybenzoyl-CoA reductase (7), rapid q from As QualOx (8), rapid type 2 borate from milk XO (9), resting q2 from Pp QuinOr (10), rapid q from Pd IsoOr (11), resting q from Pp QuinOr (12). In blue, very rapid-type signals from Pp QuinOr (13), with 2-Hydroxy-6-methylpurine from milk XO (14)," from Pd IsoOr (15), from As QualOx (16). Linear correlations between the available data sets within each family can be extrapolated and are schematically indicated by straight lines to help the reader.

Heme-coupled monooxygenases contain cytochrome P-450. They are present in microsomes and are responsible for many hydroxylation reactions, e.g. llp-hydroxylation of steroids in the adrenal cortex, 2-hydroxylation of estrc ens in the liver the liver system is especially important in the hydroxylation of drugs and xenobiotics, thus rendering them water soluble, capable of conjugation and easily excreiable. A cytochrome P-450 system responsible for the hydroxylation of camphor (a 5-exo-hydroxylase) has been purified from Pseudomonas putida, and named putidaredoxin it contains FAD, an Fc2S2CyS4 center, and a P-4S0 cytochrome substrate hydroxylation is coupled to the oxidation of NADPH. 

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