Hydroxylations with Pseudomonas putida

One 71-bond of an aromatic ring can be converted to a cyclohexadiene 1,2-diol by reaction with enzymes associated with Pseudomonas putida A variety of substituted aromatic compounds can be oxidized, including bromobenzene, chlor-obenzene, and toluene.In these latter cases, introduction of the hydroxyl... 

Considerable amount of v ork has been reported in the hydroxylation of aromatic rings. Thus, benzene on oxidation with Pseudomonas putida in presence of oxygen gives the cis-diol (Scheme 6). The cis-diol obtained could be converted by four steps into 1,2,3,4-tetrahydroxy compound, conduritol-and by five steps into the hexahydroxy compound, pinitol, an antidiabetic agent (Scheme 6)." ... 

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). 

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). 

The cytochrome / 450 responsible for the l droxylation of camphor by Pseudomonas putida has been isolated, and is. in contrast to most others, stable. However this enzyme will only hydroxylate substrates closely related to camphor, and the site of hydroxylation may vary with substrate. Thus camphor gives the exo-S-hydroxy derivative but 5,5-difluorocamphor gives the 9-hydroxy derivative. ... 

The P450 enzyme from Pseudomonas putida (P450cam or C YP101), which hydroxylates camphor to 5-exo-hydroxycamphor has been studied in detail and is regarded as a model protein for other P450s. The electron donor to P450cam is putidaredoxin, which can deliver one electron at a time. In the course of the reaction, an activated oxygen atom from the heme iron is transferred to the unactivated C H bond of the substrate. A Fe(IV)=0 porphyrin-7r-cation radical has been proposed as the key iron-oxo intermediate, compound I (Cpd I), but it has remained elusive so far in studies of the enzyme with substrate ( RH ). 

Strains of Pseudomonas putida are very versatile in metabolizing aromatic compounds, particularly to the corresponding 1,2-dihydro-l,2-diols. The hydroxylating enzyme of the P. putida mutant is not strongly substrate specific and alkyl, aryl and halogen functionalities are usually readily tolerated380. Thus, 4-bromobenzoic acid (1, R = Br) is converted to a. v-4-bro-mo-5,6-dihydroxy-l, 3-cyclohexadiene-l-carboxylic acid (2, R = Br) in 80% yield with 98% cc (determined by chiral NMR shift experiments on the 4-nitrobenzyl ester) 375. The absolute stereochemistry, (5R,6R), was determined by a single crystal X-ray analysis. 

The benzene rings in toluene [92, 1074], chlorobenzene [92], styrene [92], and phenylacetylene [92] undergo stereospecific syn hydroxylation in positions 2 and 3 with concomitant reduction to methyl-, chloro-, vinyl-, and acetylenyl-ciy-2,3-dihydroxycyclohexa-4,6-dienes when treated with a culture of Pseudomonas putida (equation 146) [92, 1074],... 

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. 

Rates of bacterial hydroxylation of substituted phenols to catechols by Pseudomonas putida correlated well with the van der Waals radii of the substituents (Paris et al. 1982), and this was also demonstrated for the biotransformation of anilines to catechols both by this strain and by a natural population of bacteria (Paris and Wolfe 1987). 

Ethylphenol oxidoreductase from Pseudomonas putida JD1 is structurally almost identical to 4-cresol oxidoreductase, but catalyzes the hydroxylation of para-alkylphe-nols with longer aliphatic chains (Table 16.3-12). The hydroxylation reactions enantioselectively produce (-R)-alcohols 64> 65l The regeneration properties of this enzyme are quite similar to 4-cresol oxidoreductase1611. 

The application of direct electrochemistry of small redox proteins is not restricted to cytochrome c. For example, the hydroxylation of aromatic compounds was possible by promoted electron transfer from p-cresol methylhydroxylase (a monooxygenase from Pseudomonas putida) to a modified gold electrode [87] via the blue copper protein azurin. All these results prove that well-oriented non-covalent binding of redox proteins on appropriate electrode surfaces increases the probability of fast electron transfer, a prerequisite for unmediated biosensors. Although direct electron-transfer reactions based on small redox proteins and modified electrode surfaces are not extensively used in amperometric biosensors, the understanding of possible electron-transfer mechanisms is important for systems with proteins bearing catalytic activity. 

The monooxygenases of the bacterium Pseudomonas putida hydroxylate camphor, while the monooxygenases from mitochondria and microsomes in the adrenal cortex cause the side chain of cholesterol to be split off with subsequent hydroxylation of the saturated C-H bonds. In the presence of liver microsomes, alkanes and aliphatic acids are hydroxylated predominantly at the terminal (co)... 

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