Camphor Pseudomonas putida

They are widely distributed across species. Bacteria possess cytochrome P450s, and P450cani (involved in the metabolism of camphor) of Pseudomonas putida is the only P450 isoform whose crystal stmcture has been established. 

The initial hydroxylation in the degradation of some terpenes the ring methylene group of camphor by Pseudomonas putida (Katagiri et al. 1968 Tyson et al. 1972 Koga et al. 1986), and the isopropylidene methyl group of linalool by a strain of P. putida (Ullah et al. 1990). 

The oxidation of f-butyl methyl ether to f-butanol (Steffan et al. 1997), which is also mediated by the cytochrome P450 from camphor-grown Pseudomonas putida CAM, but not by that from Rhodococcus rhodochrous strain 116. 

Rheinwald JG, AM Chakrabarty, C Gunsalus (1973) A transmissible plasmid controlling camphor oxidation in Pseudomonas putida. Proc Natl Acad Sci USA 70 885-889. 

Jones KH, RT Smith, PW Trudgill (1993) Diketocamphane enantiomer-specific Bayer-Villiger monooxygenases from camphor-grown Pseudomonas putida ATCC 17453. J Gen Microbiol 139 797-805. 

Ougham HJ, DG Taylor, PW Trudgill (1983) Camphor revisited involvement of a unique monooxygenase in metabolism of 2-oxo-A3-4,5,5-trimethylcyclopentenylacetic acid by Pseudomonas putida. J Bacteriol 15 140-152. 

There are two types of electron transport those involving flavoproteins and iron-sulfur proteins, and those requiring only flavoproteins. The X-ray crystal structure of the soluble cytochrome P450 from Pseudomonas putida grown on camphor (P-450-CAM) has been determined (Poulos et ah, 1985), as have several others. The haem group is deeply embedded in the hydrophobic interior of the protein, and the identity of the proximal haem iron ligand, based on earlier spectroscopic studies (Mason et ah, 1965) is confirmed as a specific cysteine residue. 

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). 

It has been clearly established that Pseudomonas putida which has been selected to grow on camphor possesses an oxidative metabolic system (54). It contains a hemoprotein, cytochrome Qrn>... 

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

Both optical isomers of camphor (2) are found widely in nature, (+)-camphor being more abundant. It is the main component of oils obtained from the camphor tree Cinnamomum camphora [26]. The hydroxylation of D-(+)-camphor by Pseudomonas putida Ci was described [112]. The substrate was hydroxylated exclusively in its 5-exo- and 6-exo-positions. 

Using camphor 1 as the only carbon source, Pseudomonas putida employs P450cam in order to catalyze the stereospecific hydroxylation at the 5-exo position 1 => 2 as a first step in a cascade of energy supplying reactions (Fig. 2). 

The camphor 5-oxygenase of Pseudomonas putida, which has been isolated in crystalline form, catalyzes the hydroxylation of camphor (equation 25).69... 

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 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... 

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 ). 

P450CAM camphor-hydroxylating P450 from Pseudomonas putida... 

Epoxidation of various olefins by cytochrome P-450 enzymes has been studied using rat liver microsomes [29,30] as well as using enzymes from microbial origin. For example, Ruettinger and Fulco [31] reported the epoxidation of fatty acids such as palmitoleic acid by a cytochrome P-450 from Bacillus megaterium. Their results indicate that both the epoxidation and the hydroxylation processes are catalyzed by the same NADPH-dependent monooxygenase. More recently, other researchers demonstrated that the cytochrome P-450cam from Pseudomonas putida, which is known to hydroxylate camphor at a non-activated carbon atom, is also responsible for stereoselective epoxidation of cis- -methylstyrene [32]. The (lS,2R)-epoxide enantiomer obtained showed an enantiomeric purity (ee) of 78%. This result fits the predictions based on a theoretical approach (Fig. 2). 

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. 

Although reductive pathways are discussed in Section 6.4.4, examples involving both oxidative and reductive pathways have been observed, for example, in Pseudomonas putida strain G-786 in which the synthesis of cytochrome P-450 monooxygenase was induced by growth with camphor. 

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