Enzymes camphor hydroxylase

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... 

This enzyme [EC 1.14.15.1], also known as camphor 5-exo-methylene hydroxylase, and cytochrome P450-cam, catalyzes the reaction of (+)-camphor with putidare-doxin and dioxygen to generate (-F)-exo-5-hydroxy-camphor, oxidized putidaredoxin, and water. A heme-thiolate acts as a cofactor. The enzyme can also utilize ( )-camphor as a substrate, and l,2A-campholide will result in the formation of 5-exo-hydroxy-l,2-campholide. V. Ullrich W. Duppel (1975) The Enzymes, 3rd ed., 12, 253. 

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). 

P450cam was further engineered to an alkane hydroxylase by step-by-step adaptation of the enzyme to smaller n-alkanes beginning with hexane [198], then proceeding to butane and propane [199], and finally to ethane (47) [200]. The best mutant, with eight substitutions, oxidized propane at 500min with 86% coupling, which was comparable with that of the wild-type enzyme toward camphor (10) [200]. 

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