Chemotaxis by Pseudomonas putida (ATCC 17453) towards camphor involves cytochrome P450cam (CYP101A1)

The camphor-degrading microorganism, Pseudomonas putida strain ATCC 17453, is an aerobic, gram-negative soil bacterium that uses camphor as its sole carbon and energy source. The genes responsible for the catabolic degradation of camphor are encoded on the extra-chromosomal CAM plasmid. A monooxygenase, cytochrome P450_cam, mediates hydroxylation of camphor to 5-exo-hydroxycamphor as the first and committed step in the camphor degradation pathway, requiring a dioxygen molecule (O₂) from air. Under low O₂ levels, P450_cam catalyzes the production of borneol via an unusual reduction reaction. We have previously shown that borneol downregulates the expression of P450_cam. To understand the function of P450_cam and the consequences of down-regulation by borneol under low O₂ conditions, we have studied chemotaxis of camphor induced and non-induced P. putida strain ATCC 17453. We have tested camphor, borneol, oxidized camphor metabolites and known bacterial attractants (d)-glucose, (d) - and (l)-glutamic acid for their elicitation chemotactic behavior. In addition, we have used 1-phenylimidazole, a P450_cam inhibitor, to investigate if P450_cam plays a role in the chemotactic ability of P. putida in the presence of camphor. We found that camphor, a chemoattractant, became toxic and chemorepellent when P450_cam was inhibited. We have also evaluated the effect of borneol on chemotaxis and found that the bacteria chemotaxed away from camphor in the presence of borneol. This is the first report of the chemotactic behaviour of P. putida ATCC 17453 and the essential role of P450_cam in this process.     

Structural and Dynamic Implications of an Effector-induced Backbone Amide cis-trans Isomerization in Cytochrome P450cam

Experimental evidence has been provided for a functionally relevant cis-trans isomerization of the Ile88-Pro89 peptide bond in cytochrome P450_cam (CYP101). The isomerization is proposed to be a key element of the structural reorganization leading to the catalytically competent form of CYP101 upon binding of the effector protein putidaredoxin (Pdx). A detailed comparison of the results of molecular dynamics simulations on the cis and trans conformations of substrate- and carbonmonoxy-bound ferrous CYP101 with sequence-specific Pdx-induced structural perturbations identified by nuclear magnetic resonance is presented, providing insight into the structural and dynamic consequences of the isomerization. The mechanical coupling between the Pdx binding site on the proximal face of CYP101 and the site of isomerization is described.     

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

Fatty acid binding and oxidation kinetics for wild type P450_BM3 (CYP102A1) from Bacillus megaterium have been found to display chain length-dependent homotropic behavior. Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths. Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate, pH 7.4, but sigmoidal kinetics (with an apparent intercept) in low ionic strength buffers and at physiological phosphate concentrations. In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites, with high cooperativity for the binding of the fourth palmitate molecule, and the full-length enzyme showing tighter palmitate binding than the heme domain. The first flavin-to-heme electron transfer is faster for laurate, myristate and palmitate in 100 mmol/L phosphate than in 50 mmol/L Tris (pH 7.4), yet each substrate induces similar high-spin heme content. For palmitate in low phosphate buffer concentrations, the rate constant of the first electron transfer is much larger than k_cat. The results suggest that phosphate has a specific effect in promoting the first electron transfer step, and that P450_BM3 could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.