Universität Stuttgart
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Item Open Access Microbial P450 enzymes in biotechnology(2004) Urlacher, Vlada B.; Lutz-Wahl, Sabine; Schmid, Rolf D.Oxidations are key reactions in chemical syntheses. Biooxidations using fermentation processes have already conquered some niches in industrial oxidation processes, since they allow the introduction of oxygen even into non-activated carbon atoms in a sterically and optically selective manner which is difficult or impossible to achieve by synthetic organic chemistry. Biooxidation using isolated enzymes is limited to oxidases and dehydrogenases. Surprisingly, cytochrome P450 monooxygenases (CYPs) have scarcely been studied for use in biooxidations, although they are one of the largest known superfamilies of enzyme proteins. Their gene sequences have been identified in various organisms such as humans, bacteria, algae, fungi and plants. The reactions catalyzed by P450s are quite diverse and range from biosynthetic pathways (e.g. those of animal hormones and secondary plant metabolites) to the activation or biodegradation of hydrophobic xenobiotic compounds (e. g. those of various drugs in the liver of higher animals). From a practical point of view, the great potential of P450s is limited by their functional complexity, low activity, and limited stability. In addition, P450-catalyzed reactions require a constant supply of NAD(P)H which makes continuous cell-free processes very expensive. Quite recently, several groups have started to investigate cost-efficient ways which could allow the continuous supply of electrons to the heme iron. These include, for example, the use of electron mediators, direct electron supply from electrodes and enzymatic approaches. In addition, methods of protein design and directed evolution have been applied in an attempt to enhance the activity of the enzymes and improve their selectivity. The promising application of bacterial P450s as catalyzing agents in biocatalytic reactions and recent progress made in this field are covered in this review.Item Open Access Reconstitution of beta-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase(2006) Momoi, Kyoko; Hofmann, Ute; Schmid, Rolf D.; Urlacher, Vlada B.CYP175A1 is a thermostable P450 Monooxygenase from Thermus thermophilus HB27, demonstrating in vivo activity towards -carotene. Activity of CYP175A1 was reconstituted in vitro using artificial electron transport proteins. First results were obtained in the mixture with a crude E. coli cell extract at 37°C. In this system -carotene was hydroxylated to -cryptoxanthin. The result indicated the presence of electron transport enzymes among the E. coli proteins, which are suitable for CYP175A1. However, upon in vitro reconstitution of CYP175A1 activity with purified recombinant flavodoxin and flavodoxin reductase from E. coli, only very low -cryptoxanthin production was observed. Remarkably, with another artificial electron transport system, putidaredoxin and putidaredoxin reductase from Pseudomonas putida, purified CYP175A1 enzyme hydroxylated -carotene at 3- and also 3’-positions, resulting in -cryptoxanthin and zeaxanthin. Under the optimal reaction conditions, the turnover rate of the enzyme reached 0.23 nmol -cryptoxanthin produced per nmol P450 per min.Item Open Access Selective hydroxylation of highly branched fatty acids and their derivatives by CYP102A1 from Bacillus megaterium(2006) Budde, Michael; Morr, Michael; Schmid, Rolf D.; Urlacher, Vlada B.Highly branched fatty acids, representing the main component of the preen gland wax of the domestic goose, and their derivatives are promising chiral precursors for the synthesis of macrolid antibiotics. The key step in utilisation of these compounds is the regioselective hydroxylation, which can not be done in a classical chemical approach. Three P450 monooxygenases CYP102A1, CYP102A2 and CYP102A3, demonstrating high turnover numbers in hydroxylation of iso and anteiso fatty acids (>400 min-1), were tested for their activity towards these substrates. CYP102A1 from Bacillus megaterium as well as its A74G F87V L188Q triple mutant hydroxylate a variety of these substrates with high activity and regioselectivity. In all cases the triple mutant showed much higher activities than the wild type enzyme. The binding constants, determined for CYP102A1 wild type and the triple mutant were >200 µM and ~23 µM, respectively, when tetramethyl nonanol was used as substrate. The data derived from binding analysis supports the differences in activity found for the CYP102A1 wild type and the triple mutant. Surprisingly the CYP102A2 and CYP102A3 from Bacillus subtilis did not show activity at all. Substrate binding spectra, recorded to investigate substrate accessibility to the enzyme’s active site, revealed that the substrates either could not access the active site of the Bacillus subtilis monooxygenases, or did not reach the heme proximity.