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- Gustafsson, Mattias, et al.
(författare)
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Expression, purification, and characterization of Bacillus subtilis cytochromes P450CYP102A2 and CYP102A3: Flavocytochronie homologues of P450BM3 from Bacillus megaterium
- 2004
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:18, s. 5474-5487
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Tidskriftsartikel (refereegranskat)abstract
- The cyp102A2 and cyp102A3 genes encoding the two Bacillus subtilis homologues (CYP102A2 and CYP102A3) of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium have been cloned, expressed in Escherichia coli, purified, and characterized spectroscopically and enzymologically. Both enzymes contain heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors and bind a variety of fatty acid molecules, as demonstrated by conversion of the low-spin resting form of the heme iron to the high-spin form induced by substrate-binding. CYP102A2 and CYP102A3 catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduction of artificial electron acceptors at high rates. Binding of carbon monoxide to the reduced forms of both enzymes results in the shift of the heme Soret band to 450 nm, confirming the P450 nature of the enzymes. Reverse-phase high-performance liquid chromatography (HPLC) of products from the reaction of the enzymes with myristic acid demonstrates that both catalyze the subterminal hydroxylation of this substrate, though with different regioselectivity and catalytic rate. Both P450s 102A2 and 102A3 show kinetic and binding, preferences for long-chain unsaturated and branched-chain fatty acids over saturated fatty acids, indicating that the former two molecule types may be the true substrates. P450s 102A2 and 102A3 exhibit differing substrate selectivity profiles from each other and from P450 BM3, indicating that they may fulfill subtly different cellular roles. Titration curves for binding and turnover kinetics of several fatty acid Substrates with P450s 102A2 and 102A3 are better described by sigmoidal (rather than hyperbolic) functions, suggesting binding of more than one molecule of substrate to the P450s, or possibly cooperativity in substrate binding. Comparison of the amino acid sequences of the three flavocytochromes shows that several important amino acids in P450 BM3 are not conserved in the B. subtilis homologues, pointing to differences in the binding modes for the substrates that may explain the unusual sigmoidal kinetic and titration properties.
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- Wikström, Sofia A., et al.
(författare)
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Establishment of the exotic species Fucus evanescens C. Ag. (Phaeophyceae) in Öresund, Southern Sweden
- 2002
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Ingår i: Botanica Marina. - 0006-8055 .- 1437-4323. ; 45:6, s. 510-517
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Tidskriftsartikel (refereegranskat)abstract
- The spread and establishment of the exotic species Fucus evanescens in öresund, Southern Sweden was documented through inventories in 1966–72 and 2000. The species spread fast from the first known introduction in 1955, colonising harbours along the coast. After this expansion the further spread has been limited and the species is still largely confined to harbour areas, where it occurs in the same depth zone as its native relatives, F. vesiculosus, F. serratus and Ascophyllum nodosum. It is more common in the northern part of the area and has not expanded into the Baltic Sea proper. Tests of the attachment strength of F. evanescens plants suggest that the restriction to harbours is not due to the direct effects of wave exposure on adults. In the laboratory, reproductive success of F. evanescens decreased from 99% in 24 psu to 12% in 10 psu and at lower salinity reproduction failed. Growth of embryonic recruits was similarly affected by salinity. Hence, low salinity explains the limited success of F. evanescens in southern Öresund, where the salinity is low and fluctuating, and its failure to colonise the Baltic Sea proper.
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