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Expression, purification and characterization of Bacillus subtilis cytochromes P450 102A2 and 102A3: Flavocytochrome homologs of P450 BM3 from Bacillus megaterium. Biochemistry

Författare:
Publiceringsår: 2004
Språk: Engelska
Sidor: 5474-5487
Publikation/Tidskrift/Serie: Biochemistry
Volym: 43
Nummer: 18
Dokumenttyp: Artikel
Förlag: ACS Publications

Sammanfattning

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.

Disputation

Nyckelord

  • Biology and Life Sciences

Övriga

Published
Yes
  • ISSN: 1520-4995 (Online)
  • ISSN: 0006-2960 (Print)

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