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Biochemical Characterization of StyAB from Pseudomonas sp . Strain VLB120 as a Two-Component Flavin-Diffusible Monooxygenase. Katja Otto, 2004.Pseudomonas sp . VLB120 uses styrene as a sole source of carbon and energy . The first step in this metabolic pathway is catalyzed by an oxygenase (StyA) and a NADH-flavin oxidoreductase (StyB) . Both components have been isolated from wild-type Pseudomonas strain VLB120 as well as from recombinant Escherichia coli . StyA from both sources is a dimer, with a subunit size of 47 kDa, and catalyzes the enantioselective epoxidation of C  C
double bonds . Styrene is exclusively converted to S-styrene
oxide with a specific activity of 2.1 U mg–1 (kcat
= 1.6 s–1) and Km values for styrene of
0.45 ± 0.05 mM (wild type) and 0.38 ± 0.09 mM (recombinant) . The
epoxidation reaction depends on the presence of a NADH-flavin adenine
dinucleotide (NADH-FAD) oxidoreductase for the supply of reduced FAD .
StyB is a dimer with a molecular mass of 18 kDa and a NADH oxidation
activity of 200 U mg–1 (kcat [NADH] = 60 s–1) .
Steady-state kinetics determined for StyB indicate a mechanism of
sequential binding of NADH and flavin to StyB . This enzyme reduces
FAD as well as flavin mononucleotide and riboflavin . The NADH
oxidation activity does not depend on the presence of StyA . During
the epoxidation reaction, no formation of a complex of StyA and
StyB has been observed, suggesting that electron transport between
reductase and oxygenase occurs via a diffusing flavin .
Defined Inactive FecA Derivatives Mutated in Functional Domains of the Outer Membrane Transport and Signaling Protein of Escherichia coli K-12. Annette Sauter, 2004.The FecA outer membrane protein of Escherichia coli functions as a transporter of ferric citrate and as a signal receiver and signal transmitter for transcription initiation of the fec transport genes . Three FecA regions for which functional roles have been predicted from the crystal structures were mutagenized: (i) loops 7 and 8, which move upon binding of ferric citrate and close the entrance to the ferric citrate binding site; (ii) the dinuclear ferric citrate binding site; and (iii) the interface between the globular domain and the ß-barrel . Deletion of loops 7 and 8 abolished FecA transport and induction activities . Deletion of loops 3 and 11 also inactivated FecA, whereas deletion of loops 9 and 10 largely retained FecA activities . The replacement of arginine residue R365 or R380 and glutamine Q570, which are predicted to serve as binding sites for the negatively charged dinuclear ferric citrate, with alanine resulted in inactive FecA, whereas the binding site mutant R438A retained approximately 50% of the FecA induction and transport activities . Residues R150, E541, and E587, conserved among energy-coupled outer membrane transporters, are predicted to form salt bridges between the globular domain and the ß-barrel and to contribute to the fixation of the globular domain inside the ß-barrel . Mutations E541A and E541R affected FecA induction and transport activity slightly, whereas mutations E587A and E587R more strongly reduced FecA activity . The double mutations R150A E541R and R150A E587R nearly abolished FecA activity . Apparently, the salt bridges are less important than the individual functions these residues seem to have for FecA activity . Comparison of the properties of the FecA, FhuA, FepA, and BtuB transporters indicates that although they have very similar crystal structures, the details of their functional mechanisms differ .
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