ABSTRACT
The lux-operon of bioluminescent bacteria contains the genes coding for the enzymes required for light emission. Some species of Photobacteria feature an additional gene, luxF, which shows similarity to luxA and luxB, the genes encoding the heterodimeric luciferase. Isolated dimeric LuxF binds four molecules of an unusually derivatized flavin, i.e., 6-(3'-(R)-myristyl)-FMN (myrFMN). In the present study we have heterologously expressed LuxF in Escherichia coli BL21 in order to advance our understanding of the protein's binding properties and its role in photobacterial bioluminescence. Structure determination by X-ray crystallography confirmed that apo-LuxF possesses four preorganized binding sites, which are further optimized by adjusting the orientation of amino acid side chains. To investigate the binding properties of recombinant LuxF we have isolated myrFMN from Photobacterium leiognathi S1. We found that LuxF binds myrFMN tightly with a dissociation constant of 80±20 nM demonstrating that the purified apo-form of LuxF is fully competent in myrFMN binding. In contrast to LuxF, binding of myrFMN to luciferase is much weaker (Kd=4.0±0.4 µM) enabling LuxF to prevent inhibition of the enzyme by scavenging myrFMN. Moreover, we have used apo-LuxF to demonstrate that myrFMN occurs in all Photobacteria tested, irrespective of the presence of luxF indicating that LuxF is not required for myrFMN biosynthesis.
Subject(s)
Apoproteins/chemistry , Bacterial Proteins/chemistry , Flavin Mononucleotide/chemistry , Luciferases/chemistry , Myristic Acid/chemistry , Photobacterium/chemistry , Amino Acid Sequence , Apoproteins/genetics , Apoproteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Luciferases/genetics , Luciferases/metabolism , Luminescence , Models, Molecular , Molecular Sequence Data , Photobacterium/enzymology , Protein Binding , Protein Conformation , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , ThermodynamicsABSTRACT
Anthranoyl-CoA monooxygenase/reductase (ACMR) participates in an unusual pathway for the degradation of aromatic compounds in Azoarcus evansii. It catalyzes the monooxygenation of anthranoyl-CoA to 5-hydroxyl-2-aminobenzoyl-CoA and the subsequent reduction to the dearomatized product 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. The two reactions occur in separate domains, termed the monooxygenase and reductase domain. Both domains were reported to utilize FAD as a cofactor for hydroxylation and reduction, respectively. We have heterologously expressed ACMR in Escherichia coli BL21 and found that the monooxygenase domain contains FAD. However, the reductase domain utilizes FMN and not FAD for the reduction of the intermediate 5-hydroxyl-2-aminobenzoyl-CoA. A homology model for the reductase domain predicted a topology similar to the Old Yellow Enzyme family, which exclusively bind FMN, in accordance with our results. Binding studies with 2-aminobenzoyl-CoA (AbCoA) and p-hydroxybenzaldehyde (pHB) as probes for the monooxygenase and reductase domain, respectively, indicated that two functionally distinct and independent active sites exist. Given the homodimeric quartenary structure of ACMR and the compact shape of the dimer as determined by small-angle X-ray scattering experiments we propose that the monooxygenase and reductase domain of opposite peptide chains are involved in the transformation of anthranoyl-CoA to 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA.
