STRUCTURAL REFINEMENT OF THE NON-FLUORESCENT FLAVOPROTEIN FROM PHOTOBACTERIUM LEIOGNATHI AT 1.60 ANGSTROM RESOLUTION

摘要

The crystallographically-determined structure of the non-fluorescent flavoprotein (NFP) from Photonbacterium leiognathi, a homolog of the bacterial luciferase subunits, has been refined to a conventional R-factor (R = SigmaF-o - F-c/SigmaF-o) of 0.175 using synchrotron data between 10.0 and 1.60 Angstrom resolution. The molecular structure is a homodimer of beta/alpha domains, the monomer having structural similarities to (beta alpha)(8) barrel proteins. However, one beta-strand and three alpha-helices of a typical (beta alpha)(8) domain are not present in the NFP structure. The refined structure of NFP consists of the 228 amino acid polypeptide, 191 water molecules, a sulfate ion, and two flavin mononucleotides (FMNs), each with a covalently-attached myristate (C-14 fatty acid). Both flavin adducts are well-ordered and have exceptional electron density for both the FMN and the myristate moieties. Each flavin mononucleotide-myristate adduct is characterized by a stereospecific linkage (the S enantiomer) between C-6 of the flavin isoalloxazine ring and the C-3' atom of the fatty acyl chain. The stereospecific nature of this flavin-fatty acid linkage suggests that it is the result of an enzyme-catalyzed reaction, most likely the bioluminescence reaction itself. The myristate chains are buried from solvent in hydrophobic pockets in the interior of the Protein. Four amino acid side-chains of the NFP polypeptide have been modeled with alternate conformations. Five of the protein's seven a-helices have classical C-capping boxes. NFP is dimeric and many of the extensive contacts at the dimer interface are mediated by hydrogen-bonded water molecules as well as by hydrophobic interactions. One of the myristate acyl chains sits between NFP monomers and contributes a significant portion of the hydrophobic interactions at the NFP dimer interface. [References: 50]