Amines play an important role in global cycling of carbon and nitrogen in nature, they are apparently involved in the production of greenhouse gases and are widespread contaminants of water and soil. Despite the fact that many amines are normal constituents of human blood, they have been connected with cases of poisoning and have been shown to cause vascular tissue pathology. Enzymes participating in amine oxidation are widespread in both eukaryotes and prokaryotes. One group of enzymes oxidizing amines are quinoproteins with quinone groups synthesized posttranslationally from a tyrosine or tryptophan residue of the polypeptide chain. The periplasmic methylamine dehydrogenase (MADH, encoded by the mau-genes) is the only known member of this unique class of quinoproteins containing tryptophan tryptophylquinone (TTQ) as a cofactor, and the enzyme has so far been found only in prokaryotes. The long term objective of this proposal is the study of TTQ cofactor biosynthesis in methylotrophic bacteria.
The specific aims of the initial portion of this long-term work is to generate and characterize mutants in the mau genes to identify those that may be involved in TTQ synthesis and to develop an in vivo expression system in order to reconstruct events of TTQ biosynthesis in a heterologous host. A series of chemically induced Mau- mutants of Methylobacillus flagellatum KT will be isolated and the impaired genes will be identified in all of the mutants. Those genes that do not map within the known mau gene cluster will be cloned. Site-specific mutagenesis will also be carried out on MADH structural genes to determine targets and temporal order for modifications. Physiological and biochemical studies of all mutants will be carried out to elucidate their participation in specific steps of MADH assembly and cofactor biosynthesis. The following characteristics of Mau- mutants will be studied: l. activities of MADH, its electron acceptor and NAD(P) and dye-linked formaldehyde and formate dehydrogenases. This will allow the elimination of some of the mutants as not pertaining to MADH assembly and TTQ synthesis. 2. the presence of the MADH small and large subunit polypeptides in the mutants, the form of each (mature or premature), and the cell compartment in which they are present. 3. the presence of a quinone in MADH after SDS-PAGE separation of the mutant enzymes (by specific staining). MADHs from the mutants that appear to be defective in cofactor biosynthesis or MADH assembly will be isolated and characterized by visible spectroscopy and the presence of the TTQ cross- link will be assessed by proteolytic analysis of the small subunit. These studies should delineate the steps in cofactor biosynthesis and their temporal and spatial characteristics. The second approach will be to develop in vivo expression systems for mau genes. Two systems will be considered: P(L)-dependent expression of the mau genes from M.flagellatum KT in E. coli and expression of the mau genes from Methylobacterium extorquens AM1 in other Methylobacterium strains which do not grow on methylamine naturally. The information obtained will allow us to refine the existing working model of TTQ biosynthesis, to determine the pathway for TTQ biosynthesis, and to begin to define its place in the process of MADH assembly as a whole. This knowledge will facilitate studying the synthesis and assembly of other post-translationally modified quinoproteins including those of medical importance.
