RepAM of the Amycolatopsis methanolica integrative element pMEA300 belongs to a novel class of replication initiator proteins.

Accessory genetic elements, such as plasmids and integrative elements, are widespread amongst actinomycetes, but little is known about their functions and mode of replication. The conjugative element pMEA300 from Amycolatopsis methanolica is present mostly in an integrated state at a single specific site in the chromosome, but it can also replicate autonomously. Complete nucleotide sequencing, in combination with deletion studies, has revealed that orfB of pMEA300 is essential for autonomous replication in its host. In this study, it was shown that purified OrfB protein binds specifically to the 3' end of its own coding sequence. Within this short sequence, a putative hairpin structure is located, which contains several direct and inverted repeats, and a nucleotide stretch that resembles the nicking site of the pC194 family of rolling circle replicating plasmids. Additional binding studies revealed that OrfB binds to an 8 bp inverted repeat that occurs three times within the hairpin structure. The data presented show that OrfB is the replication initiator (Rep) protein of pMEA300, and is therefore termed RepAM. Surprisingly, RepAM lacks significant sequence similarity with known prokaryotic Rep proteins, but it is highly similar to a number of yet uncharacterized ORFs that are located on integrative and conjugative elements of other actinomycetes. It is concluded that RepAM and its homologues are members of a novel class of Rep proteins.

Organization of the teicoplanin gene cluster in Actinoplanes teichomyceticus.

The glycopeptide teicoplanin is used for the treatment of serious infections caused by Gram-positive pathogens. The tcp gene cluster, devoted to teicoplanin biosynthesis in the actinomycete Actinoplanes teichomyceticus, was isolated and characterized. From sequence analysis, the tcp cluster spans approximately 73 kb and includes 39 ORFs participating in teicoplanin biosynthesis, regulation, resistance and export. Of these, 34 ORFs find a match in at least one of the five glycopeptide gene clusters previously characterized. Putative roles could be assigned for most of the tcp genes. The two glycosyltransferases responsible for attaching amino sugars to amino acids 4 and 6 of the teicoplanin aglycon were overexpressed in Escherichia coli and characterized. They both recognize N-acetylglucosamine as the substrate. tGtfA can add a sugar residue in the presence or absence of N-acetylglucosamine at amino acid 4, while tGtfB can only glycosylate the teicoplanin aglycon.

Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus.

The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed.

Molecular, biochemical, and functional characterization of a Nudix hydrolase protein that stimulates the activity of a nicotinoprotein alcohol dehydrogenase.

The cytoplasmic coenzyme NAD(+)-dependent alcohol (methanol) dehydrogenase (MDH) employed by Bacillus methanolicus during growth on C(1)-C(4) primary alcohols is a decameric protein with 1 Zn(2+)-ion and 1-2 Mg(2+)-ions plus a tightly bound NAD(H) cofactor per subunit (a nicotinoprotein). Mg(2+)-ions are essential for binding of NAD(H) cofactor in MDH protein expressed in Escherichia coli. The low coenzyme NAD(+)-dependent activity of MDH with C(1)-C(4) primary alcohols is strongly stimulated by a second B. methanolicus protein (ACT), provided that MDH contains NAD(H) cofactor and Mg(2+)-ions are present in the assay mixture. Characterization of the act gene revealed the presence of the highly conserved amino acid sequence motif typical of Nudix hydrolase proteins in the deduced ACT amino acid sequence. The act gene was successfully expressed in E. coli allowing purification and characterization of active ACT protein. MDH activation by ACT involved hydrolytic removal of the nicotinamide mononucleotide NMN(H) moiety of the NAD(H) cofactor of MDH, changing its Ping-Pong type of reaction mechanism into a ternary complex reaction mechanism. Increased cellular NADH/NAD(+) ratios may reduce the ACT-mediated activation of MDH, thus preventing accumulation of toxic aldehydes. This represents a novel mechanism for alcohol dehydrogenase activity regulation.