Structural studies on KijD1, a sugar C-3'-methyltransferase.
Kijanimicin is an antitumor antibiotic isolated from Actinomadura kijaniata. It is composed of three distinct moieties: a pentacyclic core, a monosaccharide referred to as d-kijanose, and a tetrasaccharide chain composed of l-digitoxose units. d-Kijanose is a highly unusual nitro-containing tetradeoxysugar, which requires at least ten enzymes for its production. Here we describe a structural analysis of one of these enzymes, namely KijD1, which functions as a C-3'-methyltransferase using S-adenosylmethionine as its cofactor. For this investigation, two ternary complexes of KijD1, determined in the presence of S-adenosylhomocysteine (SAH) and dTDP or SAH and dTDP-3-amino-2,3,6-trideoxy-4-keto-3-methyl-d-glucose, were solved to 1.7 or 1.6 Å resolution, respectively. Unexpectedly, these structures, as well as additional biochemical analyses, demonstrated that the quaternary structure of KijD1 is a dimer. Indeed, this is in sharp contrast to that previously observed for the sugar C-3'-methyltransferase isolated from Micromonospora chalcea. By the judicious use of site-directed mutagenesis, it was possible to convert the dimeric form of KijD1 into a monomeric version. The quaternary structure of KijD1 could not have been deduced based solely on bioinformatics approaches, and thus this investigation highlights the continuing need for experimental validation.
Department of Biochemistry, University of Wisconsin, Madison, WI, 53706.
Garrett T
GT
Dow
Department of Biochemistry, University of Wisconsin, Madison, WI, 53706.
James B
JB
Thoden
Department of Biochemistry, University of Wisconsin, Madison, WI, 53706.
Hazel M
HM
Holden
D039903:Actinobacteria / Q000201:enzymology*; D000617:Aminoglycosides / Q000096:biosynthesis / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D008780:Methyltransferases / Q000737:chemistry*; D000072417:Protein Domains; D020836:Protein Structure, Quaternary
D000617:Aminoglycosides; D001426:Bacterial Proteins; C033276:kijanimicin; D008780:Methyltransferases
Actinomadura kijaniata; C-3′-methyltransferase; antitumor antibiotic; d-kijanose; d-tetronitrose; kijanimicin