Structure and mechanism of the rebeccamycin sugar 4'-O-methyltransferase RebM.
The 2.65-angstroms crystal structure of the rebeccamycin 4'-O-methyltransferase RebM in complex with S-adenosyl-l-homocysteine revealed RebM to adopt a typical S-adenosylmethionine-binding fold of small molecule O-methyltransferases (O-MTases) and display a weak dimerization domain unique to MTases. Using this structure as a basis, the RebM substrate binding model implicated a predominance of nonspecific hydrophobic interactions consistent with the reported ability of RebM to methylate a wide range of indolocarbazole surrogates. This model also illuminated the three putative RebM catalytic residues (His140/141 and Asp166) subsequently found to be highly conserved among sequence-related natural product O-MTases from GC-rich bacteria. Interrogation of these residues via site-directed mutagenesis in RebM demonstrated His140 and Asp166 to be most important for catalysis. This study reveals RebM to be a member of the general acid/base-dependent O-MTases and, as the first crystal structure for a sugar O-MTase, may also present a template toward the future engineering of natural product MTases for combinatorial applications.
Singh
Shanteri
S
Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53705, USA.
McCoy
Jason G
JG
Zhang
Changsheng
C
Bingman
Craig A
CA
Phillips
George N
GN
Thorson
Jon S
JS
D002227:Carbazoles / Q000737:chemistry / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000378:metabolism; D019311:Staurosporine / Q000737:chemistry / Q000378:metabolism
D002227:Carbazoles; D011994:Recombinant Proteins; C053339:rebeccamycin; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases; C531981:rebeccamycin 4'-O-methyltransferase, Lechevalieria aerocolonigenes; D019311:Staurosporine