2.1.1.292: carminomycin 4-O-methyltransferase

site-directed mutagenesis, the mutation greatly reduces the methylation activity on flavonoids

R303K

site-directed mutagenesis, the DnrK R1.2 mutant shows altered substrate specificity, the mutant displays greatly reduced activities for both 4-O-methylation and 10-hydroxylation. The 10-hydroxylation activity is completely lost in the R303Q mutant, whereas only trace activities remain in the R303K mutant when 8 is used as a substrate. The 4-O-methylation activity is also affected, although the mutant still harbors about 10% of its activity

R303Q

site-directed mutagenesis, the DnrK R1.2 mutant shows altered substrate specificity, the mutant displays greatly reduced activities for both 4-O-methylation and 10-hydroxylation. The 10-hydroxylation activity is completely lost in the R303Q mutant. The 4-O-methylation activity is also affected, although the mutant still harbors about 10% of its activity

S297F

site-directed mutagenesis, the DnrK R1.2 mutant shows altered substrate specificity

Y142W

48% of the rate of the recombinant native enzyme with 4-methoxy-epsilon-rhodomycin T

722641

additional information

insertion of a single serine residue at position 297 to DnrK is sufficient for introduction of a monooxygenation activity. The inserted serine S297 resides in an alpha-helical segment adjacent to the substrate, but in a manner where the side chain points away from the active site. The shift in activity is mediated by rotation of a preceding phenylalanine F296 toward the active site, which blocks a channel to the surface of the protein that is present in native DnrK. The 10-decarboxylation activity of DnrK is the basis of evolution of a RdmB, an atypical 10-hydroxylase that requires SAM of the rhodomycin pathways, which has 10-hydroxylation ability. For analysis of the origin of the 10-hydroxylation activity in the DnrK R1 chimera, the R1 region is divided into two segments corresponding to the loop region and the following alpha16 helix, which results in two additional mutants denoted as DnrK R1.1 and DnrK R1.2, respectively. The 10-hydroxylation activity can be attributed solely to the alpha16 helix, because the activity of DnrK R1.2 is similar to that of DnrK R1 (i.e. both methylation and hydroxylation of aclacinomycin T and hydroxylation of triglycosylated aclacinomycin A), whereas DnrK R1.1 behaves like native DnrK (i.e. methylation of aclacinomycin T and no activity with triglycosylated aclacinomycin A). Inspection of the amino acid sequences of the R1.2 region reveals that the RdmB sequence contains an additional serine insertion in this area in comparison with DnrK. Fusion of the dimerization domain of DnrK onto the catalytic domain of RdmB generates the enzyme variant RdmB-CT, the activity of RdmB-CT is not altered and the enzyme catalyzes exclusively 10-hydroxylation. Creation of chimeric enzymes by interchanging key subdomain regions ranging from 4-O-methyl-15-decarboxyaclacinomycin T to 4-O-methyl-11-deoxy-beta-rhodomycin T aa to probe the functional differentiation of the enzyme pair