Site directed mutagenesis of L-pyrrolysine in dimethylamine methyltransferase
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Date
2009-03
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The Ohio State University
Abstract
Methanosarcina barkeri is an archaeon and is able to use methylamines as growth substrates for methanogenesis. The methylamine methyltransferases that are involved in initiating methanogenesis from the methylamines have an in-frame amber codon (UAG) that does not terminate translation, but in fact encodes for the 22nd amino acid L-pyrrolysine. This thesis attempts to examine the rationale for the presence of pyrrolysine in one of these three methylamine methyltransferases.
A system that allowed the recombinant expression of the dimethylamine methyltransferase gene, mtbB, in the native organism Methanosarcina acetivorans, was developed (9). Site directed mutagenesis of mtbB was performed wherein UAG, which encodes for pyrrolysine, was substituted by GCA, which encodes for alanine (Jodie Lee). This allowed for functional studies of MtbB vs. the mutant MtbB, dubbed MtbB(O356A). An activity assay, which measured the ability of MtbB and MtbB(O356A) to methylate hydroxocobalamin, showed that MtbB(O356A) had lost its functionality. This indicated that pyrrolysine is an essential residue in MtbB and that it may play a catalytic role. However, preliminary data from native gel electrophoresis showed that the ability of MtbB(O356A) to bind its cognate corrinoid cofactor protein, MtbC, was greatly reduced; whilst the wild-type MtbB was able to form a complex with MtbC. This indicated that pyrrolysine is required for proper folding of MtbB and/or it is required for binding MtbC.
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Keywords
pyrrolysine, Methanosarcina barkeri, amber codon, Methanosarcina