Expression and assay of an N-methyltransferase involved in the biosynthesis of a vancomycin group antibiotic

Chemical Communications

Volumn , Issue 1, 2000, Pages 103-104

  O'Brien, Dominic P ^a   Kirkpatrick, Peter N ^a   O'Brien, Simon W ^a   Staroske, Thomas ^a   Richardson, Timothy I ^b   Evans, David A ^b   Hopkinson, Andrew ^c   Spencer, Jonathan B ^a   Williams, Dudley H ^a  

^a UNIVERSITY OF CAMBRIDGE   (United Kingdom)

^b HARVARD UNIVERSITY   (United States)

^c UNILEVER RESEARCH   (United Kingdom)

Author keywords

[No Author keywords available]

Indexed keywords

METHYLTRANSFERASE; VANCOMYCIN;

ARTICLE; CHEMICAL ANALYSIS; CROSS LINKING; DRUG SYNTHESIS; HIGH PERFORMANCE LIQUID CHROMATOGRAPHY; MOLECULAR WEIGHT; PROTEIN EXPRESSION;

EID: 0034614460     PISSN: 13597345     EISSN: None     Source Type: Journal    
DOI: 10.1039/a907953j     Document Type: Article

References (14)

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10. This peptide was synthesised using a combination of solution phase and solid phase methodology. Phpg and Dhpg refer to 4-hydroxyphenylglycine and 3,5-dihydroxyphenylglycine, respectively.
11. Equimolar mixtures of (R)-N-methylleucine and (R) leucine, vancomycin and N-demethylvancomycin and vancomycin aglycone and N-demethyvancomycin aglycone were subjected to FT-ICR mass spectrometry. For a given solution, the intensities of the singly and doubly charged parent ion peaks of each component were the same to within 10%.
12. Fragmentation peaks corresponding to N-demethylvancomycin, N-demethylvancomycin aglycone and (R)-leucine were not observed in the FT-ICR spectra of vancomycin, vancomycin aglycone and (R)-N-methylleucine, respectively.
13. Solutions of leucine and N-methylleucine were subjected to reverse phase HPLC and fractions were collected at intervals of 30 s. Each fraction was then subjected to FT-ICR mass spectrometry to test for the presence of the corresponding compound. By progressively narrowing the time interval between collections the retention time of the compounds was determined to within five seconds. For each reaction assay, samples corresponding to these retention times were collected and subjected to FT-ICR mass spectrometry.
14. 60% of 100 μ;M substrate 3 is methylated in the presence of 100 μM (S)-adenosyl-L-methionine and MtfA, whereas 95% is methylated under the same conditions with 200 μM (S)-adenosyl-L-methionine. Hence, a 1:1 mixture of (S)-adenosyl-L-methionine and substrate provides a cofactor concentration which limits the extent of conversion.