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Title: Biosynthesis and mutasynthesis of the 3-methylglutamate residue of the nonribosomal lipopeptide antibiotics
Author: Nakeeb, Majid M. Mohammed Al
ISNI:       0000 0004 2678 8422
Awarding Body: The University of Manchester
Current Institution: University of Manchester
Date of Award: 2008
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Calcium dependent-antibiotic (CDA) is an acidic cyclic lipopeptide with a lactone core consist of 11 amino acid residues and possess aN-terminal 2,3-epoxyhexanoyl fatty acid side chain produced non ribosomally by nonribosomal peptide synthetases, CDA was isolated from Streptomyces coelicolor. CDA is similar in structure to the lipopeptide daptomycin from Streptomyces roseosporus. The structural similarities between CDA and daptomycin suggest that they have similar mode of action. Daptomycin (Cubicin) was approved in 2003 by the US FDA to be used as a treatment for many infections caused by Gram positive pathogens such as MRSA. Daptomycin has gone on to become one of the best selling intravenous antibiotics in US history ( CDA contains many nonproteinogenic amino acids such as penultimate C-terminal 3-methyl glutamate (3-MeGlu) residue. The work described in this study focused on 3-MeGlu residue found at position lOin CDA lactone ring. A stereocontrolled synthesis has been developed to obtain (2S,3R)- and (2S,3S)-3-MeGlu diastereoisomers, this synthesis can also be adapted to prepare analogues of 3-MeGlu. Synthetic methods were developed to prepare the racemic mixture of 3-MeGlu and 3-methyl-2-oxoglutarate. A mutasynthesis approach was developed which involved feeding synthetic 3-methyl-2-oxoglutarate and (2S,3R)-3-MeGlu to S. coelicolor mutant t:.glmT. This re-established the wild type phenotype (with 3-MeGlu containing CDA4b as a major variant) indicating GlmT is involved in 3-MeGlu biosynthesis. No CDA4a was evident by feeding synthetic (2S,3S)-3-MeGlu. This indicates that methylation is specific for the (2S,3R)-3MeGlu stereoisomer. Comparisons between natural 3-MeGlu (from hydrolysis of CDA and daptomycin) and synthetic 3-MeGlu revealed the absolute stereochemistry of 3-MeGlu in CDA as 2S,3R. The biosynthetic origin of 3-MeGlu in CDA was elucidated by further mechanistic analysis, which suggest that GlmT catalyses the SAM-dependent methylation of a-ketoglutarate to give (3R)-methyl-2-oxoglutarate and then transaminated to (2S,3R)-3MeGlu. Racemic 3-trifluoromethylglutamic acid and 3-ethylglutamic acid were also synthesised. Precursor-directed biosynthesis of CDAs with modified 3-trifluoromethyl and 3ethyl glutamate residues was achieved by feeding these synthetic glutamate analogues to the t:.glmT mutant strain of S. coelicolor, resulting in several novel CDA variants. Synthesis of (2S,3R)-3-methylglutamine were developed and stabilized by dipeptide coupling. Producing novel antibiotics with improved antibiotic activity and reduced toxicity are particularly important due to the continuous resistance to antibiotics by the pathogenic bacteria.
Supervisor: Not available Sponsor: Not available
Qualification Name: Not available Qualification Level: Doctoral
EThOS ID:  DOI: Not available