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Title: Molecular and biochemical characterisation of key enzymes involved in mycolic acid biosynthesis from Mycobacterium tuberculosis
Author: Brown, Alistair Karl
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2004
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Mycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall, providing the basis for its lipid-rich permeability barrier. These oc-alkyl, P-hydroxy fatty acids are thought to be formed by the Claisen-typec ondensationo f a long C56m eromycolic acid and a shorter C24-C2f6a tty acid. These componentf atty acids are producedv ia a combination of type I and 11f atty acid synthase( FAS) systems.T he C16-C2f6a tty acyl products of FAS-I are elongated by FAS-II with simultaneous modification to form meromycolic acids, which are then condensedw ith the C24-C26fa tty acyl chain. These studies aimed to characterisek ey enzymes of FAS-11 (mtFabH, KasA) and enzymes possibly involved in the Claisen-type condensationr eactiont o form mycolatic acids (Accl) enzymes,P ksl3, FadD32). The P-ketoacyl ACP synthase (KAS) III (mtFabH) is proposed to link FAS-I and FAS-11, catalyzing the condensation of FAS-1-derived acyl-CoA with malonyl-Acyl Carrier Protein (ACP). The acyl-CoA chain length specificity of mtFabH was assessedin vitro . When using E. coll, the preferreds ubstratesw ere C12-a ndC 14-CoAH. owever, with the mycobacterialA CP (AcpM), the enzyme was able to utilise longer (up tp C2o) acyl-CoA chains. The substitution of residues implicated in acyl-CoA chain length specificity totally abrogated overall KAS activity and reduced the transacylation activity of the enzyme. Mutation of the proposed catalytic triad residues confirmed that Cys122 is essential for transacylation and His258 is essential for malonyl-AcpM decarboxylation. KasA, which belongs to the FAS-11 system, utilises palmitoyl-ACP rather than short-chain acyl-ACP primers. Purified recombinant KasA had in vitro KAS activity that was highly sensitive to cerulenin, a well-known KAS inhibitor. Mutation of proposed catalytic residues Cys 17 1, His31 1, Lys340 and His345 inactivated the enzyme completely. Four putative accD genes were found in Corynebacterium glutamicum. Overexpression of each gene resulted in increased acyl-CoA dependent 14CO2 fixation in vitro, providing evidence that the accD genes encode a family of carboxyltransferases. Disruption of either accD2 or accD3 led to complete and specific loss of mycolic acids. These two carboxyltransferasesa re also retained in all Corynebacterianeaei,n cluding M. leprae, and probably provide a carboxylated. intennediate for condensation of the mero-chain and CCbranch directed by thepks]3-encoded polyketide synthase.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available