Slayden RA, Ramaswamy S, Musser JM, Barry CE; American Society for Microbiology. General Meeting.

Abstr Gen Meet Am Soc Microbiol. 1999 May 30-Jun 3; 99: 646 (abstract no. U-65).

NIAID, NIH, Rockville, MD.

Isonicotinic acid hydrazide (INH ), a first-line drug, has recently been shown to inhibit mycolic acid biosynthesis in M. tuberculosis by disrupting FAS II activity via the formation of a covalent trimolecular complex containing an acyl carrier protein, AcpM, and a b-keto acyl synthase, KasA. AcpM was shown to carry fatty acids ranging from 16-50 carbons in length. However, in the presence of INH, AcpM accumulated with only short (16-26 carbon) fatty acids attached. Further analysis of these genes using the genome sequence revealed that they are part of a five gene operon. acpM is located proximal to two b-keto acyl synthases (kasA and kasB) which are an integral part of the biosynthetic machinery involved in mero-mycolate lipogenesis. It is generally accepted that short-chain lipid precursors (C16- C26) are the result of the pre-described FAS type I. At which point the acyl chain is transferred to AcpM and further elongation up to C56 as a result of combined KasA and KasB activity. To demonstrate the function of these FAS II enzymes, two approaches that utilize C16 as the synthetic precursor have been undertaken. One approach was in vitro synthesis of mero-mycolate fatty acids by hyper-production of either KasA or KasA/KasB. This approach has established the biosynthetic activity of KasA and KasB and upon addition of INH KasA activity can be diminished. Furthermore kasA genes containing mutations where cloned from INH clinical isolates and shown to be resistant to INH in vitro. The second approach exploits the promiscuity of FAS II proteins; through operon reconstruction and co-ordinate production of FabD, AcpM, KasA and KasB in the host, E. coli was used to achieve meromycolate like fatty acid synthesis. It has been established that AcpM is post-transitionallv modified with a phosphopantotheine prosthetic group and charged by the metabolic machinery of the host resulting in the synthesis of acyl-AcpM. These studies are the initial steps in conclusively identifying the key enzymatic steps involved in mycolic acid lipogenesis by producing these actinomycete- specific lipids in vitro and in an unrelated host system such as E. coli.

Publication Types:

• Meeting Abstracts

Keywords:

• Acyl Carrier Protein
• Base Sequence
• Fatty Acids
• In Vitro
• Isoniazid
• Mycolic Acids
• Operon
• Tuberculosis
• biosynthesis
• chemical synthesis
• enzymology
• genetics

Other ID:

• 20712283

UI: 102195813

From Meeting Abstracts