Characterization of The β‐Keto‐Acyl‐ACP Synthase, KasA from Mycobacterium Tuberculosis

Abstract

Isoniazid (INH) is a frontline drug used to treat tuberculosis, an infectious disease that kills more than 2 million people annually. While INH is known to inhibit the synthesis of long chain mycolic acids, there is still controversy over the exact molecular target for this drug. Evidence exists that implicates both KasA, the β-keto-acyl-ACP synthase, and InhA, the enoyl-ACP reductase from the fatty acid biosynthesis pathway (FASII), as targets for INH. Clinical mutations in both InhA and KasA correlate with resistance to INH, however the InhA mutants I21V and I47T have the same affinity as wild-type InhA for the active drug. These observations are explained by postulating that InhA and KasA interact with each other in the cell, possibly through the formation of a multienzyme complex involving other components of the FASII pathway. In order to investigate whether InhA and KasA directly interact, KasA is being expressed and purified. Attempts to obtain KasA from E. coli have been unsuccessful and currently this protein is being expressed in Mycobacterium smegmatis. Acyl-CoA substrates, commonly used with other FASII enzymes, are much less active with KasA. Consequently we have developed a procedure for synthesizing and purifying substrates based on the mycobacterial acyl carrier protein (AcpM). Currently we are studying whether KasA is inactivated by INH.