The Mycobacterium tuberculosis LipB enzyme functions as a cysteine/lysine dyad acyltransferase

Abstract

Lipoic acid is essential for the activation of a number of protein complexes involved in key metabolic processes. Growth of Mycobacterium tuberculosis relies on a pathway in which the lipoate attachment group is synthesized from an endogenously produced octanoic acid moiety. In patients with multiple-drug-resistant M. tuberculosis, expression of one gene from this pathway, lipB, encoding for octanoyl-[acyl carrier protein]-protein acyltransferase is considerably up-regulated, thus making it a potential target in the search for novel antiinfectives against tuberculosis. Here we present the crystal structure of the M. tuberculosis LipB protein at atomic resolution, showing an unexpected thioether-linked activesite complex with decanoic acid. We provide evidence that the transferase functions as a cysteine͞lysine dyad acyltransferase, in which two invariant residues (Lys-142 and Cys-176) are likely to function as acid͞base catalysts. Analysis by MS reveals that the LipB catalytic reaction proceeds by means of an internal thioesteracyl intermediate. Structural comparison of LipB with lipoate protein ligase A indicates that, despite conserved structural and sequence active-site features in the two enzymes, 4-phosphopantetheine-bound octanoic acid recognition is a specific property of LipB.catalytic dyad ͉ lipoic acid ͉ x-ray structure ͉ thioester formation ͉ mass spectrometry S everal multicomponent enzyme complexes that catalyze key metabolic reactions in the citric acid cycle and single-carbon metabolism are posttranslationally modified by attachment to lipoic acid (1). These systems share a domain that covalently binds lipoic acid by means of an amide bond to the -amino group of a conserved exposed lysine residue. In many organisms, lipoylation is catalyzed by two separate enzymes, lipoyl protein ligase A (LplA) or octanoyl-[acyl carrier protein]-protein transferase (LipB; see Fig. 5, which is published as supporting information on the PNAS web site). Although LplA uses exogenous lipoic acid, LipB transfers endogenous octanoic acid, which is attached by means of a thioester bond to the 4Ј-phosphopantetheine cofactor of acyl carrier protein (ACP) onto lipoyl domains (2-4). These octanoylated domains are converted into lipoylated derivatives by the S-adenosyl-L-methioninedependent enzyme, lipoyl synthase (LipA), which catalyzes the insertion of sulfur atoms into the six-and eight-carbon positions of the corresponding fatty acid (5-7). This process bypasses the requirement for an exogenous supply of lipoic acid.In bacteria, enzymes involved in lipoylation have gained increasing attention because of their implication in pathogenicity. For instance, a Listeria monocytogenes mutant strain lacking LplA has been found to be defective in its ability to grow in the host cytosol and is less virulent in animals because of its dependence on host-derived lipoic acid (8). Mice Lias Ϫ/Ϫ (LipA) null variants die during embryogenesis, indicating that the mammalian pathway is related to the bacterial LipB͞LipA pathway and is essenti...

Ohr (Organic Hydroperoxide Resistance Protein) Possesses a Previously Undescribed Activity, Lipoyl-dependent Peroxidase

“…This enzyme is responsible for lipoylation of enzymes using exogenous lipoic acid as substrate (45). However, we found all of the three genes involved in the biosynthetic pathway of lipoic acid: LipA (lipoyl synthase, XF1050), LipB (lipoyltranferase, XF1051), and ACP (acyl carrier protein, XF0672) (46,47). This finding is in line with the observation that the plant pathogen X. fastidiosa colonizes an environment with poor availability of nutrients.…”

Conservation and variability of the pore-lining helices in P-loop channels

“…The NH-S hydrogen bonds are seen in X-ray structures of both small molecules and proteins. [23][24][25][26] Besides the likely H-bond N Xi20 -M (X-1)i30 , the large sidechains M (X-1)i30 form close inter-subunit contacts with V Xi27 , I Xi23 , and G Xi24 . Furthermore, alanine residues A (X-1) i29 , which are far from the conserved asparagines N Xi20 , form tight intersegment contacts with G Xk10 , M Xk11 , and A Xk14 .…”

Reverse docking study unravels the potential Mycobacterium tuberculosis enzyme targets of Agelasine F

“…20 Lipoate-protein ligase B (LipB), also known as octanoyltransferase, catalyzes the first committed step in the biosynthesis of lipoyl cofactor, which is essential for the function of several key enzymes involved in the oxidative metabolism of M. tuberculosis. 21 Moreover, a relatively new putative Mtb target, 7,8-diaminopelargonic acid synthase (DAPAS), the second enzyme in the biotin biosynthetic pathway, is necessary for de novo biotin biosynthesis. 22 It has been shown that the disruption of the Mtb biotin biosynthesis machinery results in rapid bacterial clearance.…”