Mechanism and regulation of mycobactin fatty acyl-AMP ligase FadD33.
Mycobacterial siderophores are critical components for bacterial virulence in the host. Pathogenic mycobacteria synthesize iron chelating siderophores named mycobactin and carboxymycobactin to extract intracellular macrophage iron. The two siderophores differ in structure only by a lipophilic aliphatic chain attached on the ε-amino group of the lysine mycobactin core, which is transferred by MbtK. Prior to acyl chain transfer, the lipophilic chain requires activation by a specific fatty acyl-AMP ligase FadD33 (also known as MbtM) and is then loaded onto phosphopantetheinylated acyl carrier protein (holo-MbtL) to form covalently acylated MbtL. We demonstrate that FadD33 prefers long chain saturated lipids and initial velocity studies showed that FadD33 proceeds via a Bi Uni Uni Bi ping-pong mechanism. Inhibition experiments suggest that, during the first half-reaction (adenylation), fatty acid binds first to the free enzyme, followed by ATP and the release of pyrophosphate to form the adenylate intermediate. During the second half-reaction (ligation), holo-MbtL binds to the enzyme followed by the release of products AMP and acylated MbtL. In addition, we characterized a post-translational regulation mechanism of FadD33 by the mycobacterial protein lysine acetyltransferase in a cAMP-dependent manner. FadD33 acetylation leads to enzyme inhibition, which can be reversed by the NAD(+)-dependent deacetylase, MSMEG_5175 (DAc1). To the best of our knowledge, this is the first time that bacterial siderophore synthesis has been shown to be regulated via post-translational protein acetylation.
From the Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
Olivia
O
Vergnolle
Hua
H
Xu
John S
JS
Blanchard
D000107:Acetylation; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D000263:Adenylate Kinase / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D011088:DNA Ligases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D005810:Multigene Family; D020102:Mycobacterium smegmatis / Q000201:enzymology / Q000235:genetics; D010080:Oxazoles / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017262:Siderophores / Q000737:chemistry; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D010080:Oxazoles; D017262:Siderophores; C018608:mycobactins; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase; C076545:Ampligase; D011088:DNA Ligases
Acetylation; Cyclic AMP (cAMP); Enzyme Mechanisms; FadD33; Kinetics; Mycobacteria; Mycobactin; Ping-Pong; Siderophores