Mechanistic analysis of Mycobacterium tuberculosis Rv1347c, a lysine Nε-acyltransferase involved in mycobactin biosynthesis

Abstract

Mycobactin acylation plays a crucial role in the ability of Mycobacterium tuberculosis to acquire intracellular iron during infection. M. tuberculosis Rv1347c, the lysine N(epsilon)-acyltransferase responsible for mycobactin acylation, represents a valid target for the development of novel anti-tubercular agents. Here we investigate the substrate specificity of Rv1347c, evaluate its kinetic mechanism and probe the contributions of active-site residues to catalysis. Our results confirm that Rv1347c demonstrates a preference for longer acyl-chains and suggest that mycobactin acylation occurs subsequent to mycobactin core assembly. Steady-state bisubstrate kinetics and dead-end inhibitor studies support a random sequential kinetic mechanism. Analysis of the pH dependence of k(cat)/K(m) revealed the presence of two groups that must be deprotonated for efficient catalysis. Mutagenesis of His(130) and Asp(168) indicated that both residues are critical for acyltransferase activity and suggests that His(130) is responsible for general base activation of the epsilon-amino group of lysine.

Mechanism and Regulation of Mycobactin Fatty Acyl-AMP Ligase FadD33

“…Apparent K m values of FadD33 for fatty acids decrease as the chain length increases from C6 to C16. According to the k cat /K m values, the enzyme prefers the fatty acids with longer length acyl chain as observed previously for the lysine acetyl N ⑀ -acyltransferase (MbtK), which is the enzyme transferring the ACP bound fatty acyl chain onto the N ⑀ -lysine of the mycobactin core (25). The preference for the C16 substrate indicates that FadD33 is primarily involved in the biosynthesis of the lipophilic mycobactin but raises the following question.…”

Analyses of MbtB, MbtE, and MbtF Suggest Revisions to the Mycobactin Biosynthesis Pathway in Mycobacterium tuberculosis

“…The previously reported reaction conditions for Rv1347c catalysis were used to catalyze N 6 acylation of L-Lys and N 6 -hydroxy-L-Lys (11,21). Briefly, 3.3 g of Rv1347c was added to a 350-l reaction mixture of 100 mM Tris-HCl, pH 8.0, 1 mM decanoyl-coenzyme A (decanoyl-CoA), and 500 M L-Lys or N 6 -hydroxy-L-Lys.…”

“…Briefly, the progression of the N 6 -hydroxy-L-Lyscontaining reaction was monitored using an established DTNB-based assay that monitors the release of free CoA from decanoyl-CoA after Rv1347c-dependent acylation of N 6 -hydroxy-L-Lys (11,21). The amount of N 6 -acyl-N 6 -hydroxy-L-Lys formed was determined using the molar extinction coefficient of 5-thio-2-nitrobenzoate (14,150 M Ϫ1 cm Ϫ1 ), which is formed when free CoA reacts with DTNB (31).…”

“…This suggests that the N 6 -hydroxylation of L-Lys occurs prior to N 6 -acylation. While prior analyses of Rv1347c investigated its amino acid specificity (11,21), neither Fig. S1 and S2 in the supplemental material for the Michaelis-Menten plots.…”

“…5A). The decanoyl group was chosen as a mimic of the various acyl groups found on MBT, and prior work on Rv1347c showed that this enzyme efficiently recognized decanoyl-CoA as a surrogate substrate (11). The enzymatically generated substrates (at 15 M final concentration) were used in ATP-PP i exchange assays, and it was determined that MbtE efficiently activated N 6 -decanoyl-N 6 -hydroxy-L-Lys, but only weakly activated N 6 -decanoyl-L-Lys (Fig.…”