Role of Serine140 in the mode of action of Mycobacterium tuberculosis β-ketoacyl-ACP Reductase (MabA)

BMC Research Notes

Diógenes S Santos¹  Luiz A Basso¹  Walter Filgueira de Azevedo¹  Rafael Andrade Caceres²  Leonardo A Rosado¹ 

[1]Programa de Pós-Graduação em Biologia Celular e Molecular, PUCRS, Av. Ipiranga 6681 – Tecnopuc – Prédio 92A, ZIP CODE 90619-900, Porto Alegre, RS, Brazil

[2]Faculdade de Biociências, Laboratório de Bioquímica Estrutural, PUCRS, Av. Ipiranga 6681, Porto Alegre, RS, 90619-900, Brazil

关键词: Molecular modeling;    Fluorescence spectroscopy;    Enzyme activity;    Site-directed mutagenesis;    MabA;    β-Ketoacyl-ACP Reductase;    Mycobacterium tuberculosis;   

Others  :  1165604
DOI  :  10.1186/1756-0500-5-526

 received in 2012-05-07, accepted in 2012-09-14,  发布年份 2012

【 摘 要 】

Background

Tuberculosis (TB) still remains one of the most deadly infectious diseases in the world. Mycobacterium tuberculosis β-ketoacyl-ACP Reductase (MabA) is a member of the fatty acid elongation system type II, providing precursors of mycolic acids that are essential to the bacterial cell growth and survival. MabA has been shown to be essential for M. tuberculosis survival and to play a role in intracellular signal transduction of bacilli.

Findings

Here we describe site-directed mutagenesis, recombinant protein expression and purification, steady-state kinetics, fluorescence spectroscopy, and molecular modeling for S140T and S140A mutant MabA enzymes. No enzyme activity could be detected for S140T and S140A. Although the S140T protein showed impaired NADPH binding, the S140A mutant could bind to NADPH. Computational predictions for NADPH binding affinity to WT, S140T and S140A MabA proteins were consistent with fluorescence spectroscopy data.

Conclusions

The results suggest that the main role of the S140 side chain of MabA is in catalysis. The S140 side chain appears to also play an indirect role in NADPH binding. Interestingly, NADPH titrations curves shifted from sigmoidal for WT to hyperbolic for S140A, suggesting that the S140 residue may play a role in displacing the pre-existing equilibrium between two forms of MabA in solution. The results here reported provide a better understanding of the mode of action of MabA that should be useful to guide the rational (function-based) design of inhibitors of MabA enzyme activity which, hopefully, could be used as lead compounds with anti-TB action.

【 授权许可】

   
2012 Rosado et al.; licensee BioMed Central Ltd.

【 图 表 】

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