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Biochemical characterization of acyl-coenzyme A synthetases involved in mycobacterial steroid side-chain catabolism and molecular design: synthesis of an anti-mycobacterial agent

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Abstract

The metabolism of host cholesterol by Mycobacterium tuberculosis is an important factor for both its virulence and pathogenesis. However, the rationale for this cholesterol metabolism has not been fully understood yet. In the present study, we characterized several previously undescribed acyl-CoA synthetases that are involved in the steroid side-chain degradation in Mycobacterium smegmatis, and an analogue of intermediate from steroid degradation, 5′-O-(lithocholoyl sulfamoyl) adenosine (LCA-AMS), was successfully designed and synthesized to be used as a specific anti-mycobacterial agent. The acyl-CoA synthetases exhibited strong preferences for the length of side chain. FadD19 homologs, including FadD19 (MSMEG_5914), FadD19-2 (MSMEG_2241), and FadD19-4 (MSMEG_3687), are unanimously favorable cholesterol with a C8 alkanoate side chain. FadD17 (MSMEG_5908) and FadD1 (MSMEG_4952) showed high preferences for steroids, containing a C5 alkanoate side chain. FadD8 (MSMEG_1098) exhibited specific activity toward cholestenoate with a C8 alkanoate side chain. An acylsulfamoyl analogue of lithocholate, 5′-O-(lithocholoyl sulfamoyl) adenosine (LCA-AMS), was designed and synthesized. As expected, the intermediate analogue not only specifically inhibited those steroid-activated acyl-CoA synthetases, but also selectively inhibited the growth of mycobacterial species, including M. tuberculosis, M. smegmatis, and Mycobacterium neoaurum. Overall, our research advanced our understanding of mycobacterial steroid degradation and provided new insights to develop novel mechanism-based anti-mycobacterial agents.

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Acknowledgements

This study was funded by the National Natural Science Foundation of China (no. 31571285), the Sichuan provincial Science & Technology Department (2012JY0067), and the Sichuan Provincial Department of Education (17ZA0323), College student innovation, and entrepreneurship training program (201310636026).

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Author notes

  1. Yang Niu, Fanglan Ge and Yongzhi Yang have contributed equally to this work.

Authors and Affiliations

  1. College of Life Sciences, Sichuan Normal University, Chengdu, 610068, People’s Republic of China

    Yang Niu, Fanglan Ge, Yongzhi Yang, Yao Ren, Wei Li, Guiying Chen, Dongmei Wen, Fuhong Liu & Li Xiong

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  1. Yang Niu
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  2. Fanglan Ge
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Contributions

YN, FG, and YY did all the biological and analytical work on acyl-coenzyme A synthetases. YR and XZ did the synthetic work. WL conceived and designed the project. GC, DW, FL, and XY prepared the purified acyl-coenzyme A synthetases. All the authors participated in writing the manuscript.

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Correspondence to Wei Li.

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13205_2019_1703_MOESM1_ESM.jpg

Fig. S1 Synthesis of LCA-AMS. TBDSC = tert-butyldimethylsilyl chloride; CDI = 1,1-carbonyldiimidazole; DBU =1,8-diazabicyclo [5.4.0] undec-7-ene; TBAF = tetrabutylammonium; THF = tetrahydrofuran (JPEG 63 kb)

Fig. S2 TOF-MS map of LCA-AMS (JPEG 683 kb)

Fig. S3 TOF-MS characterization of steroid-CoA thioesters. A: cholest-4-en-26-oyl-CoA, B: cholyl-CoA (JPEG 323 kb)

Fig. S4 HPLC characterization of steroid-CoA thioesters (JPEG 435 kb)

Fig. S5 Activation of fatty acids and steroids by different FadDs of M. smegmatisem (JPEG 2098 kb)

Fig.S6 qRT-PCR analyses of M. smegmatismc2155 FadD genes during growth in different media (JPEG 708 kb)

Table S1 Primers used in the cloning of FadD genes (DOCX 19 kb)

Table S2 The primers used for qRT-PCR analyses (DOC 41 kb)

Table S3 Acyl-coenzyme A synthetase orthologs and related genes from the several mycobacterial genomes (DOCX 19 kb)

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Niu, Y., Ge, F., Yang, Y. et al. Biochemical characterization of acyl-coenzyme A synthetases involved in mycobacterial steroid side-chain catabolism and molecular design: synthesis of an anti-mycobacterial agent. 3 Biotech 9, 169 (2019). https://doi.org/10.1007/s13205-019-1703-y

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