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Comprehensive characterization of sphingolipid ceramide N-deacylase for the synthesis and fatty acid remodeling of glycosphingolipids

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Abstract

Sphingolipid ceramide N-deacylase (SCDase) catalyzes reversible reactions in which the amide linkage in glycosphingolipids is hydrolyzed or synthesized. While SCDases show great value for the enzymatic synthesis of glycosphingolipids, they are relatively poorly characterized enzymes. In this work, the enzymatic properties of SCDase from Shewanella alga G8 (SA_SCD) were systematically characterized and compared with the commercially available SCDase from Pseudomonas sp. TK4 (PS_SCD). The optimal pH values for the hydrolytic and synthetic activity of SA_SCD were pH 6.0 and pH 7.5, respectively. Both activities were strongly inhibited by Zn2+ and Cu2+, while Fe2+, Co2+, Ni2+, Mn2+, Ca2+, and Mg2+ promoted the hydrolytic activity but inhibited the synthetic activity. SA_SCD showed very broad substrate specificity both in hydrolysis and synthesis. Importantly, SA_SCD has a broader specificity for acyl donor acceptance than does PS_SCD, especially for unsaturated fatty acids and fatty acids with very short or long acyl chains. Further kinetic analysis revealed that the k cat/K M value for the hydrolytic activity of SA_SCD was 8.9-fold higher than that of PS_SCD for GM1a, while the values for the synthetic activity were 38-fold higher for stearic acid and 23-fold higher for lyso-GM1a (d18:1) than those of PS_SCD, respectively. The broad fatty acid specificity and high catalytic efficiency, together with the ease of expression of SA_SCD in Escherichia coli, make it a better biocatalyst than is PS_SCD for the synthesis and structural remodeling of glycosphingolipids.

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Acknowledgments

This work was supported by National Basic Research Program of China (973 Program), the Natural Science Foundation of China (grant number 31070056 and 31470788), the Natural Sciences and Engineering Research Council of Canada (NSERC), and the Canadian Institutes for Health Research (CIHR). The authors would thank Wei Zhang and Xinde Zhu in the Instrument and Service Center of School of Life Sciences and Biotechnology, Shanghai Jiao Tong University for technical assistance.

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The authors have no conflict of interest.

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Authors and Affiliations

  1. State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China

    Yun-Bin Han, Lie Wu, Feng-Tao Huang, Yan Feng & Guang-Yu Yang

  2. State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China

    Lie Wu

  3. Department of Chemistry, University of British Columbia, Vancouver, British Columbia, V6T 1Z1, Canada

    Jamie R. Rich & Stephen G. Withers

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  1. Yun-Bin Han
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  4. Feng-Tao Huang
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  6. Yan Feng
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  7. Guang-Yu Yang
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Correspondence to Yan Feng or Guang-Yu Yang.

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Han, YB., Wu, L., Rich, J.R. et al. Comprehensive characterization of sphingolipid ceramide N-deacylase for the synthesis and fatty acid remodeling of glycosphingolipids. Appl Microbiol Biotechnol 99, 6715–6726 (2015). https://doi.org/10.1007/s00253-015-6421-8

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  • DOI: https://doi.org/10.1007/s00253-015-6421-8

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