title: Comprehensive characterization of sphingolipid ceramide N-deacylase for the synthesis and fatty acid remodeling of glycosphingolipids.
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 Zn(2+) and Cu(2+), while Fe(2+), Co(2+), Ni(2+), Mn(2+), Ca(2+), and Mg(2+) 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.
authors: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors: Yun-Bin
authors: YB
authors: Han
authors: Lie
authors: L
authors: Wu
authors: Jamie R
authors: JR
authors: Rich
authors: Feng-Tao
authors: FT
authors: Huang
authors: Stephen G
authors: SG
authors: Withers
authors: Yan
authors: Y
authors: Feng
authors: Guang-Yu
authors: GY
authors: Yang
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000378:metabolism*; D015870:Gene Expression; D006028:Glycosphingolipids / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology*; D020592:Shewanella / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D002413:Cations, Divalent; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D005227:Fatty Acids; D006028:Glycosphingolipids; D008670:Metals; D000581:Amidohydrolases; C096410:sphingolipid ceramide N-deacylase