Conserved domain of neutral ceramidases.

<p>DNAMAN alignment of the neutral ceramidases from a variety of species showing the highly conserved hexapeptide sequence GDVSPN within the broader conserved amidase domain NXGDVSPNXXGXXC that is crucial for ceramidase activity.</p

Expression of AmCDase in the yeast double knockout strain Δypc1Δydc1.

<p>a) Determination of whole-cell lysates (20 μg protein per lane) by Western blot with anti-His antibody at different time points. b) D-erythro-C12-NBD-ceramide content in different groups after the reaction. Values are the means ± SD of three replicates from each independent experiment.</p

Biochemical characterization of the purified recombinant AmCDase.

<p>Hydrolysis of D-erythro-C12-NBD-ceramide was measured by the method described in the “Materials and Methods”. The purified recombinant AmCDase was used for enzyme activity detection, and the reaction continued for 1 h. Values are the means ± SD of three replicates from each independent experiment. (a) Michaelis–Menten represented AmCDase activity by increasing concentrations of D-erythro-C12-NBD-ceramide. (b) Lineweaver-Burk plots for AmCDase. (c) Effects of different cations on AmCDase activity. (d) pH optimum of AmCDase. The buffers were used as described in the experimental procedures.</p

Primers for amplifying cDNA of AmCDase.

<p>Primers for amplifying cDNA of AmCDase.</p

Proposed metabolic pathway of sphingolipids in <i>A</i>. <i>muelleri</i>.

<p>Serine palmitoyltransferase (SPT), 3-Dehydrosphinganine reductase (DSR), sphingosine kinase (SPHK), sphinganine C4-monooxygenase (SUR2), sphinganine-1-phosphate aldolase (SPL), acyl-CoA-dependent ceramide synthase (LAG1), neutral ceramidase (ASAH2), dihydroceramidase (DHCDase), beta-galactosidase (GLB1), sphingolipid delta-4 desaturase (DEGS), non-lysosomal glucosylceramidase (GBA2), alpha-galactosidase (GalA), ceramide kinase (CERK), cellulose synthase-like A (CSLA), cellulose synthase-like D (CSLD), GDPD-pyrophosphorylase (GGP).</p

Phylogenetic tree of neutral ceramidases from different species.

<p>The MrBayes analyses were performed with a mixed amino acid model. Species names are (<i>Amorphophallus muelleri; Jatropha curcas; Prunus persica; Sesamum indicum; Arabidopsis thaliana; Anthurium amnicola; Oryza sativa Japonica; Hordeum vulgare; Triticum aestivum; Dicchanthelium oligosanthes; Human; Fukomys damarensis; Microcebus murinus; Castor Canadensis</i>).</p