Table 1.
Primers for amplifying cDNA of AmCDase.
Fig 1.
Gene function classification (GO).
Fig 2.
Pathway annotation and classification of contigs (KEGG).
Fig 3.
Proposed metabolic pathway of sphingolipids in A. muelleri.
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).
Fig 4.
Phylogenetic tree of neutral ceramidases from different species.
The MrBayes analyses were performed with a mixed amino acid model. Species names are (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).
Fig 5.
Conserved domain of neutral ceramidases.
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.
Fig 6.
Expression of AmCDase in the yeast double knockout strain Δypc1Δydc1.
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.
Fig 7.
Biochemical characterization of the purified recombinant AmCDase.
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.