Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyzes the decarboxylative condensation of L-serine with palmitoyl-CoA to form 3-ketodihydrosphingosine. SPT belongs to the fold type I family of the pyridoxal 5'-phosphate (PLP)-dependent enzyme. SPT is different from most of the fold type I enzymes in that its re face of the PLP-Lys aldimine is occupied by a His residue (His159) instead of an aromatic amino acid residue. His159 was changed into alanine or aromatic amino acid residues in order to examine its role during catalysis. All mutant SPTs formed the PLP-L-serine aldimine and catalyzed the abortive transamination of L-serine. Only H159A SPT retained activity, and showed a prominent 505-nm absorption band of the quinonoid species during catalysis. Based on the results obtained from the kinetic analyses, we propose a novel mechanism, in which His159 plays multiple roles by exploiting the stereochemistry of the Dunathan's conjecture.