The pyridoxal 5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) catalyses the first step of de novo sphingolipid biosynthesis. The core human enzyme is a membrane-bound heterodimer composed of two subunits (hLCB1 and hLCB2a/b), and mutations in both hLCB1 (e.g., C133W and C133Y) and hLCB2a (e.g., V359M, G382V, and I504F) have been identified in patients with hereditary sensory and autonomic neuropathy type I (HSAN1), an inherited disorder that affects sensory and autonomic neurons. These mutations result in substrate promiscuity, leading to formation of neurotoxic deoxysphingolipids found in affected individuals. Here we measure the activities of the hLCB2a mutants in the presence of ssSPTa and ssSPTb and find that all decrease enzyme activity. High resolution structural data of the homodimeric SPT enzyme from the bacterium Sphingomonas paucimobilis (Sp SPT) provides a model to understand the impact of the hLCB2a mutations on the mechanism of SPT. The three human hLCB2a HSAN1 mutations map onto Sp SPT (V246M, G268V, and G385F), and these mutant mimics reveal that the amino acid changes have varying impacts; they perturb the PLP cofactor binding, reduce the affinity for both substrates, decrease the enzyme activity, and, in the most severe case, cause the protein to be expressed in an insoluble form.