The external aldimine form of serine palmitoyltransferase

external aldimine form of serine palmitoyltransferase: structural, kinetic, and spectroscopic analysis of the wild-type enzyme and HSAN1 mutant mimics' General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Both of these authors contributed equally to this work. Abbreviations: HSAN1 – hereditary sensory autonomic neuropathy type 1; SPT – serine palmitoyltransferase; PLP – pyridoxal phosphate; KDS – 3-ketodihydrosphingosine; DTNB – 5,5′-dithiobis-2-nitrobenzoic acid; r.m.s. – root mean square; AONS – 8-amino-7-oxononanoate synthase; ALAS – 5-aminolevulinate synthase; KBL – 2-amino-3-ketobutyrate-CoA ligase; PEG – polyethylene glycol. The external aldimine form of serine palmitoyltransferase: structural, kinetic, and spectroscopic analysis of the wild-type enzyme and HSAN1 mutant mimics. Abstract Sphingolipid biosynthesis begins with the condensation of l-serine and palmitoyl-CoA catalyzed by the PLP-dependent enzyme serine palmitoyltransferase (SPT). Mutations in human SPT cause hereditary sensory autonomic neuropathy type 1, a disease characterized by loss of feeling in extremities and severe pain. The human enzyme is a membrane-bound hetereodimer, and the most common mutations are located in the enzymatically incompetent monomer, suggesting a " dominant " or regulatory effect. The molecular basis of how these mutations perturb SPT activity is subtle and is not simply loss of activity. To further explore the structure and mechanism of SPT, we have studied the homodimeric bacterial enzyme from Sphingomonas paucimobilis. We have analyzed two mutants (N100Y and N100W) engineered to mimic the mutations seen in hereditary sensory autonomic neuropathy type 1 as well as a third mutant N100C designed to mimic the wild-type human SPT. The N100C mutant appears fully active, whereas both N100Y and N100W are significantly compromised. The structures of the holoenzymes reveal differences around the active site and in neighboring secondary structure that transmit across the dimeric interface in both N100Y and N100W. Comparison of the l-Ser external aldimine structures of both native and N100Y reveals significant differences that hinder the movement of a catalytically important Arg 378 …