The external aldimine form of serine palmitoyltransferase: structural, kinetic, and spectroscopic analysis of the wild-type enzyme and HSAN1 mutant mimics.
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) residue into the active site. Spectroscopic analysis confirms that both N100Y and N100W mutants subtly affect the chemistry of the PLP. Furthermore, the N100Y and R378A mutants appear less able to stabilize a quinonoid intermediate. These data provide the first experimental insight into how the most common disease-associated mutations of human SPT may lead to perturbation of enzyme activity.
From EaStChem, School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ.
Marine C C
MCC
Raman
EaStChem, Scottish Structural Proteomics Facility, and Centre for Biomolecular Science, University of St. Andrews, Edinburgh KY16 9RH, Scotland, United Kingdom.
Kenneth A
KA
Johnson
From EaStChem, School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ.
Beverley A
BA
Yard
From EaStChem, School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ.
Jonathan
J
Lowther
EaStChem, Scottish Structural Proteomics Facility, and Centre for Biomolecular Science, University of St. Andrews, Edinburgh KY16 9RH, Scotland, United Kingdom.
Lester G
LG
Carter
EaStChem, Scottish Structural Proteomics Facility, and Centre for Biomolecular Science, University of St. Andrews, Edinburgh KY16 9RH, Scotland, United Kingdom. Electronic address: naismith@st-andrews.ac.uk.
James H
JH
Naismith
From EaStChem, School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ. Electronic address: Dominic.Campopiano@ed.ac.uk.
Dominic J
DJ
Campopiano
D019943:Amino Acid Substitution; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000235:genetics; D009477:Hereditary Sensory and Autonomic Neuropathies / Q000201:enzymology / Q000235:genetics; D006801:Humans; D007097:Imines / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D018716:Molecular Mimicry; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051102:Serine C-Palmitoyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D020578:Sphingomonas / Q000201:enzymology / Q000235:genetics; D055672:Static Electricity; D013379:Substrate Specificity
D017931:DNA Primers; D007097:Imines; D011994:Recombinant Proteins; D051102:Serine C-Palmitoyltransferase