Inhibition of the PLP-dependent enzyme serine palmitoyltransferase by cycloserine: evidence for a novel decarboxylative mechanism of inactivationThis article is part of the 2010 Molecular BioSystems 'Emerging Investigators' issue: highlighting the work of outstanding young scientists at the chemical- and systems-biology interfaces.The atomic coordinates and structure factors (code 2XBN) of the structure reported in this paper have been deposited in the Protein Data Bank (http://www.pdb.org).Elec

• Main Authors: Lowther, Jonathan, Yard, Beverley A, Johnson, Kenneth A, Carter, Lester G, Bhat, Venugopal T, Raman, Marine C. C, Clarke, David J, Ramakers, Britta, McMahon, Stephen A, Naismith, James H, Campopiano, Dominic J
• Format: Article
• Language: English
• ISSN: 1742-206X; 1742-2051
• DOI: 10.1039/c003743e

Cycloserine (CS, 4-amino-3-isoxazolidone) is a cyclic amino acid mimic that is known to inhibit many essential pyridoxal 5′-phosphate (PLP)-dependent enzymes. Two CS enantiomers are known; d -cycloserine (DCS, also known as Seromycin) is a natural product that is used to treat resistant Mycobacterium tuberculosis infections as well as neurological disorders since it is a potent NMDA receptor agonist, and l -cycloserine (LCS) is a synthetic enantiomer whose usefulness as a drug has been hampered by its inherent toxicity arising through inhibition of sphingolipid metabolism. Previous studies on various PLP-dependent enzymes revealed a common mechanism of inhibition by both enantiomers of CS; the PLP cofactor is disabled by forming a stable 3-hydroxyisoxazole/pyridoxamine 5′-phosphate (PMP) adduct at the active site where the cycloserine ring remains intact. Here we describe a novel mechanism of CS inactivation of the PLP-dependent enzyme serine palmitoyltransferase (SPT) from Sphingomonas paucimobilis . SPT catalyses the condensation of l -serine and palmitoyl-CoA, the first step in the de novo sphingolipid biosynthetic pathway. We have used a range of kinetic, spectroscopic and structural techniques to postulate that both LCS and DCS inactivate SPT by transamination to form a free pyridoxamine 5′-phosphate (PMP) and β-aminooxyacetaldehyde that remain bound at the active site. We suggest this occurs by ring opening of the cycloserine ring followed by decarboxylation. Enzyme kinetics show that inhibition is reversed by incubation with excess PLP and that LCS is a more effective SPT inhibitor than DCS. UV-visible spectroscopic data, combined with site-directed mutagenesis, suggest that a mobile Arg 378 residue is involved in cycloserine inactivation of SPT. Spectroscopic and structural approaches reveal a new inactivation mechanism of a key sphingolipid biosynthetic enzyme.