Absence of a catalytic water confers resistance to the neurotoxin gabaculine.
Gabaculine is a potent inhibitor of the vitamin B6-dependent key enzyme in chlorophyll biosynthesis, glutamate-1-semialdehyde aminomutase (GSAM). The inhibition effect is caused by an enzymatic deprotonation of the neurotoxin and requires the aldimine (PLP) form of the cofactor at the active site. In this study, we show that a single-point mutation confers resistance to gabaculine. A combined functional and structural analysis of wild-type GSAM in complex with gabaculine and the GSAM(M248I) form allowed us to decipher in atomic detail the molecular basis of this unique resistance. Interestingly, the gabaculine tolerance is caused by the absence of an essential water molecule that has a dual functional role. It serves as a nucleophilic shuttle for the hydroxyl anion along the reaction pathway and holds active-site Lys273 in a catalytically competent conformation. The single-point mutant is not able to fix this catalytic water between the beta-branched side chain of Ile248 and Lys273. As a consequence, the mutant enzyme is trapped in a gabaculine-insensitive but still enzymatically active amine (PMP) form.
Orriss
George L
GL
Department of Chemistry, University of Manitoba, 144 Dysart Rd, Winnipeg, R3T 2N2, Manitoba, Canada.
Patel
Trushar R
TR
Sorensen
John
J
Stetefeld
Jörg
J
D000595:Amino Acid Sequence; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003509:Cyclohexanecarboxylic Acids / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D004351:Drug Resistance / Q000502:physiology; D019143:Evolution, Molecular; D019751:Intramolecular Transferases / Q000037:antagonists & inhibitors* / Q000235:genetics*; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D017354:Point Mutation; D011487:Protein Conformation; D046940:Synechococcus / Q000201:enzymology; D014867:Water / Q000737:chemistry* / Q000502:physiology
D003509:Cyclohexanecarboxylic Acids; D014867:Water; C012987:gabaculine; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase; D008239:Lysine