Investigating the conservation pattern of a putative second terpene synthase divalent metal binding motif in plants.
Terpene synthases (TPS) require divalent metal ion co-factors, typically magnesium, that are bound by a canonical DDXXD motif, as well as a putative second, seemingly less well conserved and understood (N/D)DXX(S/T)XXXE motif. Given the role of the Ser/Thr side chain hydroxyl group in ligating one of the three catalytically requisite divalent metal ions and the loss of catalytic activity upon substitution with Ala, it is surprising that Gly is frequently found in this 'middle' position of the putative second divalent metal binding motif in plant TPS. Herein we report mutational investigation of this discrepancy in a model plant diterpene cyclase, abietadiene synthase from Abies grandis (AgAS). Substitution of the corresponding Thr in AgAS with Ser or Gly decreased catalytic activity much less than substitution with Ala. We speculate that the ability of Gly to partially restore activity relative to Ala substitution for Ser/Thr stems from the associated reduction in steric volume enabling a water molecule to substitute for the hydroxyl group from Ser/Thr, potentially in a divalent metal ion coordination sphere. In any case, our results are consistent with the observed conservation pattern for this putative second divalent metal ion binding motif in plant TPS.
Zhou
Ke
K
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Molecular Biology Building, Rm. 4216, Ames, IA 50011, United States.
Peters
Reuben J
RJ
D028202:Abies / Q000201:enzymology*; D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D002942:Circular Dichroism; D007535:Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D010940:Plant Proteins; D007535:Isomerases; C088741:abietadiene cyclase