Structural basis for catalysis in a CDP-alcohol phosphotransferase

Abstract

The CDP-alcohol phosphotransferase (CDP-AP) family of integral membrane enzymes catalyzes the transfer of a substituted phosphate group from a CDP-linked donor to an alcohol-acceptor. This is an essential reaction for phospholipid biosynthesis across all kingdoms of life, and it is catalyzed solely by CDP-APs. Here we report the 2.0 Å resolution crystal structure of a representative CDP-AP from Archaeoglobus fulgidus. The enzyme (AF2299) is a homodimer, with each protomer consisting of six transmembrane helices and an N-terminal cytosolic domain. A polar cavity within the membrane accommodates the active site, lined with the residues from an absolutely conserved CDP-AP signature motif (D1xxD2G1xxAR…G2xxxD3xxxD4). Structures in the apo, CMP-bound, CDP-bound and CDP-glycerol-bound states define functional roles for each of these eight conserved residues and allow us to propose a sequential, base-catalyzed mechanism universal for CDP-APs, in which the fourth aspartate (D4) acts as the catalytic base.

The active site of yeast phosphatidylinositol synthase Pis1 is facing the cytosol

“…Thus, substitutions were made solely based on the TOPCONS predictions. The possibility to model the Pis1 sequence on a crystal structure now allows to interpret SCAM data with higher confidence, although it should be said that the eukaryotic PI synthases are not closely related to the archeal DIPPSs [3] and the model therefore may be somewhat inaccurate, especially as DIPPSs do not use a lipid substrate. Presently, our data orient the yeast Pis1 model of Figs.…”

“…The cocrystallization of one CAPT with CDP and CMP allowed to show that D 1 , D 2 and D 3 of the CAPT motif coordinate the pyro-phosphate of CDP via a divalent cation and that the planar cytidine ring is inserted between G 1 and the conserved A 3 positions further down on the one hand, G 2 on the other hand (Fig. 1B) [3]. Thus, it is beyond doubt that the CAPT motif is part of the active site.…”

“…High resolution crystal structures have recently been reported for two CAPTs of the hyperthermophilic Archaeon Archaeoglobus fulgidus, one of unknown specificity, the other being a di-myo-inositol-1,3-phosphate-1′-phosphate synthase (AfDIPPS) catalyzing the condensation of two hydrophilic alcohols, CDP-inositol and inositol-1-phosphate [3,4]. Crystals show a novel fold formed by 6 transmembrane helices (TMs) forming a polar cavity extending from the cytosol to a depth of 8 Å into the membrane [4].…”

“…Thus, it is beyond doubt that the CAPT motif is part of the active site. D 4 of the CAPT motif is proposed to act as a catalytic base, deprotonating the second substrates' alcohol to allow its attack on the β-phosphate of CDP [3].…”

Role of Aminoalcoholphosphotransferases 1 and 2 in Phospholipid Homeostasis in Arabidopsis

“…The AAPTs, CPTs, and EPTs from animals and yeast all share a highly conserved region, which is predicted to localize on membrane surfaces (Supplemental Figure 1), by TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Since this conserved region is the only long, exposed peptide in AAPT protein, it is possible that it is responsible for substrate binding and catalytic activity, as shown in a recent structural analysis of CDP-alcohol phosphotransferase (Sciara et al, 2014). Both AAPTs were expressed in young vegetative tissue (Goode and Dewey, 1999).…”