Search

Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry, saturation-transfer difference nuclear magnetic resonance and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. In addition, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemical reaction with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress., P. L. -M. was funded by a PhD fellowship from the ‘‘la Caixa’’ Foundation and received financial support from the Universidad de Zaragoza, Fundación Bancaria Ibercaja, and Fundación CAI (CM 1/16) during his stay at the UW–Madison. We acknowledge Ministerio de Economía, Industria y Competitividad for grant AGL2015-66177 (J. A. C.), the University of Zaragoza for grant UZ-2015-BIO-01 (J. A. C.), and OTRI (University of Zaragoza), Ministerio de Ciencia, Innovación y Universidades for grants CTQ2013-44367-C2-2-P and BFU2016-75633-P (R. H. -G.) and BFU2016-78232-P (A. V. -C.), Gobierno de Aragón for grants E34_R17 and LMP58_18 (R. H. -G.) and E45_17R (J. A. C and A. V. -C.) and FEDER (2014–2020) funds for “Building Europe from Aragón”. R. H. -G. and A. V. -C. thank the ARAID foundation for support. The research leading to these results also received funding from the FP7 (2007–2013) under BioStruct-X (grant agreement N283570 and BIOSTRUCTX_5186). We also thank the ALBA synchrotron radiation source, in particular, the beamline XALOC facility. J. A. and I. D. acknowledge access to UEA Faculty of Science Research Facilities. J. A. acknowledges funding support from the Biotechnology and biological Sciences Research Council (BBSRC; BB/P010660/1) and the Spanish Ministry of Science, Innovation and Universities through grant PID2019-109395GB-100. T. H. acknowledges a BBSRC DTP studentship. Financial support from the Universidad de Sevilla (Acciones Especiales del VI Plan Propio de Investigación y Transferencia) is also acknowledged. This work was also supported by National Institutes of Health (NIH) Grant GM065386 (J. M. D.), NIH National Research Service Award T32 GM007215 (J. B.) and the National Science Foundation Graduate Research Fellowship Program (NSF-GRFP) DGE-1256259 (J. B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

69 Pags.- 4 Figs.- Supplementary Information (6 Suppl. Tabls.- 19 Suppl. Figs.). The definitive version is available at: http://www.nature.com/nchembio/index.html, Protein O-fucosyltransferase 2 (POFUT2) is an essential enzyme that fucosylates serine and threonine residues of folded thrombospondin type 1 repeats (TSRs). To date, the mechanism by which this enzyme recognizes very dissimilar TSRs has been unclear. By engineering a fusion protein, we report the crystal structure of Caenorhabditis elegans POFUT2 (CePOFUT2) in complex with GDP and human TSR1 that suggests an inverting mechanism for fucose transfer assisted by a catalytic base and shows that nearly half of the TSR1 is embraced by CePOFUT2. A small number of direct interactions and a large network of water molecules maintain the complex. Site-directed mutagenesis demonstrates that POFUT2 fucosylates threonine preferentially over serine and relies on folded TSRs containing the minimal consensus sequence C-X-X-S/T-C. Crystallographic and mutagenesis data, together with atomic-level simulations, uncover a binding mechanism by which POFUT2 promiscuously recognizes the structural fingerprint of poorly homologous TSRs through a dynamic network of water-mediated interactions., We thank ARAID, MEC (BFU2010-19504, CTQ2013-­‐ 44367-­‐C2-­‐2-­‐P, CTQ2012-36365), NIH (GM061126 and CA123071) and the DGA (B89) for financial support, and BIFI (Memento cluster) for supercomputer support. The research leading to these results has also received funding from the FP7 (2007-2013) under BioStruct-X (grant agreement N°283570 and BIOSTRUCTX_5186).