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@ARTICLE{delCaoOchoa:619752,
      author       = {del Caño-Ochoa, Francisco and Grande-García, Araceli and
                      Reverte-López, María and D’Abramo, Marco and
                      Ramón-Maiques, Santiago},
      title        = {{C}haracterization of the catalytic flexible loop in the
                      dihydroorotase domain of the human multi-enzymatic protein
                      {CAD}},
      journal      = {Biologist},
      volume       = {293},
      number       = {49},
      issn         = {0021-9258},
      address      = {Bethesda, MD},
      publisher    = {American Soc. for Biochemistry and Molecular Biology},
      reportid     = {PUBDB-2024-07887},
      pages        = {18903-18913},
      year         = {2018},
      note         = {ISSN 0021-9258 not unique: **2 hits**.},
      abstract     = {The dihydroorotase (DHOase) domain of the multifunctional
                      protein carbamoyl-phosphate synthetase 2, aspartate
                      transcarbamoylase, and dihydroorotase (CAD) catalyzes the
                      third step in the de novo biosynthesis of pyrimidine
                      nucleotides in animals. The crystal structure of the DHOase
                      domain of human CAD (huDHOase) revealed that, despite
                      evolutionary divergence, its active site components are
                      highly conserved with those in bacterial DHOases, encoded as
                      monofunctional enzymes. An important element for catalysis,
                      conserved from Escherichia coli to humans, is a flexible
                      loop that closes as a lid over the active site. Here, we
                      combined mutagenic, structural, biochemical, and molecular
                      dynamics analyses to characterize the function of the
                      flexible loop in the activity of CAD's DHOase domain. A
                      huDHOase chimera bearing the E. coli DHOase flexible loop
                      was inactive, suggesting the presence of distinctive
                      elements in the flexible loop of huDHOase that cannot be
                      replaced by the bacterial sequence. We pinpointed Phe-1563,
                      a residue absolutely conserved at the tip of the flexible
                      loop in CAD's DHOase domain, as a critical element for the
                      conformational equilibrium between the two catalytic states
                      of the protein. Substitutions of Phe-1563 with Ala, Leu, or
                      Thr prevented the closure of the flexible loop and
                      inactivated the protein, whereas substitution with Tyr
                      enhanced the interactions of the loop in the closed position
                      and reduced fluctuations and the reaction rate. Our results
                      confirm the importance of the flexible loop in CAD's DHOase
                      domain and explain the key role of Phe-1563 in configuring
                      the active site and in promoting substrate strain and
                      catalysis.},
      cin          = {EMBL-User},
      ddc          = {610},
      cid          = {I:(DE-H253)EMBL-User-20120814},
      pnm          = {6G3 - PETRA III (DESY) (POF4-6G3) / BIOSTRUCT-X -
                      Transnational access and enhancement of integrated
                      Biological Structure determination at synchrotron X-ray
                      radiation facilities (283570)},
      pid          = {G:(DE-HGF)POF4-6G3 / G:(EU-Grant)283570},
      experiment   = {EXP:(DE-H253)P-P13-20150101},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:30315107},
      UT           = {WOS:000458467400008},
      doi          = {10.1074/jbc.RA118.005494},
      url          = {https://bib-pubdb1.desy.de/record/619752},
}