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Ramon-Maiques S.,Instituto Of Biomedicina Of Valencia Consejo Superior Of Investigaciones Cientificas | Marina A.,Instituto Of Biomedicina Of Valencia Consejo Superior Of Investigaciones Cientificas | Guinot A.,Instituto Of Biomedicina Of Valencia Consejo Superior Of Investigaciones Cientificas | Gil-Ortiz F.,Instituto Of Biomedicina Of Valencia Consejo Superior Of Investigaciones Cientificas | And 3 more authors.
Journal of Molecular Biology | Year: 2010

Carbamate kinase (CK) makes ATP from ADP and carbamoyl phosphate (CP) in the final step of the microbial fermentative catabolism of arginine, agmatine, and oxalurate/allantoin. Two previously reported CK structures failed to clarify CP binding and catalysis and to reveal the significance of the protruding subdomain (PSD) that hangs over the CK active center as an exclusive and characteristic CK feature. We clarify now these three questions by determining two crystal structures of Enterococcus faecalis CK (one at 1.5 Å resolution and containing bound MgADP, and the other at 2.1 Å resolution and having in the active center one sulfate and two fixed water molecules that mimic one bound CP molecule) and by mutating active-center residues, determining the consequences of these mutations on enzyme functionality. Superimposition of the present crystal structures reconstructs the filled active center in the ternary complex, immediately suggesting in-line associative phosphoryl group transfer and a mechanism for enzyme catalysis involving N51, K209, K271, D210, and the PSD residue K128. The large respective increases and decreases in Km CP and kcat triggered by the mutations N51A, K128A, K209A, and D210N corroborate the ternary complex active-site architecture and the catalytic mechanism proposed. The extreme negative effects of K128A demonstrate a key role of the PSD in substrate binding and catalysis. The crystal structures reveal large rigid-body movements of the PSD towards the enzyme body that place K128 next to CP and bury the CP site. A mechanism that connects CP site occupation with the PSD approach, involving V206-I207 in the CP site and P162-S163 in the PSD stem, is identified. The effects of the V206A and V206L mutations support this mechanism. It is concluded that the PSD movement allows CK to select against the abundant CP/carbamate analogues acetylphosphate/acetate and bicarbonate, rendering CK highly selective for CP/carbamate. © 2010 Elsevier Ltd. Source


Sevilla L.M.,Instituto Of Biomedicina Of Valencia Consejo Superior Of Investigaciones Cientificas | Latorre V.,Instituto Of Biomedicina Of Valencia Consejo Superior Of Investigaciones Cientificas | Sanchis A.,Instituto Of Biomedicina Of Valencia Consejo Superior Of Investigaciones Cientificas | Perez P.,Instituto Of Biomedicina Of Valencia Consejo Superior Of Investigaciones Cientificas
Journal of Investigative Dermatology | Year: 2013

The glucocorticoid (GC) receptor (GR) mediates the effects of physiological and pharmacological GC ligands and has a major role in cutaneous pathophysiology. To dissect the epithelial versus mesenchymal contribution of GR in developing and adult skin, we generated mice with keratinocyte-restricted GR inactivation (GR epidermal knockout or GREKO mice). Developing and early postnatal GREKO mice exhibited impaired epidermal barrier formation, abnormal keratinocyte differentiation, hyperproliferation, and stratum corneum (SC) fragility. At birth, GREKO epidermis showed altered levels of epidermal differentiation complex genes, proteases and protease inhibitors which participate in SC maintenance, and innate immunity genes. Many upregulated genes, including S100a8/a9 and Tslp, also have increased expression in inflammatory skin diseases. Infiltration of macrophages and degranulating mast cells were observed in newborn GREKO skin, hallmarks of atopic dermatitis. In addition to increased extracellular signal-regulated kinase activation, GREKO newborn and adult epidermis had increased levels of phosphorylated signal transducer and activator of transcription 3, a feature of psoriasis. Although adult GREKO epidermis had a mild phenotype of increased proliferation, perturbation of skin homeostasis with detergent or phorbol ester triggered an exaggerated proliferative and hyperkeratotic response relative to wild type. Together, our results show that epidermal loss of GR provokes skin barrier defects and cutaneous inflammation. © 2013 The Society for Investigative Dermatology. Source

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