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Dysfunction of the dopaminergic pathway is linked to numerous diseases of the nervous system. The D1-D2 receptor heteromer is known to play a role in certain neuropsychiatric disorders, such as depression. Here, we synthesized an eight amino acid residue peptide, EAARRAQE, derived from the third intracellular loop of the D2 receptor and show that the peptide binds to the D1 receptor with comparable efficiency as that of the full-length D2 receptor protein. Moreover, immunoprecipitation studies show the existence of a heteromeric complex formed both in vitro and in total protein derived from temporal and frontal lobe tissue from normal and depressed subjects. The efficiency of the peptide to block the D1-D2 heteromeric complex was comparable in all the samples tested., (© 2019 Federation of European Biochemical Societies.)

Dihydroorotase (DHOase) is involved in the de novo synthesis of pyrimidine in virtually all organisms, and it is usually associated with two other enzymes found in this biosynthetic pathway, carbamylphosphate synthetase and/or aspartate transcarbamylase (ATCase). In the hyperthermophilic bacterium Aquifex aeolicus, ATCase and DHOase are noncovalently associated. Upon dissociation, ATCase keeps its activity entirely while DHOase is totally inactivated. It was previously shown that high pressure fully restores the activity of this isolated DHOase. On the basis of kinetic studies, site-directed mutagenesis and the use of peptides mimicking loop A, a loop that appears to block access to the active site, was proposed that this pressure-induced reactivation was due to the decrease in the volume of the system, -ΔV, resulting from the disruption of known ionic interactions between the loop and the main part of the protein. In this study, this interpretation is more precisely demonstrated by the determination of the crystallographic structure of isolated DHOase under pressure. In addition to the loop displacements, pressure induces a discrete rearrangement of the catalytic site aspartate 305, an effect that might additionally contribute to the reactivation of this enzyme., (© 2019 Federation of European Biochemical Societies.)

The genome of the major intestinal archaeon Methanobrevibacter smithii contains a complex gene system coding for carbamoyl phosphate synthetase (CPSase) composed of both full-length and reduced-size synthetase subunits. These ammonia-metabolizing enzymes could play a key role in controlling ammonia assimilation in M. smithii, affecting the metabolism of gut bacterial microbiota, with an impact on host obesity. In this study, we isolated and characterized the small (41 kDa) CPSase homolog from M. smithii. The gene was cloned and overexpressed in Escherichia coli, and the recombinant enzyme was purified in one step. Chemical cross-linking and size exclusion chromatography indicated a homodimeric/tetrameric structure, in accordance with a dimer-based CPSase activity and reaction mechanism. This small enzyme, MS-s, synthesized carbamoyl phosphate from ATP, bicarbonate, and ammonia and catalyzed the same ATP-dependent partial reactions observed for full-length CPSases. Steady-state kinetics revealed a high apparent affinity for ATP and ammonia. Sequence comparisons, molecular modeling, and kinetic studies suggest that this enzyme corresponds to one of the two synthetase domains of the full-length CPSase that catalyze the ATP-dependent phosphorylations involved in the three-step synthesis of carbamoyl phosphate. This protein represents the smallest naturally occurring active CPSase characterized thus far. The small M. smithii CPSase appears to be specialized for carbamoyl phosphate metabolism in methanogens., (Copyright © 2012 S. Karger AG, Basel.)