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CAD is a 1.5 MDa particle formed by hexameric association of a 250 kDa protein divided into different enzymatic domains, each catalyzing one of the initial reactions for de novo biosynthesis of pyrimidine nucleotides: glutaminase‐dependent Carbamoyl phosphate synthetase, Aspartate transcarbamoylase, and Dihydroorotase. The pathway for de novo pyrimidine synthesis is essential for cell proliferation and is conserved in all living organisms, but the covalent linkage of the first enzymatic activities into a multienzymatic CAD particle is unique to animals. In other organisms, these enzymatic activities are encoded as monofunctional proteins for which there is abundant structural and biochemical information. However, the knowledge about CAD is scarce and fragmented. Understanding CAD requires not only to determine the three‐dimensional structures and define the catalytic and regulatory mechanisms of the different enzymatic domains, but also to comprehend how these domains entangle and work in a coordinated and regulated manner. This review summarizes significant progress over the past 10 years toward the characterization of CAD's architecture, function, regulatory mechanisms, and cellular compartmentalization, as well as the recent finding of a new and rare neurometabolic disorder caused by defects in CAD activities.

CAD, the multienzymatic protein that initiates and controls the de novo biosynthesis of pyrimidines, plays a major role in nucleotide homeostasis, cell growth and proliferation. Despite its interest as a potential antitumoral target, there is a lack of understanding on CAD's structure and functioning mechanisms. Although mainly identified as a cytosolic complex, different studies support the translocation of CAD into the nucleus, where it could have a yet undefined function. Here, we track the subcellular localization of CAD by using fluorescent chimeras, cell fractionation and immunoblotting with specific antibodies. Contradicting previous studies, we demonstrate that CAD is exclusively localized at the cytosol and discard a possible translocation to the nucleus.

13 páginas, 6 figuras., Aspartate transcarbamoylase (ATC), an essential enzyme for de novo pyrimidine biosynthesis, is uniquely regulated in plants by feedback inhibition of uridine 5-monophosphate (UMP). Despite its importance in plant growth, the structure of this UMP-controlled ATC and the regulatory mechanism remain unknown. Here, we report the crystal structures of Arabidopsis ATC trimer free and bound to UMP, complexed to a transition-state analog or bearing a mutation that turns the enzyme insensitive to UMP. We found that UMP binds and blocks the ATC active site, directly competing with the binding of the substrates. We also prove that UMP recognition relies on a loop exclusively conserved in plants that is also responsible for the sequential firing of the active sites. In this work, we describe unique regulatory and catalytic properties of plant ATCs that could be exploited to modulate de novo pyrimidine synthesis and plant growth., This work was supported by funding from the Spanish Ministry of Science, Innovation and Universities (BFU2016-80570-R and RTI2018-098084-B-100; AEI/FEDER, UE) to S.R.-M., DFG (Mo 1032/4-1 and CRC175-B08) to T.M., DFG-IRTG 1830 to T.M. and L.B., and Bayer AG Crop Science (Grants for Targets 2017-02-005) to S.R.-M. and T.M. We thank the staff from ALBA (Barcelona, Spain) and ESRF (Grenoble, France) synchrotron facilities for help during crystallographic data collection; R. Campos-Olivas and C.M. Santiveri for support with SEC-MALS analyses; S. Niopek-Witz and N. Navaseelan for support with ATC expression and kinetic analysis; L. Ohler, A. John, and A. Grande-García for support during protein purification, mutant generation, and crystallization trials; and M. Moreno-Morcillo for support with crystal structure analysis.

8 pages, 4 figures, 1 table. The online version of this article (https://doi.org/10.1038/s41436-020-0833-2) contains supplementary material, which is available to authorized users., Purpose: Pathogenic autosomal recessive variants in CAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size of CAD with over 1000 missense variants and the nonspecific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy. Methods: Using CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype. Results: We identified 25 individuals with biallelic variants in CAD and a phenotype consistent with a CAD deficit. We used the CAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype. Conclusions: We designed a cell-based assay to test the pathogenicity of CAD variants, identifying 11 CAD-deficient individuals who could benefit from uridine therapy., This work was supported by The Rocket Fund, by R01DK99551 to H.H.F., and by grants BFU2016–80570-R and RTI2018–098084- B-100 from the Spanish Ministry of Science and Innovation (AEI/ FEDER, UE) and with partial support from U54 NS115198. The University of Washington Center for Mendelian Genomics for exome sequencing and analysis of CDG-0117

Pyrimidine nucleotides are essential to plant development. We proved that Arabidopsis growth can be inhibited or enhanced by down- or upregulating aspartate transcarbamoylase (ATC), the first committed enzyme forde novobiosynthesis of pyrimidines in plants. To understand the unique mechanism of feedback inhibition of this enzyme by uridine 5-monophosphate (UMP), we determined the crystal structure of the Arabidopsis ATC trimer free and bound to UMP, demonstrating that the nucleotide binds and blocks the active site. The regulatory mechanism relies on a loop exclusively conserved in plants, and a single-point mutation (F161A) turns ATC insensitive to UMP. Moreover, the structures in complex with a transition-state analog or with carbamoyl phosphate proved a mechanism in plant ATCs for sequential firing of the active sites. The disclosure of the unique regulatory and catalytic properties suggests new strategies to modulate ATC activity and to controlde novopyrimidine synthesis and plant growth.

CAD is a 1.5 MDa particle formed by hexameric association of a 250 kDa protein that carries the enzymatic activities for the first three steps in the de novo biosynthesis of pyrimidine nucleotides: glutamine-dependent Carbamoyl phosphate synthetase, Aspartate transcarbamoylase and Dihydroorotase. This metabolic pathway is essential for cell growth and proliferation and is conserved in all living organisms. However, the fusion of the first three enzymatic activities of the pathway into a single multienzymatic protein only occurs in animals. In prokaryotes, by contrast, these activities are encoded as distinct monofunctional enzymes that function independently or by forming more or less transient complexes. Whereas the structural information about these enzymes in bacteria is abundant, the large size and instability of CAD has only allowed a fragmented characterization of its structure. Here we retrace some of the most significant efforts to decipher the architecture of CAD and to understand its catalytic and regulatory mechanisms.

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.

Pyrimidines are essential precursors for DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) synthesis, protein glycosylation and lipid synthesis. In resting cells, pyrimidines are largely obtained through salvage pathways, but in proliferating cells, particularly in tumours, the synthesis of pyrimidines de novo is indispensable to fuel the high demand of nucleic acids and other cellular components. In animals, the de novo pathway is initiated and controlled by CAD, a ∼240-kDa multifunctional protein with four different enzymatic domains: glutaminase (GLN), carbamoyl phosphate synthetase (CPS), dihydroorotase (DHO) and aspartate transcarbamoylase (ATC). In contrast, in bacteria, archaeans and plants, GLN, CPS, DHO and ATC are distinct monofunctional proteins. The structures of a number of these enzymes from bacteria and archaea are known, but until recently, there was no structural information about CAD other than that it self-assembles into ∼1.5-megaDa hexamers. Key Concepts De novo synthesis of pyrimidine nucleotides is essential for cell growth and proliferation. In animals, the multifunctional protein CAD catalyses the first three reactions of de novo pyrimidine synthesis. CAD is a 243-kDa polypeptide with four enzymatic domains [glutaminase (GLN), carbamoyl phosphate synthetase (CPS), dihydroorotase (DHO) and aspartate transcarbamoylase (ATC)] that oligomerises into 1.5-megaDa hexamers. In bacteria, GLN, CPS, DHO and ATC are individual proteins for which structural information is available. The crystal structures of the DHO and ATC domains of human CAD were recently reported. The GLN and CPS domains of CAD are expected to be similar to the Escherichia coli CPS and human mitochondrial CPS1 crystal structures. A model of CAD is proposed that sets the DHO and ATC domains as the central framework of the hexameric particles. Keywords: nucleotide metabolism; multifunctional protein; glutaminase; carbamoyl phosphate synthetase; dihydroorotase; aspartate transcarbamoylase; URA2; protein structure; X-ray crystallography

SummaryUpregulation of CAD, the multifunctional protein that initiates and controls the de novo biosynthesis of pyrimidines in animals, is essential for cell proliferation. Deciphering the architecture and functioning of CAD is of interest for its potential usage as an antitumoral target. However, there is no detailed structural information about CAD other than that it self-assembles into hexamers of ∼1.5 MDa. Here we report the crystal structure and functional characterization of the dihydroorotase domain of human CAD. Contradicting all assumptions, the structure reveals an active site enclosed by a flexible loop with two Zn2+ ions bridged by a carboxylated lysine and a third Zn coordinating a rare histidinate ion. Site-directed mutagenesis and functional assays prove the involvement of the Zn and flexible loop in catalysis. Comparison with homologous bacterial enzymes supports a reclassification of the DHOase family and provides strong evidence against current models of the architecture of CAD.

DHO is a zinc-dependent enzyme that catalyzes the third step of the pathway for de novo biosynthesis of pyrimidine nucleotides, the reversible cyclization of N-carbamoyl-l-aspartate (CA-asp) to dihydroorotate (CA-asp2− + H+ dihydroorotate− + H2O). In animals, DHO is integrated into CAD, a multienzymatic complex catalyzing the first three steps of the de novo pyrimidine pathway. CAD is a 1.5 MDa homohexamer formed by the self-association of a 243 kDa polypeptide composed of four contiguous functional domains: glutaminase (GLN), carbamoyl phosphate synthetase (CPS; EC 6.3.5.5), aspartate transcarbamoylase (ATC; EC 2.1.3.2), and DHO. 3D Structure Cartoon representation of the structure of the DHO domain of human CAD. Human DHO forms dimers and each subunit is shown in a different color. Three Zn2+ ions bound per protein subunit are represented as cyan spheres. PDB code: 4C6C. Dashed lines indicate the linkers connecting the N- and C-termini of DHO to the CPS and ATC domains, respectively. The structure representations are produced with program PyMOL (The PyMOL Molecular Graphics System, Schrodinger, LLC). Keywords: CAD; pyrimidine nucleotides; carbamoyl phosphate synthetase; aspartate transcarbamoylase

CAD, the multifunctional protein initiating and controlling de novo biosynthesis of pyrimidines in animals, self-assembles into ∼1.5 MDa hexamers. The structures of the dihydroorotase (DHO) and aspartate transcarbamoylase (ATC) domains of human CAD have been previously determined, but we lack information on how these domains associate and interact with the rest of CAD forming a multienzymatic unit. Here, we prove that a construct covering human DHO and ATC oligomerizes as a dimer of trimers and that this arrangement is conserved in CAD-like from fungi, which holds an inactive DHO-like domain. The crystal structures of the ATC trimer and DHO-like dimer from the fungus Chaetomium thermophilum confirm the similarity with the human CAD homologs. These results demonstrate that, despite being inactive, the fungal DHO-like domain has a conserved structural function. We propose a model that sets the DHO and ATC complex as the central element in the architecture of CAD., Spanish Ministry of Economy and Competitiveness (MINECO; BFU2013-48365-P and BFU2016-80570-R)

CAD is a 243 kDa eukaryotic multifunctional polypeptide that catalyzes the first three reactions ofde novopyrimidine biosynthesis: glutamine-dependentcarbamyl phosphate synthetase,aspartate transcarbamylase anddihydroorotase (DHO). In prokaryotes, these activities are associated with monofunctional proteins, for which crystal structures are available. However, there is no detailed structural information on the full-length CAD protein or any of its functional domains apart from that it associates to form a homohexamer of ∼1.5 MDa. Here, the expression, purification and crystallization of the DHO domain of human CAD are reported. The DHO domain forms homodimers in solution. Crystallization experiments yielded small crystals that were suitable for X-ray diffraction studies. A diffraction data set was collected to 1.75 Å resolution using synchrotron radiation at the SLS, Villigen, Switzerland. The crystals belonged to the orthorhombic space groupC2221, with unit-cell parametersa= 82.1,b= 159.3,c= 61.5 Å. The Matthews coefficient calculation suggested the presence of one protein molecule per asymmetric unit, with a solvent content of 48%.