scholarly journals Inter-Active Site Communication Mediated by the Dimer Interface β-Sheet in the Half-the-Sites Enzyme, Thymidylate Synthase

Biochemistry ◽  
2019 ◽  
Vol 58 (30) ◽  
pp. 3302-3313 ◽  
Author(s):  
Paul J. Sapienza ◽  
Konstantin I. Popov ◽  
David D. Mowrey ◽  
Bradley T. Falk ◽  
Nikolay V. Dokholyan ◽  
...  
Keyword(s):  
Thymidylate Synthase ◽  
Active Site ◽  
Dimer Interface ◽  
Β Sheet

scholarly journals Crystal structures of non-oxidative decarboxylases reveal a new mechanism of action with a catalytic dyad and structural twists

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Matthias Zeug ◽  
Nebojsa Markovic ◽  
Cristina V. Iancu ◽  
Joanna Tripp ◽  
Mislav Oreb ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Enzyme Engineering ◽  
Base Catalysis ◽  
Oxidative Decarboxylation ◽  
Transition State Stabilization ◽  
Major Bottleneck ◽  
Hydroxybenzoic Acids ◽  
Potassium Binding ◽  
Β Sheet

AbstractHydroxybenzoic acids, like gallic acid and protocatechuic acid, are highly abundant natural compounds. In biotechnology, they serve as critical precursors for various molecules in heterologous production pathways, but a major bottleneck is these acids’ non-oxidative decarboxylation to hydroxybenzenes. Optimizing this step by pathway and enzyme engineering is tedious, partly because of the complicating cofactor dependencies of the commonly used prFMN-dependent decarboxylases. Here, we report the crystal structures (1.5–1.9 Å) of two homologous fungal decarboxylases, AGDC1 from Arxula adenivorans, and PPP2 from Madurella mycetomatis. Remarkably, both decarboxylases are cofactor independent and are superior to prFMN-dependent decarboxylases when heterologously expressed in Saccharomyces cerevisiae. The organization of their active site, together with mutational studies, suggests a novel decarboxylation mechanism that combines acid–base catalysis and transition state stabilization. Both enzymes are trimers, with a central potassium binding site. In each monomer, potassium introduces a local twist in a β-sheet close to the active site, which primes the critical H86-D40 dyad for catalysis. A conserved pair of tryptophans, W35 and W61, acts like a clamp that destabilizes the substrate by twisting its carboxyl group relative to the phenol moiety. These findings reveal AGDC1 and PPP2 as founding members of a so far overlooked group of cofactor independent decarboxylases and suggest strategies to engineer their unique chemistry for a wide variety of biotechnological applications.

scholarly journals An enzymatic activation of formaldehyde for nucleotide methylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Charles Bou-Nader ◽  
Frederick W. Stull ◽  
Ludovic Pecqueur ◽  
Philippe Simon ◽  
Vincent Guérineau ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thymidylate Synthase ◽  
Active Site ◽  
De Novo ◽  
Crystallographic Structure ◽  
De Novo Synthesis ◽  
Active Site Residues ◽  
Enzymatic Activation ◽  
Enzyme Cofactors ◽  
Unique Ability

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

scholarly journals Three Conserved Regions in Baculovirus Sulfhydryl Oxidase P33 Are Critical for Enzymatic Activity and Function

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
Wenhua Kuang ◽  
Huanyu Zhang ◽  
Manli Wang ◽  
Ning-Yi Zhou ◽  
Fei Deng ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Active Site ◽  
Salt Bridge ◽  
Sulfhydryl Oxidase ◽  
Dimer Interface ◽  
Conserved Regions ◽  
Occlusion Bodies ◽  
Budded Virus ◽  
And Function ◽  
Substrate Proteins

ABSTRACT Baculoviruses encode a conserved sulfhydryl oxidase, P33, which is necessary for budded virus (BV) production and multinucleocapsid occlusion-derived virus (ODV) formation. Here, the structural and functional relationship of P33 was revealed by X-ray crystallography, site-directed mutagenesis, and functional analysis. Based on crystallographic characterization and structural analysis, a series of P33 mutants within three conserved regions, i.e., the active site, the dimer interface, and the R127-E183 salt bridge, were constructed. In vitro experiments showed that mutations within the active site and dimer interface severely impaired the sulfhydryl oxidase activity of P33, while the mutations in the salt bridge had a relatively minor influence. Recombinant viruses containing mutated P33 were constructed and assayed in vivo. Except for the active-site mutant AXXA, all other mutants produced infectious BVs, although certain mutants had a decreased BV production. The active-site mutant H114A, the dimer interface mutant H227D, and the salt bridge mutant R127A-E183A were further analyzed by electron microscopy and bioassays. The occlusion bodies (OBs) of mutants H114A and R127A-E183A had a ragged surface and contained mostly ODVs with a single nucleocapsid. The OBs of all three mutants contained lower numbers of ODVs and had a significantly reduced oral infectivity in comparison to control virus. Crystallographic analyses further revealed that all three regions may coordinate with one another to achieve optimal function of P33. Taken together, our data revealed that all the three conserved regions are involved in P33 activity and are crucial for virus morphogenesis and peroral infectivity. IMPORTANCE Sulfhydryl oxidase catalyzes disulfide bond formation of substrate proteins. P33, a baculovirus-encoded sulfhydryl oxidase, is different from other cellular and viral sulfhydryl oxidases, bearing unique features in tertiary and quaternary structure organizations. In this study, we found that three conserved regions, i.e., the active site, dimer interface, and the R127-E183 salt bridge, play important roles in the enzymatic activity and function of P33. Previous observations showed that deletion of p33 results in a total loss of budded virus (BV) production and in morphological changes in occlusion-derived virus (ODV). Our study revealed that certain P33 mutants lead to occlusion bodies (OBs) with a ragged surface, decreased embedded ODVs, and reduced oral infectivity. Interestingly, some P33 mutants with impaired ODV/OB still retained BV productivity, indicating that the impacts on BV and on ODV/OB are two distinctly different functions of P33, which are likely to be performed via different substrate proteins.

scholarly journals Structure of undecaprenyl pyrophosphate synthase from Acinetobacter baumannii

2018 ◽  
Vol 74 (12) ◽  
pp. 765-769 ◽  
Author(s):  
Tzu-Ping Ko ◽  
Chi-Hung Huang ◽  
Shu-Jung Lai ◽  
Yeh Chen
Keyword(s):  
Acinetobacter Baumannii ◽  
Active Site ◽  
Multidrug Resistant ◽  
Structural Basis ◽  
X Ray Diffraction ◽  
X Ray ◽  
Peptidoglycan Synthesis ◽  
C Terminus ◽  
Dimeric Protein ◽  
Β Sheet

Undecaprenyl pyrophosphate (UPP) is an important carrier of the oligosaccharide component in peptidoglycan synthesis. Inhibition of UPP synthase (UPPS) may be an effective strategy in combating the pathogen Acinetobacter baumannii, which has evolved to be multidrug-resistant. Here, A. baumannii UPPS (AbUPPS) was cloned, expressed, purified and crystallized, and its structure was determined by X-ray diffraction. Each chain of the dimeric protein folds into a central β-sheet with several surrounding α-helices, including one at the C-terminus. In the active site, two molecules of citrate interact with the side chains of the catalytic aspartate and serine. These observations may provide a structural basis for inhibitor design against AbUPPS.

scholarly journals Atomic structures of the RNA end-healing 5′-OH kinase and 2′,3′-cyclic phosphodiesterase domains of fungal tRNA ligase: conformational switches in the kinase upon binding of the GTP phosphate donor

2019 ◽  
Author(s):  
Ankan Banerjee ◽  
Yehuda Goldgur ◽  
Beate Schwer ◽  
Stewart Shuman
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Fungal Pathogens ◽  
Pathogenic Fungus ◽  
Atomic Structures ◽  
Polynucleotide Kinase ◽  
Phosphate Donor ◽  
Guanine Base ◽  
Conformational Switches ◽  
Β Sheet

Abstract Fungal tRNA ligase (Trl1) rectifies RNA breaks with 2′,3′-cyclic-PO4 and 5′-OH termini. Trl1 consists of three catalytic modules: an N-terminal ligase (LIG) domain; a central polynucleotide kinase (KIN) domain; and a C-terminal cyclic phosphodiesterase (CPD) domain. Trl1 enzymes found in all human fungal pathogens are untapped targets for antifungal drug discovery. Here we report a 1.9 Å crystal structure of Trl1 KIN-CPD from the pathogenic fungus Candida albicans, which adopts an extended conformation in which separate KIN and CPD domains are connected by an unstructured linker. CPD belongs to the 2H phosphotransferase superfamily by dint of its conserved central concave β sheet and interactions of its dual HxT motif histidines and threonines with phosphate in the active site. Additional active site motifs conserved among the fungal CPD clade of 2H enzymes are identified. We present structures of the Candida Trl1 KIN domain at 1.5 to 2.0 Å resolution—as apoenzyme and in complexes with GTP•Mg2+, IDP•PO4, and dGDP•PO4—that highlight conformational switches in the G-loop (which recognizes the guanine base) and lid-loop (poised over the nucleotide phosphates) that accompany nucleotide binding.

scholarly journals Structural and Functional Characterization of the Human Thymidylate Synthase (hTS) Interface Variant R175C, New Perspectives for the Development of hTS Inhibitors

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1362
Author(s):  
Cecilia Pozzi ◽  
Stefania Ferrari ◽  
Rosaria Luciani ◽  
Maria Costi ◽  
Stefano Mangani
Keyword(s):  
Thymidylate Synthase ◽  
Active Site ◽  
Functional Characterization ◽  
Binding Mode ◽  
Anticancer Chemotherapy ◽  
Innovative Strategies ◽  
X Ray Crystallography ◽  
Site Targeting ◽  
New Strategies

Human thymidylate synthase (hTS) is pivotal for cell survival and proliferation, indeed it provides the only synthetic source of dTMP, required for DNA biosynthesis. hTS represents a validated target for anticancer chemotherapy. However, active site-targeting drugs towards hTS have limitations connected to the onset of resistance. Thus, new strategies have to be applied to effectively target hTS without inducing resistance in cancer cells. Here, we report the generation and the functional and structural characterization of a new hTS interface variant in which Arg175 is replaced by a cysteine. Arg175 is located at the interface of the hTS obligate homodimer and protrudes inside the active site of the partner subunit, in which it provides a fundamental contribution for substrate binding. Indeed, the R175C variant results catalytically inactive. The introduction of a cysteine at the dimer interface is functional for development of new hTS inhibitors through innovative strategies, such as the tethering approach. Structural analysis, performed through X-ray crystallography, has revealed that a cofactor derivative is entrapped inside the catalytic cavity of the hTS R175C variant. The peculiar binding mode of the cofactor analogue suggests new clues exploitable for the design of new hTS inhibitors.

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