Biological and Structural Characterization of Trypanosoma cruzi Phosphodiesterase C and Implications for Design of Parasite Selective Inhibitors

Trypanosoma cruzi phosphodiesterase C (TcrPDEC) is a potential new drug target for the treatment of Chagas disease but has not been well studied. This study reports the enzymatic properties of various kinetoplastid PDECs and the crystal structures of the unliganded TcrPDEC1 catalytic domain and its complex with an inhibitor. Mutations of PDEC during the course of evolution led to inactivation of PDEC in Trypanosoma brucei/Trypanosoma evansi/Trypanosoma congolense, whereas the enzyme is active in all other kinetoplastids. The TcrPDEC1 catalytic domain hydrolyzes both cAMP and cGMP with a Km of 23.8 μm and a kcat of 31 s−1 for cAMP and a Km of 99.1 μm and a kcat of 17 s−1 for cGMP, thus confirming its dual specificity. The crystal structures show that the N-terminal fragment wraps around the TcrPDEC catalytic domain and may thus regulate its enzymatic activity via direct interactions with the active site residues. A PDE5 selective inhibitor that has an IC50 of 230 nm for TcrPDEC1 binds to TcrPDEC1 in an orientation opposite to that of sildenafil. This observation, together with the screen of the inhibitory potency of human PDE inhibitors against TcrPDEC, implies that the scaffold of some human PDE inhibitors might be used as the starting model for design of parasite PDE inhibitors. The structural study also identified a unique parasite pocket that neighbors the active site and may thus be valuable for the design of parasite-specific inhibitors.

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Characterization of a novel cAMP-binding, cAMP-specific cyclic nucleotide phosphodiesterase (TcrPDEB1) from Trypanosoma cruzi

Biochemical Journal ◽

10.1042/bj20060757 ◽

2006 ◽

Vol 399 (2) ◽

pp. 305-314 ◽

Cited By ~ 19

Author(s):

Rocío Díaz-Benjumea ◽

Sunil Laxman ◽

Thomas R. Hinds ◽

Joseph A. Beavo ◽

Ana Rascón

Keyword(s):

Trypanosoma Cruzi ◽

Cyclic Nucleotide ◽

Catalytic Domain ◽

Calorimetric Technique ◽

Camp Analogue ◽

Pde Inhibitors ◽

N Terminus ◽

Trypanosomatid Parasites ◽

A Domains

Trypanosoma cruzi, the causative agent of Chagas disease, encodes a number of different cAMP-specific PDE (phosphodiesterase) families. Here we report the identification and characterization of TcrPDEB1 and its comparison with the previously identified TcrPDEB2 (formerly known as TcPDE1). These are two different PDE enzymes of the TcrPDEB family, named in accordance with the recent recommendations of the Nomenclature Committee for Kinetoplast PDEs [Kunz, Beavo, D'Angelo, Flawia, Francis, Johner, Laxman, Oberholzer, Rascon, Shakur et al. (2006) Mol. Biochem. Parasitol. 145, 133–135]. Both enzymes show resistance to inhibition by many mammalian PDE inhibitors, and those that do inhibit do so with appreciable differences in their inhibitor profiles for the two enzymes. Both enzymes contain two GAF (cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coliFhlA) domains and a catalytic domain highly homologous with that of the T. brucei TbPDE2/TbrPDEB2 family. The N-terminus+GAF-A domains of both enzymes showed significant differences in their affinities for cyclic nucleotide binding. Using a calorimetric technique that allows accurate measurements of low-affinity binding sites, the TcrPDEB2 N-terminus+GAF-A domain was found to bind cAMP with an affinity of ∼500 nM. The TcrPDEB1 N-terminus+GAF-A domain bound cAMP with a slightly lower affinity of ∼1 μM. The N-terminus+GAF-A domain of TcrPDEB1 did not bind cGMP, whereas the N-terminus+GAF-A domain of TcrPDEB2 bound cGMP with a low affinity of ∼3 μM. GAF domains homologous with those found in these proteins were also identified in related trypanosomatid parasites. Finally, a fluorescent cAMP analogue, MANT-cAMP [2′-O-(N-methylanthraniloyl)adenosine-3′,5′-cyclic monophosphate], was found to be a substrate for the TcPDEB1 catalytic domain, opening the possibility of using this molecule as a substrate in non-radioactive, fluorescence-based PDE assays, including screening for trypanosome PDE inhibitors.

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Investigation of the role of cytochrome P450 2B4 active site residues in substrate metabolism based on crystal structures of the ligand-bound enzyme

Archives of Biochemistry and Biophysics ◽

10.1016/j.abb.2006.08.024 ◽

2006 ◽

Vol 455 (1) ◽

pp. 61-67 ◽

Cited By ~ 16

Author(s):

Cynthia E. Hernandez ◽

Santosh Kumar ◽

Hong Liu ◽

James R. Halpert

Keyword(s):

Cytochrome P450 ◽

Crystal Structures ◽

Active Site ◽

Active Site Residues ◽

Substrate Metabolism ◽

Cytochrome P450 2B4

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Crystal structures of murine norovirus-1 RNA-dependent RNA polymerase

Journal of General Virology ◽

10.1099/vir.0.031104-0 ◽

2011 ◽

Vol 92 (7) ◽

pp. 1607-1616 ◽

Cited By ~ 28

Author(s):

Ji-Hye Lee ◽

Intekhab Alam ◽

Kang Rok Han ◽

Sunyoung Cho ◽

Sungho Shin ◽

...

Keyword(s):

Rna Polymerase ◽

Crystal Structures ◽

Active Site ◽

Metal Ion ◽

Health Concern ◽

Murine Norovirus ◽

Active Site Residues ◽

Rna Dependent Rna Polymerase ◽

Close Proximity ◽

Functional Features

Norovirus is one of the leading agents of gastroenteritis and is a major public health concern. In this study, the crystal structures of recombinant RNA-dependent RNA polymerase (RdRp) from murine norovirus-1 (MNV-1) and its complex with 5-fluorouracil (5FU) were determined at 2.5 Å resolution. Crystals with C2 symmetry revealed a dimer with half a dimer in the asymmetrical unit, and the protein exists predominantly as a monomer in solution, in equilibrium with a smaller population of dimers, trimers and hexamers. MNV-1 RdRp exhibited polymerization activity with a right-hand fold typical of polynucleotide polymerases. The metal ion modelled in close proximity to the active site was found to be coordinated tetrahedrally to the carboxyl groups of aspartate clusters. The orientation of 5FU observed in three molecules in the asymmetrical unit was found to be slightly different, but it was stabilized by a network of favourable interactions with the conserved active-site residues Arg185, Asp245, Asp346, Asp347 and Arg395. The information gained on the structural and functional features of MNV-1 RdRp will be helpful in understanding replication of norovirus and in designing novel therapeutic agents against this important pathogen.

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Characterization of active-site residues in diadenosine tetraphosphate hydrolase from Lupinus angustifolius

Biochemical Journal ◽

10.1042/0264-6021:3570399 ◽

2001 ◽

Vol 357 (2) ◽

pp. 399 ◽

Cited By ~ 11

Author(s):

Danuta MAKSEL ◽

Paul R. GOOLEY ◽

James D. SWARBRICK ◽

Andrzej GURANOWSKI ◽

Christine GANGE ◽

...

Keyword(s):

Active Site ◽

Lupinus Angustifolius ◽

Active Site Residues ◽

Diadenosine Tetraphosphate

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Structure based protein engineering to confer selectivity of guanine deaminase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331409562x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C437-C437

Author(s):

Aruna Bitra ◽

Ruchi Anand

Keyword(s):

Crystal Structures ◽

Active Site ◽

Catalytic Mechanism ◽

Catalytic Efficiency ◽

Structural Basis ◽

Active Site Residues ◽

X Ray ◽

Secondary Activity ◽

Guanine Deaminase ◽

Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

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Biochemical characterization of mutants in the active site residues of the β-galactosidase enzyme of Bacillus circulans ATCC 31382

FEBS Open Bio ◽

10.1016/j.fob.2014.11.002 ◽

2014 ◽

Vol 4 (1) ◽

pp. 1015-1020 ◽

Cited By ~ 17

Author(s):

Jelle B. Bultema ◽

Bas J.H. Kuipers ◽

Lubbert Dijkhuizen

Keyword(s):

Active Site ◽

Biochemical Characterization ◽

Bacillus Circulans ◽

Active Site Residues

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Characterization of Self-Processing Activities and Substrate Specificities of Porcine Torovirus 3C-Like Protease

Journal of Virology ◽

10.1128/jvi.01282-20 ◽

2020 ◽

Vol 94 (20) ◽

Author(s):

Shangen Xu ◽

Junwei Zhou ◽

Yingjin Chen ◽

Xue Tong ◽

Zixin Wang ◽

...

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Site Directed Mutagenesis ◽

Active Site Residues ◽

Hydrophobic Force ◽

Substrate Specificities ◽

Consensus Sequences ◽

Cleavage Activity ◽

Porcine Torovirus

ABSTRACT The 3C-like protease (3CLpro) of nidovirus plays an important role in viral replication and manipulation of host antiviral innate immunity, which makes it an ideal antiviral target. Here, we characterized that porcine torovirus (PToV; family Tobaniviridae, order Nidovirales) 3CLpro autocatalytically releases itself from the viral precursor protein by self-cleavage. Site-directed mutagenesis suggested that PToV 3CLpro, as a serine protease, employed His53 and Ser160 as the active-site residues. Interestingly, unlike most nidovirus 3CLpro, the P1 residue plays a less essential role in N-terminal self-cleavage of PToV 3CLpro. Substituting either P1 or P4 residue of substrate alone has little discernible effect on N-terminal cleavage. Notably, replacement of the two residues together completely blocks N-terminal cleavage, suggesting that N-terminal self-cleavage of PToV 3CLpro is synergistically affected by both P1 and P4 residues. Using a cyclized luciferase-based biosensor, we systematically scanned the polyproteins for cleavage sites and identified (FXXQ↓A/S) as the main consensus sequences. Subsequent homology modeling and biochemical experiments suggested that the protease formed putative pockets S1 and S4 between the substrate. Indeed, mutants of both predicted S1 (D159A, H174A) and S4 (P62G/L185G) pockets completely lost the ability of cleavage activity of PToV 3CLpro. In conclusion, the characterization of self-processing activities and substrate specificities of PToV 3CLpro will offer helpful information for the mechanism of nidovirus 3C-like proteinase’s substrate specificities and the rational development of the antinidovirus drugs. IMPORTANCE Currently, the active-site residues and substrate specificities of 3C-like protease (3CLpro) differ among nidoviruses, and the detailed catalytic mechanism remains largely unknown. Here, porcine torovirus (PToV) 3CLpro cleaves 12 sites in the polyproteins, including its N- and C-terminal self-processing sites. Unlike coronaviruses and arteriviruses, PToV 3CLpro employed His53 and Ser160 as the active-site residues that recognize a glutamine (Gln) at the P1 position. Surprisingly, mutations of P1-Gln impaired the C-terminal self-processing but did not affect N-terminal self-processing. The “noncanonical” substrate specificity for its N-terminal self-processing was attributed to the phenylalanine (Phe) residue at the P4 position in the N-terminal site. Furthermore, a double glycine (neutral) substitution at the putative P4-Phe-binding residues (P62G/L185G) abolished the cleavage activity of PToV 3CLpro suggested the potential hydrophobic force between the PToV 3CLpro and P4-Phe side chains.

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