Structure of Urocanate Hydratase from the protozoan Trypanosoma cruzi

Journal Pre-proof Structure of Urocanate Trypanosoma cruzi

Hydratase

from

the

protozoan

Sheila Boreiko, Marcio Silva, Raíssa de F. P. Melo, Ariel M. Silber, Jorge Iulek PII:

S0141-8130(19)38046-8

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.101

Reference:

BIOMAC 14128

To appear in:

International Journal of Biological Macromolecules

Received date:

5 October 2019

Revised date:

12 December 2019

Accepted date:

12 December 2019

Please cite this article as: S. Boreiko, M. Silva, R. de F. P. Melo, et al., Structure of Urocanate Hydratase from the protozoan Trypanosoma cruzi, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/j.ijbiomac.2019.12.101

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier.

Journal Pre-proof Structure of Urocanate Hydratase from the protozoan Trypanosoma cruzi

Sheila Boreikoa, Marcio Silvab, Raíssa de F. P. Meloc, Ariel M. Silberc and Jorge Iuleka

a

Department of Chemistry, State University of Ponta Grossa, Ponta Grossa - PR,

84030-900, Brazil. Department of Education, Federal Technological University of Paraná, Ponta

of

b

Laboratory of Biochemistry of Tryps – LaBTryps - Department of Parasitology,

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c

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Grossa - PR, 84016-210, Brazil.

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Institute of Biomedical Sciences, University of São Paulo, São Paulo - SP, 05508-

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000, Brazil.

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Abstract

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Correspondence to Jorge Iulek: [email protected]

The enzyme Urocanate Hydratase (UH) participates in the catabolic pathway of L-histidine. Trypanosoma cruzi Urocanate Hydratase (TcUH) is identified as a therapeutic molecular target in the WHO/TDR Targets Database. We report the 3D structure determination and number of features of TcUH, and compared it to other few available bacterial UH structures. Each monomer presents two domains and one NAD+ molecule. Superpositions revealed differences in the relative orientation of domains within monomers, such that TcUH monomer A resembles Urocanate Hydratase from Geobacillus kaustophilus (GkUH) (open conformation), while monomer C resembles Urocanate Hydratase from Pseudomonas putida (PpUH) and

1

Journal Pre-proof Urocanate Hydratase from Bacillus subtilis (BsUH) (closed conformations). We use the structure of TcUH to make considerations about (3) non-deleterious and (2) deleterious mutations found in human UHs: non-deleterious mutations could be accommodated without large displacements or interaction interruptions, whereas deleterious mutations in one case might disrupt an α-helix (as previously suggested) and in the other case, besides disrupting the enzyme interaction with the substrate,

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might interfere with interdomain movement.

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keywords: Trypanosoma cruzi, Chagas disease; Urocanate Hydratase; Histidine

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metabolism; Interdomain movement; Human Urocanate Hydratase deleterious

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1 Introduction

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mutations.

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American trypanosomiasis (Chagas disease) is an endemic zoonosis caused by the protozoan Trypanosoma cruzi. This disease was discovered by Carlos

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Chagas in 1909 [1] and despite advances in vector control in many countries, Chagas disease remains a significant public health problem [2]. Estimations account that about 6 million to 7 million people are infected worldwide, mostly in Latin America, over 10,000 people die each year from clinical manifestations of Chagas disease and over 25 million people are at risk for the disease [3]. Current treatment depends on two nitroheterocyclic drugs, introduced in the 1960s and 1970s, respectively: Nifurtimox, a nitrofuran and Benznidazole, a nitroimidazole derivative [4]. These medications are highly active in the acute phase of the disease [5] but their efficacy in the chronic phase, when most of cases are diagnosed, remains controversial [6] (for limitations on the use of Benzonidazole,

2

Journal Pre-proof see for example [7,8]. In addition, these medications have shown several sideeffects,

including

anorexia,

vomiting,

allergic

dermopathy

and

peripheral

polyneuropathy [9] and several interactions with the immune system (reviewed by [10]). Given that Nifurtimox and Benznidazole are far from being considered ideal as trypanocidal drugs, the search for new compounds with low toxicity and increased efficacy during the undetermined and chronic phases continues [11]. As a consequence,

some

T.

cruzi

specific

metabolic

pathways

that

involve

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proteins/enzymes have been evaluated as targets, and several drugs that are known

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to interfere with these pathways are promising potential therapies opening the

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possibility for the development of more specific and less toxic drugs for the treatment

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of Chagas disease [12].

L-histidine can play a relevant role as a supplier of intermediates of the

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Tricarboxylic acids (TCA) cycle, since it operates, in part, based on the conversion of

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histidine into glutamate and, subsequently, into α-ketoglutarate in the insect stage of T. cruzi. This fact makes of histidine an efficient energy source for the parasite. It has

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been shown as well that histidine and urocanate are increased when the parasites reach the stationary phase, which could be related with their resistance to nutritional stress [13]. The canonical biochemical pathway for the conversion of histidine into glutamate involves four enzymatic steps catalyzed sequentially by: histidine ammonia-lyase (EC 4.3.1.3), urocanate hydratase (EC 4.2.1.49), imidazolone propionase (EC 3.5.2.7) and formimineglutamase (EC 3.5.3.8). The final glutamate can, in turn, be i) converted into other amino acids, for example, glutamine [14]; ii) used as a -NH2 donor in transamination reactions, with pyruvate as the main acceptor, yielding alanine and the tricarboxylic acid cycle (TCA) intermediate α-

3

Journal Pre-proof ketoglutarate (α-KG); or iii) deaminated by a glutamate dehydrogenase (GDH) to form α-KG [15–17]. Urocanate Hydratase (UH) interconverts urocanate and 4-imidazolone-5propionate (Figure 1) [18]. This enzyme is found in different organisms and has been isolated from a number of prokaryotes and eukaryotes, including humans. Under physiological conditions, Urocanate Hydratase from Pseudomonas putida (PpUH) is a homodimer

of

and presents a tightly bound NAD+ (Nicotinamide adenine dinucleotide) in each

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monomer, [19,20] essential for its catalysis, such that addition of a nucleophile or

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reduction to NADH is inhibitory [19].

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Some of the compounds already characterized as either inhibitors or with significant inhibiting action on one UH enzyme are: potassium borohydride,

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imidazolepropionate, fumarate, phenylhydrazine, hydroxylamine, semicarbazide,

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potassium cyanide [21], 4-bromocrotonate [22], thioglycolate [23], cupric ion [24], 2mercaptoethanol, thioglycolate, dithioerythritol, 3-mercaptopropionate [23] and 2-

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fluorourocanic acid [25].

It is noteworthy that currently there are only three crystallographic UH structures available in the Protein Data Bank (PDB), (ID’s: 1UWK, 2FKN and 1X87), from bacteria Pseudomonas putida (Pp), Geobacillus kaustophilus (Gk) and Bacillus subtilis (Bs), respectively. The

elucidation

of

the

TcUH

three-dimensional

structure

by

X-ray

crystallography adds valuable information per se, such as the better understanding of the enzyme at molecular level. Identified as a therapeutic molecular target in the WHO/TDR Targets Database [26], its 3D structure might be used for in silico

4

Journal Pre-proof searches for inhibitors, that in the long term would indicate lead compounds for the development of new drugs for the control of Chagas disease. In this paper we report the 3D structure determination and a number of structural features of Urocanate Hydratase from Trypanosoma cruzi. We also compare its structure to the other available three UH structures, from bacteria, and infer how mutations affect the activity of human UH's.

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2.1 Cloning, protein expression and purification

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2 Materials and Methods

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The putative TcUH gene (Tc00.1047053504045.110) was identified from the Kinetoplastid Genomics Resource – TriTrypDB (https://tritrypdb.org/tritrypdb/). The

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TcUH coding region was amplified by PCR using T. cruzi CL14 strain genomic DNA

enzymes

EcoRI

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as a template and gene-specific primers designed with restriction sites for the and

XhoI:

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AAGAATTCATGACTTCCATGAAGAAGGTC AACTCGAGATACTTCTTAAGCACATCGG

TcUH -3’ -3’.

and PCR

forward TcUH

5’-

reverse

amplification

5’-

settings

comprised 35 cycles using the following conditions: initial denaturation cycle at 94 °C (1 min), primers annealing at 58 °C (1 min) and nucleotide chain extension at 72 °C (1 min and 30 s), plus an additional final extension at 72 °C for 5 min. A single fragment (2.028 kb) was amplified and the PCR product was purified and cloned into the pGEM-T Easy vector (Promega, Madison, WI, United States). Selected clones were sequenced, and the expected identity of the cloned DNA fragments to UH was confirmed using the BLAST software program (https://blast.ncbi.nlm.nih.gov/). The gene encoding the putative UH enzyme was further subcloned into the pET24a(+)

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Journal Pre-proof expression vector (Novagen, Madison, WI, United States), which introduces a Cterminal His6-tag and contains a kanamicin resistance gene. The construct was used to transform Escherichia coli BL21(DE3) cells, which were then cultured in standard LB medium containing 100 μg/mL Kanamycin and 5 μg/mL Tetracycline at 37 °C up to an OD600 of 0.6. The temperature was then lowered to 25 C; protein expression was readily induced by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and the system was let at continuous agitation for 16 h. For protein purification, the

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cells were harvested, resuspended in lysis buffer (20 mM Tris-HCl, pH 7.9, 500 mM

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NaCl, 5 mM imidazole and 1 mg/mL lysozyme) containing protease inhibitors and

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subjected to six sonication pulses (1 min at 40% power and 1 min on ice). The

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protein was subsequently purified by affinity chromatography on a Sepharose nickel

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column (HisTrapTM FF crude, GE Life Sciences) using essentially the protocols suggested by the manufacturers. For the elution step we used 20 mM Tris-HCl, pH

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7.9, 500 mM NaCl, 500 mM imidazole. The protein was eluted when the imidazole

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concentration was in the range 329 to 500 mM.

2.2 Protein activity

Activity measurements were performed by real-time spectrophotometric assays, monitoring the decay of absorbance at 277 nm, corresponding to urocanate depletion [27]. The reaction was carried out as follows: 50 μM of urocanate, 100 mM potassium phosphate buffer, pH 7.0, recombinant enzyme (100 μg) and water to complete a final volume of 1.5 mL. Each reaction blank was the same reagent combination previously cited without the addition of the enzyme. Absorbance was monitored for 3 min at 28 °C with continuous stirring. Decays on absorbance values

6

Journal Pre-proof were converted to amount of substrate consumed using the urocanate molar extinction coefficient (CEM = 18,800 M-1cm-1).

2.3 Crystallization and X-ray data collection To improve the quality of the crystals previously obtained, [28] new crystallization assays were manually prepared using the hanging-drop vapordiffusion method [29] changing the enzyme concentration to 8.0, 9.5 and 12.0

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mg/mL, as quantified by the Bradford method [30]. Drops contained 2 µL reservoir

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reservoir solution in 24-well plates at 18 °C.

ro

solution mixed to 2 µL protein solution, which were equilibrated against 500 μL

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X-ray diffraction data was collected at a wavelength of 1.458 Å (at 100 K) using a synchrotron radiation source, at the W01B-MX2 station in the Brazilian

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Synchrotron Light Laboratory (LNLS), with a PILATUS 2M detector (Dectris). The

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crystal was harvested with a cryoloop and flash frozen directly in a nitrogen stream

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at 100 K since the crystallization condition was already cryoprotectant.

2.4 Processing, structure determination and validation Indexing, integration and scaling were performed with the X-ray Detector Software, XDS suite [31].

To estimate initial phase information, molecular replacement was performed using an edited model of the structure of Urocanate Hydratase from Pseudomonas putida (PpUH), 36% sequence identity for 81% coverage - PDB ID: 1UWK [20]. A better result was obtained when the template structure was divided into 2 search models (core domain, residues 5-141 and 344-557, and NAD domain, residues 142-

7

Journal Pre-proof 343), because the TcUH monomers present different interdomain orientation, as commented below. For structure refinement and modeling, the programs used were Phenix [32] and Coot [33], in which the model was adjusted to the electron density with preferential use of maps 2mFo-DFc [34] and mFo-DFc [35]. For structure validation, the programs used were: Procheck [36], Whatcheck [37], Validation PDB [38] and Molprobity [39]. For the calculation of the indices of the

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2.5 Structural Analysis and comparisons

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(RSCC), the program used was Mapman [40].

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electron density fit, real-space residual (RSR) and real-space correlation coefficient

Structural superpositions between TcUH monomers and domains for the

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calculation of the root mean square deviation (RMSD) between Cα’s were performed

na

using the program Lsqkab [41]. Otherwise, to highlight the difference on the relative orientation between TcUH domains, observed when the monomers are superposed,

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the program Multiprot [42] and Protein Domain Motion (DynDom) [43] were used. The indication of the electrostatic interactions hydrogen bonds, HB, and salt bridges, SB, as well as of the interface area, between domains was done with the program PISA [44]. Analysis of α-helical coiled coil was performed with the program TWISTER [45]. Homologous protein structures for structural comparisons were selected based on a BLAST tool search [46]. When there was more than one structure available of the same protein, higher resolution and the presence of ligands were attributes preferred. Therefore, structures with PDB ID’s: 1UWK (PpUH), 1X87 (GkUH) and 2FKN (BsUH) were selected for comparisons. Superposition of these to

8

Journal Pre-proof TcUH was performed also with the program Multiprot [42] and the RMSD values calculated with it are reported.

2.6 Alignment and analyses of the contacts with ligands The alignment of the amino acid sequences, for comparison studies, was performed with the UH sequences from PpUH, GkUH and BsUH cited above and the HsUH obtained from the UniProt database (code NM_001165974.1).

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The alignment was performed with the M-Coffee package [47] as an extension

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of the T-Coffee program [48] and had minimal manual edition considering the

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structural alignment. It was visualized with the program ALINE [49], in which the

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secondary structure elements were annotated as estimated with the DSSP program [50]. The relative residue conservation was colored according to the convention of

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ALSCRIPT Calcons [51].

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Contacts with NAD+ were accounted with the program Ncont of the Collaborative Computational Project Number 4 package (CCP4) considering a

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distance cutoff of 3.5 Å. Contacts with the urocanate in the homologous structure (PDB ID: 1UWK) were determined in the same way and at the sequence alignment to TcUH equivalent residues were presumed.

3 Results and Discussion

3.1 Protein activity In order to obtain soluble protein, TcUH was subcloned into the bacterial expression vector pET-24a(+), which also provided a C-terminal His6 tag for purification. The resulting purified protein had an expected molecular mass of

9

Journal Pre-proof approximately 72 kDa as shown in Figure 2A. TcUH expressed from E. coli was used to test its enzymatic activity in a time-course mode. It was possible to observe an increase of hydrolysed urocanate as a function of time in the presence of the purified enzyme (Figure 2B).

3.2 Crystallization The best crystal was obtained in 0.04 M potassium dihydrogen phosphate,

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16.0% (m/V) polyethylene glycol 8000 and 24.0% (V/V) glycerol, using a protein

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concentration of 9.5 mg/mL (Figure S1), slightly lower than previously reported [28].

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Indeed, in these assays, crystals could be observed at all protein concentrations

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tested.

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3.3 Structure refinement and description

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We report here the structure obtained from best dataset (2.16 Å), deposited in the PDB as entry 6UEK. The final processing, refinement statistics and some

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validation data are shown in Table 1.

TcUH is a relatively large protein (675 aminoacids per protomer) that crystallized as homotetramer in the asymmetric unit (Figure 3). Each monomer presents two domains, namely, a core domain (residues 1–224 and 443–675 for TcUH, according to DynDom results), that forms the interfaces between protomers, and the sequence inserted NAD–binding domain (residues 225–442 for TcUH, according to Dyndom results). In TcUH structure, there are four NAD+ molecules, one per monomer. Nevertheless, no NAD+ was added to the crystallization drops (when it was previously added, no crystals were formed), and its presence in the TcUH structure agrees with the previous observations that, since it cannot be

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Journal Pre-proof incorporated in vitro, it must be present during protein folding [52]. NAD+ is known to act as an electrophile for UH [18]. During refinement/modeling, it was verified that the quality of the electronic density in several portions of the monomers A and B is superior to the corresponding ones of the monomers C and D, especially in the NAD–binding domain. In the final model, 376 residues had their side chains not completely modeled due to the absence of convincing electron density. Due to the same reason, some

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main chain residues were not modeled: (a) monomer A, 1-2, 422-431, 673-675; (b)

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monomer B, 1-2, 24, 418-431, 672-675; (c) monomer C, 1-2, 332-335, 422-440, 667-

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675 and (d) monomer D, 1-7, 17-18, 23-30, 293-294, 313-314, 329-337, 394-399,

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417-437, 475-488, 498-505, 668-675.

Ser172, Gln209 and Arg559 are either in the generously favorable or

lP

unfavorable regions of the Ramachandran graph, but one observes that, in all

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monomers, the electron density corroborates their conformations. In the structures of the homologous proteins (Urocanate Hydratase from Pseudomanas Putida, PpUH -

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PDB ID: 1UWK, Urocanate Hydratase from Geobacillus kaustophilus, GkUH - PDB ID: 1X87 and Urocanate Hydratase from Bacillus subtilis, BsUH - PDB ID: 2FKN) these residues are in the generously allowed regions, except for GkUH (residue numbers Gln126 and Arg450), in which they were not modeled. Gln209 and Arg559 are involved in contacts with NAD+ and conserved within the homologous structures, while Ser172 is involved in a hydrogen bond network through its Oγ to Thr158-Oγ1 and Arg178-Nη2 and through its N to His156, in a region close to the tetramer core. In the homologous structures, Thr (Figure S2) residues occupy this position, which, nevertheless, present similar interactions.

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Journal Pre-proof 3.4 Analyses of the active site and contacts with the ligands (NAD+ and urocanate) Although TcUH and its homologues folding are similar, there are differences in the interacting residues, which can be observed in the alignment (Figure S2), at the NAD+ binding site, considering a distance of up to 3.5 Å to any heavy atom of the ligand. A Gly in PpUH (residue 179), GkUH (residue 174) and BsUH (residue 175) is changed to Ser258 in TcUH. Also, an Ala in PpUH (residue 244), GkUH (residue 239) and BsUH (residue 240) is changed to Val323 in TcUH. Yet, the hydrophobic

of

residues in PpUH (residue 275), GkUH (residue 270) and BsUH (residue 271), Leu,

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Ile and Val, respectively, correspond to a Tyr356 in TcUH. It is worth mentioning that

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these different residues in TcUH present their side chains relatively close to NAD+,

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especially Ser258, to which it forms a hydrogen bond through its Oγ. Nevertheless, when compared to the Urocanate Hydratase from Homo sapiens (HsUH - eukaryote)

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sequence, these residues are identical to the ones in TcUH.

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Concerning the contacts to urocanate, the alignment (Figure S2) shows that this site is totally conserved among all the enzymes. The conformation of these

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residues is also conserved, with the exception of Met257, which in the TcUH assumes a different conformation (Figure 4), compared to the other structures in which it was modeled.

3.5 Analyses and comparisons between TcUH monomers, domains and homologous protein structure The monomer pairs A:B and C:D present higher intermonomer interfacial area, number of hydrogen bonds and number of salt bridges, than any other monomer pair combinations (data not shown). This can be considered an indication

12

Journal Pre-proof that the enzyme might also be a homodimer under physiological conditions, as also observed for the enzyme PpUH [20]. Superposition among the four TcUH monomers reveal differences in the relative orientation of the domains, what leads to an RMSD between monomers of up to 3.572 Å, namely, between monomers A and C (Figure 5A). Nevertheless, when single domains are superposed, RMSD values decrease steeply, suggesting that the domains are structurally conserved within each other (Figure 5B and 5C)

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(Table 2).

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An analysis made with the program DynDom, that compares two monomers at

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a time, highlighted the significant difference in domain orientation between

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monomers A and C, mainly (B and C, and B and D, show practically the same differences, given that A and B, between them, do not present significant difference).

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At the comparison between monomers A and C, the program defined two "bending

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regions", involving residues 220-225 and 442-443, which are a "point of support" for the relative rotation between domains and, in this structure, are α-helix terminals and

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its vicinities. The estimated rotation and translation between domains are 22.9° and 0.1 Å (Figure 6A and B), respectively. When the comparison was made with monomers A and D, interdomain orientation difference is still significant, although lower, involving approximately the same "bending regions", with estimated rotation and translation of 8.6° and 0.1 Å, respectively. Monomer A, which has an "open" conformation (Figure 5A), presents 23 electrostatic interactions (hydrogen bonds + salt bridges), as estimated by PISA [44], between the domains. Yet, monomer C presents 11 electrostatic interactions between the domains (Table 3); the only interaction (hydrogen bond) present in both monomers is Gly222-N → Gly219-O.

13

Journal Pre-proof These

numbers

of

electrostatic

interactions

might

seem

incoherent

considering the general proximity between the domains. As a matter of fact, monomer C presents more interactions (total of 8 against 2 of monomer A) far from the "link region", id est, the "bending region" plus especially helix 221-238, that seems to mediate interactions between the domains, but monomer A presents many more interactions (total of 21 against 3 of monomer C) at this "link region". It seems that to promote domain orientation change (or as a result of this), a number of

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hydrogen bonds and salt bridges must be disrupted and these involve mainly helix

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221-238, positioned in a sort of central interface between the domains. As another

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consequence of domain orientation change, the coiled coil fold between part

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(residues 222-231) of this helix and the one that comprises residues 258-267 (in the NAD+ domain) is changed, with average pitches calculated by the Twister program

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as 49.7 Å for mononer A and 60.1 Å for monomer C. Besides that, in monomer C,

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the side chains of many interacting residues between these former two helices were disordered, what must correlate with the difference in the interfacial area between

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them, calculated as 244.6 and 185.8 Å2 for monomer A and C, respectively. On the other hand, helix 221-238 of monomer A presents additional electrostatic interactions also with helix 485-504, yet its C-terminal region is closer to the N-terminal region of the latter than in monomer C. It is worth mentioning that the interactions Arg232-Nη2 → Ser492-Oγ and Arg232-Nε → Asp493-Oδ1 are present only in the TcUH structure, because in the homologous enzymes this Arg is not conserved (Figure S2). Yet, other interactions observed only in monomer A, between residues Lys237Asp440, Tyr238-Asp440 and Asp403-Tyr437, should not be present in monomer C because, in the latter, residues Tyr437 and Asp440 are rather far from its partners. As a matter of fact, they are disordered in the crystal structure probably due to the

14

Journal Pre-proof absence of these interactions. Otherwise, the interactions between residues Gln221Ala512, Gln221-Arg513 and Gln221-Ile514 are not found in monomer C probably due to mere side chain disorder of its residue number Gln221. Finally, the interaction Asn233-Nδ2 → Gln490-Oε1 is not present in monomer C because of the large distance between their Cα's, 10.62 Å, whereas in monomer A this distance is only 8.28 Å. The interaction Tyr217-Oη → Gly256-O, although in the "link region", is

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present only in monomer C (monomer A presents a larger Cα distance between

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these two residues); it is noteworthy that residue Tyr217 should interact with the

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substrate urocanate (according to homologous structure) and residue Gln256 is part

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of a conserved sequence rich in glycine residues (253-259), therefore, these different structural conformations might be relevant during reaction. Still, the

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interaction Gly222-Arg454, that involves the central interfacing helix (residues 221-

504 in this monomer.

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238), is present only in monomer C, since residue Gly222 is closer to the helix 485-

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Figure 7 depicts the differences in electrostatic interactions between monomers and the regions of the structure where they are concentrated. This conformation difference between monomers allows one to observe that when at the closed conformation (monomer C), the bound NAD+ molecule, through its nicotinamide and ribose moieties, is brought to close contact to loop 595-603, especially Gly597, as already pointed in PpUH [20]. This loop presents yet other glycine rich region (595-601, that has five glycine residues and therefore must probably be highly flexible), highly conserved (Figure S2) with the point exception of residue Thr598 in TcUH (which has a Thr in place of Val, this latter present in the homologues and even in the human enzyme). As a matter of fact, a more extended

15

Journal Pre-proof region, which includes the surrounding β-strands and beyond, is well conserved among these sequences, though some pontual, generally conservative, mutations are present. Superposition among representative monomers of the homologous structures and monomer A of TcUH indicated that the fold is quite conserved, with the exception of 2 extra α-helices that exist in TcUH (Figure 8) (and probably, in the human enzyme, Figure S2). These helices comprise residues 42 to 53 and 61 to 75,

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located in the N-terminal region, which is longer in TcUH (Figure S2).

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Another significant difference is that the contiguous helices 462-479 and 485-

-p

504 are longer in TcUH and probably in the human protein (Figure S2), which also

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presents several insertions, compared to the homologous structures. Comparing the RMSD values of superposed structures (Table 4), one

lP

observes that TcUH monomer A resembles GkUH conformation in entry 1X87, while

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monomer C resembles PpUH and BsUH conformations in the 1UWK and 2FKN entries. Therefore, we conclude that UH structures might be found in either the

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closed or the open conformations, or even intermixed in the crystalline state, what corroborates the importance that such movement between domains must confer for protein function, for which a number of structural features and interactions implicated to achieve different conformations could be observed at TcUH structure elucidation.

3.6 Mutations of human Urocanate Hydratases and correlations to the TcUH structure TcUH is the first UH from eukaryotes to have its structure solved and the closest one to the human Urocanate Hydratases (isoforms 1 and 2). We use its

16

Journal Pre-proof structure as a surrogate to make considerations on mutations found on the human enzymes. Three non-deleterious mutations in human Urocanate Hydratase 1 are listed in the Domain Mapping of Disease Mutations (DMDM) database [53], Arg188Trp, Ser311Thr and Arg429Cys. These residues correspond to Arg193, Ser316, Arg433 in TcUH sequence (Figure 9 and Figure S2). In TcUH structure, one observes that these positions could apparently accommodate these mutations without large

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displacements or interaction disruptions.

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According to the alignment (Figure S2), TcUH Arg193 corresponds to a Trp in

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bacteria, therefore, one might expect this to be a non-deleterious mutation in both T.

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cruzi and human enzymes. Concerning Ser316 in T. cruzi, we observe that this residue Oγ makes a hydrogen bond with the Met292 main chain N, therefore an

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interaction to be maintained at a Thr mutation considering that there is enough room

na

in the structure to accommodate the extra Cγ. Regarding Arg433, it is generally disordered in TcUH structure, such that it could be modeled only on monomers A

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and B and, even in this case, not the complete side chains. Two deleterious mutations were reported by [54] Leu70Pro and Arg450Cys. These residues correspond to Glu74 and Arg454 in TcUH sequence (Figure S2, and Figure 8). In their article, residue Leu70 was predicted to be part of an α-helix, what we can observe now at TcUH (Glu74, Figure S2), but it is only the penultimate residue at its C-terminal. It well known that Pro residues can disrupt helices [55], but considering this mutation location we could not devise the consequences of this disruption to the TcUH structure and function alteration. Regarding mutation Arg450Cys (TcUH Arg454), although we did not find substrate urocanate in TcUH structure, superposition of PpUH and TcUH structures shows that this arginine side

17

Journal Pre-proof chain conformation is maintained. The interaction of this residue to urocanate (observed in PpUH structure) [54] and its obvious importance, and we can supplement now its probable role to provide the closed/open conformations (between the domains), such that interaction Gly222-O → Arg454-Nη is disrupted when this transition occurs, a switch unavailable at mutation Arg454Cys.

Acknowledgments

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S.B. thanks the Brazilian Biosciences National Laboratory (LNBio/Robolab),

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beamline W01B-MX2 of the Brazilian Synchrotron Light Laboratory staff for help with

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instrumentation and Complexo de Laboratórios Multiusuários (C-LABMU) at State

re

University of Ponta Grossa for equipment usage.

J.I. thanks INBEQMeDI (National Institute of Structural Biotechnology and Medicinal

lP

Chemistry in Infectious Diseases) for financial resources (CNPq, FAPESP and the

na

Ministry of Health, 573607/2008-7 and 08/57910-0) and CAPES for program PROAP.

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A.M.S. thanks Fundação de Amparo à Pesquisa do Estado de São Paulo for grant 2016/06034-2, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for grants 308351/2013-4 and 404769/2018-7 and Research Council United Kingdom Global Challenges Research Fund under grant agreement “A Global Network for Neglected Tropical Diseases” (grant MR/P027989/1).

Author contributions Conceived and designed the experiments: S.B, M.S., A.M.S. and J.I. Performed the experiments: S.B., J.I. and R.F.P.M. Analyzed the data: S.B. and J.I.

18

Journal Pre-proof Wrote the paper: S.B., J.I. and R.F.P.M.

Figure Legends

Figure 1. Reaction catalyzed by Urocanate Hydratase. Urocanate is hydrated and converted to 4-imidazolone-5-propionate.

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Figure 2. Purification of recombinant TcUH and determination of its enzymatic

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activity. (A) The recombinant protein was analyzed by SDS-PAGE using 10% (v/v)

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polyacrylamide gels under reducing conditions and visualized by Coomassie Blue

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staining. S: Supernatant of lysed bacterial culture overexpressing TcUH; FT: Supernatant after flowing through the column; W1 and W2: Samples of column

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washes with a buffer containing 60 mM imidazole; E1 to E3: Elution fractions

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performed with a buffer containing 500 mM imidazole; M: Molecular mass marker. (B) TcUH activity as a function of time. Activity measurement was performed by

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monitoring the decrease in absorbance at 277 nm, corresponding to total consumed urocanate. The reaction consists of: 100 mM potassium phosphate buffer pH = 7, 50 µM urocanate and water to complete a final volume of 1.5 mL.

Figure 3. Three-dimensional structure of TcUH. Monomer A in green, monomer B in cyan, monomer C in magenta and monomer D in yellow, the NAD + domain in a lighter shade, and the NAD+ molecules by red sticks. This figure and other structure figures were prepared using the program PyMOL [56].

19

Journal Pre-proof Figure 4. Comparison of Met257 (TcUH, green) with its corresponding ones (PpUH, blue, BsUH, yellow) in the homologous structures.

Figure 5. Superposition of TcUH monomers. Monomer A in green, monomer B in cyan, monomer C in magenta and monomer D in yellow, the NAD + domain in a lighter shade. (A) Superposition of the entire monomers, (B) Superposition of the

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single core domains and (C) Superposition of the single NAD+ domains.

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Figure 6. Relative orientations between domains. (A) Monomer A and (B) Monomer

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C. The central ("fixed") domains are shown in blue and the NAD + ("mobile") domains

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are shown in red, "bending regions" are shown in green and the positions of the perpendicular rotation axis are pointed in magenta. Figure produced by Dyndom

na

lP

[43].

Figure 7: Superposition between TcUH monomers A (green) and C (magenta) in

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which the NAD+ domains are in light colors. Electrostatic interactions present only in monomer A are colored blue and the ones present only in monomer C are colored red.

Figure 8. Superposition of TcUH monomer A to homologue structures. TcUH (monomer A) in green, 1UWK (monomer A) in yellow, 1X87 (monomer A) in purple and 2FKN (monomer A) in blue. The contouring red ellipse highlights the two extra αhelices in TcUH.

20

Journal Pre-proof Figure 9. Non-deleterious (blue) and deleterious (red) human UH mutations in their corresponding positions at TcUH (most similar structure to date).

Figure Legends (supplementary)

Figure S1. The crystal indicated by an arrow, with approximate dimensions of 240 ×

of

50 μm, provided the best dataset.

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Figure S2. Structural alignment of the amino acid sequences of the enzymes TcUH,

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PpUH, GkUH and BsUH together with the sequence of HsUH. Violet rectangles

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indicate regions that were not modeled in the respective crystallographic structures. Turquoise squares indicate residues that are mutated in PpUH structure. Blue stars

lP

indicate residues that make contacts with NAD+ in the 4 monomers. Yellow stars

na

indicate the residues that make contacts with the NAD+ in the homologues and that are different in the TcUH. Green triangles indicate residues that make contacts with

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the urocanate in PpUH. β-strands and the α-helices are indicated.

Tables Legends

Table 1. Statistics and additional values of data collection and structure refinement. Data in parenthesis are for the highest-resolution shell.

Table 2. RMSD values (Å) between Cα’s of the superposed structures. In the cells, the values are for: complete monomer (underline), core domain (italics) and NADbinding domain (regular).

21

Journal Pre-proof Table 3. Interface area and number of electrostatic interactions between domains (NAD+ and core).

Table 4. RMSD values of the superposition of TcUH monomers A and C to Pseudomonas putida (PDB ID: 1UWK), Geobacillus kaustophilus (PDB ID: 1X87) and Bacillus subtilis (PDB ID: 2FKN) structures. The number of residues selected

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with the program is also indicated.

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Declaration of interests

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The authors declare no competing interests.

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Journal Pre-proof

P21 84.61, 134.79, 113.40 90, 93.25, 90

0.2181 0.2546

-p

ro

0.002 0.544

na

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113.22 - 2.16 (2.22-2.16) 128119 (9011) 7.3 (73.7) 15.52 (2.05) 94.3 (84.2) 5.7 (4.1)

51.825 52.132 42.145 51.819

re

lP

Crystal Parameters Space group Unit. Cell dimension a, b, c (Å) α, β, γ (°) Data Colletion Resolution range (Å) Unique reflections Rmerge Mean [I/σ(I)] Completeness (%) Multiplicity Refinament Rwork Rfree Root-mean-square deviation Length bonds (Å) Bond angles (°) Average B-factor (Å2) Overall Macromolecules Solvent Ligands Ramachandran statistics (%) Most favored Additional allowed Generously allowed Outliers

90.1 9.3 0.3 0.2

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Table 1. Statistics and additional values of data collection and structure refinement. Data in parenthesis are for the highest-resolution shell.

31

Journal Pre-proof Monomer D C B 1.144 - 0.382 - 0.526 3.572 - 0.392 - 0.684 0.582 - 0.460 - 0.320 A 0.991 - 0.468 - 0.528 3.345 - 0.420 - 0.667 B 3.258 - 0.460 - 0.827 C -

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Table 2. RMSD values (Å) between Cα’s of the superposed structures. In the cells, the values are for: complete monomer (underline), core domain (italics) and NADbinding domain (regular).

32

Journal Pre-proof Monomer A B C D

Interface area (Å) 1043.1 1121.5 1261.0 532.9

Number of electrostatic interactions (HB + SB) 23 16 11 2

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Table 3. Interface area and number of electrostatic interactions between domains (NAD+ and core).

33

Journal Pre-proof RMSD (Å) / N. Residues Monomer A Monomer C 1.505 / 417 1.216 / 506 1.275 / 451 1.490 / 337 1.521 / 414 1.293 / 513

Enzyme / Organism PpUH (monomer A) GkUH (monomer A) BsUH (monomer A)

Jo ur

na

lP

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Table 4. RMSD values of the superposition of TcUH monomers A and C to Pseudomonas putida (PDB ID: 1UWK), Geobacillus kaustophilus (PDB ID: 1X87) and Bacillus subtilis (PDB ID: 2FKN) structures. The number of residues selected with the program is also indicated.

34

Journal Pre-proof Sheila Boreiko: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing - Original draft preparation, Writing - Review & Editing, Visualization. Marcio Silva: Conceptualization, Methodology, Validation, Resources,

Supervision.

Ariel

M.

Silber:

Conceptualization,

Methodology,

Validation, Resources, Writing - Original draft preparation, Writing - Review & Editing, Supervision, Project administration, Funding acquisition. Raíssa F. P. Melo: Conceptualization, Methodology, Validation, Investigation, Writing - Original draft Writing

-

Review

&

Editing,

Visualization.

of

preparation,

Jorge

Iulek:

ro

Conceptualization, Methodology, software, Validation, Formal analysis, Investigation,

-p

Data Curation, Resources, Writing - Original draft preparation, Writing - Review &

Jo ur

na

lP

re

Editing, Supervision, Project administration, Funding acquisition.

35

Journal Pre-proof Highlights 

TcUH is the first UH from eukaryotes to have its structure solved



Each TcUH monomer has two domains and one NAD+ molecule



Crystal structure reveals different conformations of monomers



Different monomer conformations should reflect protein dynamics in catalysis



Inferences to human UH deleterious and non-deleterious mutants could be

Jo ur

na

lP

re

-p

ro

of

made

36

Figure 1

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Figure 11