Structural studies of Old Yellow Enzyme of Leishmania braziliensis in solution
Graphical abstract
Introduction
The Old Yellow Enzymes (OYE, EC 1.6.99.1) are flavin-dependent NAD(P)H (reduced nicotinamide adenine dinucleotide) oxidoreductases, which has been found in organisms such as fungi, bacteria, protozoa and plants, however, it is absent in mammals. The presence of canonical OYE in pathogenic protozoa, which can infect humans, make OYE a relevant molecular target for drug design against parasitic diseases [1].
The OYEs fold into an (α/β)8 barrel with the Flavin Mononucleotide (FMN) prosthetic group located in the major cavity of the active site, at the bottom of the barrel and accessible to the solvent (Fig. 1). The members of the OYE family basically differ by variations in the top barrel (capping subdomain – see Fig. 1), which controls the substrate accessibility to the active site [1]. They have low substrate specificity, and this makes it difficult to identify the genuine metabolic substrates. The lack of substrate selectivity is indicative of the detoxification role of OYE during oxidative stress. The flexibility of its active site plays a key role in allowing the active site to interact with molecules such as menadione, phenazine and β-lapachone [2,3], which are small compounds, and with large compounds such as komaroviquinone [4], 12-oxophytodionate, a Prostaglandin H2 (PGH2) substrate analog [5], as well as NAD(P)H that works as a coenzyme [6]. Although the physiological substrates are not known for most of the OYEs, these enzymes have been shown to catalyze the olefinic reduction of α,β-unsaturated carbonyl compounds by the use of NAD(P)H as a reducing agent [7]. Thus, different metabolic functions have been suggested for several OYE orthologues, such as response to oxidative stress in yeasts and biosynthesis of jasmonic acid in plants [1,[8], [9], [10]], among others [11].
The crystal structure of OYE of Trypanosoma cruzi (TcOYE) at 1.27 Å (PDB acc. no. 4E2D) and structures of other OYEs present a conserved structural scaffold and differences in the active-site, which has a high-mobility capping subdomain allowing interactions with large ligands [3,6]. Similar to other OYEs, TcOYE has 4 characteristic structural elements (Fig. 1). The N-terminus contains a β-hairpin (residues 10 to 19; TcOYE numbering) located in the lower portion of the barrel. Residues 105 to 164 (TcOYE numbering) constitute the capping subdomain, which participates in the formation of the largest active site pocket. Some OYE structures have an α-helix motif (residues 196 to 222; TcOYE numbering), which is related to substrate recognition. The C-terminus contains an inner α-helix (residues 336 to 341; TcOYE numbering) that also contributes to FMN binding [12] (Fig. 1). As highlighted by Murakami et al. [6], there is considerable divergence in the regions belonging to the capping subdomain of the TcOYE and other OYE family members. This divergence may reflect the versatility of the family members to the types and sizes of substrates identified for the OYEs [6].
In addition to the flexible structure, the versatility of the OYEs depends on the FMN prosthetic group, which is reduced by NAD(P)H and promotes a transfer of one or two electrons to the substrate during oxidative half-reaction. The bi-bi ping-pong mechanism allows the transfer of a hydride from reduced FMN and a proton from the solvent mediated by conserved histidine residues (H195-H/N198, TcOYE numbering) during asymmetrical reduction of C=C bonds [[13], [14], [15]]. OYEs are known for their high technological potential as biocatalysts promoting the enantioselective reduction of activated C=C bonds to generate up to two stereogenic centers [14,16].
Neglected tropical diseases (NTDs) are present in 149 countries. These diseases mainly affect the populations living in extreme poverty, without adequate sanitation and in close contact with infectious vectors and domestic and wild animals [17]. Leishmaniasis and trypanosomiasis are both listed as NTDs with recurrent frequency throughout the Brazilian territory. The development of more effective and less toxic drugs for the treatment of these diseases has been an ambitious goal of many research groups around the world [[18], [19], [20]]. However, finding a specific target in these parasites is a challenge. An encouraging course of action is the functional and molecular study of the OYE protein family. Thus, we present here a detailed structural description of Leishmania braziliensis OYE (LbOYE), the first OYE of the genus Leishmania to be described. In addition, we also present a structural characterization in comparison with TcOYE.
Section snippets
Sequence analysis
Alignment of the LbOYE protein sequence (XP_001563130) was performed with other members of the OYE family. Seven proteins were listed for this analysis, five belonging to the classical subfamily (LmOYE, TcOYE, OYE1, OPR1 and MR) and two belonging to the thermophilic-like subfamily (Chr-OYE3 and YqjM). The alignment was done using the Promals3D platform (http://prodata.swmed.edu/promals3d/promals3d.php) and edited by ESprit software [21].
Expression and purification
The LbOYE coding DNA was cloned into the pET28a expression
Analysis of protein sequences
The sequence alignment of LbOYE and TcOYE with some orthologue proteins from classical and thermophilic-like subfamilies is depicted in Fig. 2, and the matrix identity among these proteins is shown in Table 1. The identities in protein alignment were higher within the same OYE subfamily, as expected. The description of new members of the OYE family has grown rapidly in recent years, so there has been a need to regroup various homologues into subfamilies [10,31]. In this paper, we describe the
Conclusions
Here, we describe the structural characterization of the recombinant LbOYE in solution, the first OYE of the genus Leishmania to be described. In our studies, we set out to evaluate LbOYE in comparison with the TcOYE protein, highlighting similarities and structural differences. We showed that LbOYE and TcOYE share 46% identity in the amino acid sequence, a relatively moderate to high value, mainly when compared with other orthologous proteins. In addition, the secondary structure content
Acknowledgements
We are greatly indebted to FAPESP (2011/23110-0; 2014/07206-6 and 2017/07335-9), CNPq (471415/2013-8 and 303129/2015-8) and CAPES for financial support. We thank LNBio and LNLS for making available to us the AUC device and SAXS beamline, respectively.
References (36)
- et al.
Structural studies of the trypanosoma cruzi old yellow enzyme: insights into enzyme dynamics and specificity
Biophys. Chem.
(2013) - et al.
Characterization of YqjM, an old yellow enzyme homolog from Bacillus subtilis involved in the oxidative stress response
J. Biol. Chem.
(2003) - et al.
A novel stress-inducible 12-oxophytodienoate reductase from Arabidopsis thaliana provides a potential link between brassinosteroid-action and jasmonic-acid synthesis
J. Plant Physiol.
(2000) - et al.
X-ray structure of 12-Oxophytodienoate reductase 1 provides structural insight into substrate binding and specificity within the family of OYE
Structure
(2001) - et al.
Bisphosphonates as inhibitors of Trypanosoma cruzi hexokinase: kinetic and metabolic studies
J. Biol. Chem.
(2007) - et al.
Vaccine development against trypanosoma cruzi and chagas disease
Adv. Parasitol.
(2011) - et al.
Low resolution structural characterization of the Hsp70-interacting protein - Hip - from Leishmania braziliensis emphasizes its high asymmetry
Arch. Biochem. Biophys.
(2012) - et al.
Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems
Biophys. J.
(2002) Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing
Biophys. J.
(1999)- et al.
Calculation of hydrodynamic properties of globular proteins from their atomic-level structure
Biophys. J.
(2000)
PtII, PdIIand AuIII complexes with a thiosemicarbazone derived from diacethylmonooxime: structural analysis, trypanocidal activity, cytotoxicity and first insight into the antiparasitic mechanism of action
Eur. J. Med. Chem.
New developments in ’ene’-reductase catalysed biological hydrogenations
Curr. Opin. Chem. Biol.
The 1.3 Å crystal structure of the flavoprotein YqjM reveals a novel class of old yellow enzymes
J. Biol. Chem.
“New uses for an old enzyme” - the Old Yellow Enzyme family of flavoenzymes
Microbiology
A key role for old yellow enzyme in the metabolism of drugs by Trypanosoma cruzi
J. Exp. Med.
Structure of the inhibitor complex of old yellow enzyme from Trypanosoma cruzi
J. Synchrotron Radiat.
Antichagasic activity of komaroviquinone is due to generation of reactive oxygen species catalyzed by
Society
Structural insight into the stereoselective production of PGF2?? by old yellow enzyme from trypanosoma cruzi
J. Biochem.
Cited by (3)
Interaction between diterpene icetexanes and old yellow enzymes of Leishmania braziliensis and Trypanosoma cruzi
2024, International Journal of Biological MacromoleculesHeterobimetallic nickel(II) and palladium(II) complexes derived from S-benzyl-N- (ferrocenyl)methylenedithiocarbazate: Trypanocidal activity and interaction with Trypanosoma cruzi Old Yellow Enzyme (TcOYE)
2019, European Journal of Medicinal ChemistryCitation Excerpt :The ability of the ligand to block the hydride transfer to menadione has been verified by an assay in which the HFedtc ligand is added to the reaction medium after re-oxidation of FMN by menadione. In this assay, the ligand concentration used was 34 μM for HFedtc, which represents approximately twice the KD value for such ligand, and 50 μM for menadione, which is of the same order of the KD value of TcOYE for menadione [37]. The ligand HFedtc was obtained from the condensation reaction between benzylthiocarbazate and ferrocenocarboxaldehyde in equimolar amounts under reflux conditions in ethanol.