Structural studies of Old Yellow Enzyme of Leishmania braziliensis in solution

https://doi.org/10.1016/j.abb.2018.11.009Get rights and content

Highlights

  • LbOYE is an α-helical, monomeric and subtly elongated protein.

  • A low-resolution model for a whole LbOYE is proposed.

  • LbOYE shares structural properties with TcOYE orthologue.

  • LbOYE has a lower structural stability than TcOYE.

  • Menadione interacts with LbOYE and TcOYE with similar affinities.

Abstract

First described in yeast in 1932 by Christian & Warburg, the Old Yellow Enzyme (OYE) (EC 1.6.99.1) has aroused the interest of the scientific community regarding its high ability to catalyze stereoselective reactions of α/β-unsaturated carbonyl compounds with important industrial applications. In addition, the OYE family of proteins has been found in different organisms, such as plants, bacteria and protozoa, but not in mammals, which makes it an excellent candidate for a functional and molecular study aimed at more effective therapies with fewer undesirable side effects. Several OYE orthologues have been characterized; however, the real physiological role for most members of this family of proteins remains a mystery. In this paper, we present the structural studies of the OYE of Leishmania braziliensis. The findings are discussed in comparison with OYE of Trypanosoma cruzi, revealing some biophysical differences. The main differences are related to their chemical and thermal stabilities and behavior in solution. In addition, the L. braziliensis OYE shape is more elongated than that of the T. cruzi orthologue. Despite this, the active sites of these enzymes do not appear to have major differences, since their interactions with the substrate menadione occur with an affinity of the same order of magnitude, revealing that the binding sites in both proteins are essentially similar.

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.

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