Characterization of a Novel Putative S-Adenosylmethionine Decarboxylase-Like Protein from <i>Leishmania donovani</i>

<div><p>In addition to the S-adenosylmethionine decarboxylase (AD) present in all organisms, trypanosomatids including <i>Leishmania spp.</i> possess an additional copy, annotated as the putative S-adenosylmethionine decarboxylase-like proenzyme (ADL). Phylogenetic analysis confirms that ADL is unique to trypanosomatids and has several unique features such as lack of autocatalytic cleavage and a distinct evolutionary lineage, even from trypanosomatid ADs. In <i>Trypanosoma</i> ADL was found to be enzymaticaly dead but plays an essential regulatory role by forming a heterodimer complex with AD. However, no structural or functional information is available about ADL from <i>Leishmania spp</i>. Here, in this study, we report the cloning, expression, purification, structural and functional characterization of <i>Leishmania donovani</i> (<i>L. donovani</i>) ADL using biophysical, biochemical and computational techniques. Biophysical studies show that, <i>L. donovani</i> ADL binds S-adenosylmethionine (SAM) and putrescine which are natural substrates of AD. Computational modeling and docking studies showed that in comparison to the ADs of other organisms including human, residues involved in putrescine binding are partially conserved while the SAM binding residues are significantly different. <i>In silico</i> protein-protein interaction study reveals that <i>L. donovani</i> ADL can interact with AD. These results indicate that <i>L. donovani</i> ADL posses a novel substrate binding property and may play an essential role in polyamine biosynthesis with a different mode of function from known proteins of the S-adenosylmethionine decarboxylase super family.</p></div

Phylogenetic analysis showing evolutionary patterns of AD and ADL in trypanosomatids.

<p>Amino acid sequences were retrieved from the Swiss-Prot gene data base and phylogenetic tree was constructed using MEGA 5.0 software showing the different clusters of AD and ADL.</p

Multiple sequence alignment of the amino acid sequences of AD and ADLs of <i>L.</i> donovani, L. infantum, L. major, L. brazilensis, T. brucei and T. cruzi and human AD.

<p>Based on this alignment, the residues of human AD involved in autocatalysis are shown by a green asterisk; SAM positioning by red asterisk; SAM binding by brown asterisk and putrescine binding shown in cyan asterisk. Molecular docking studies of SAM and putrescine with homology model of L. <i>donovani</i> ADL suggest that SAM binding residues are not conserved while putrescine binding residues are found partially conserved. The residue comprising SAM binding pocket and involved in H-bond interaction with SAM in <i>L. donovani</i> ADL are enclosed in blue boxes. Putrescine binding residues of <i>L. donovani</i> ADL are represented by black boxes. Alignment is made with the help of Espript 2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065912#pone.0065912-Gouet1" target="_blank">[54]</a>.</p

Conformation profiles of ADL as analysed by fluorescence and far-UV CD spectroscopy.

<p>(A) Intrinsic tryptophan fluorescence profile of <i>L. donovani</i> ADL shows emission maxima at 341 nm, indicating tryptophans are partially exposed. (B) Far-UV CD spectra (260 nm-200 nm) of <i>L. donovani</i> ADL protein shows ADL is comprised of mixture of α-helix and β-sheet. (C) Thermal denaturation curve (θ₂₂₂) of <i>L. donovani</i> ADL showing co-operative unfolding with midpoint at ∼70°C. (D) Far-UV CD spectra of <i>L. donovani</i> ADL at pH range (4–9) showing maximum stability near to biological pH (7.0).</p

Putrescine binding analysis by fluorescence and far-UV CD spectroscopy.

<p>(A) Effect of increasing concentration of putrescine on tryptophan fluorescence shows putrescine quench <i>L. donovani</i> ADL in non-interpretable manner. (B) Far-UV CD spectra with increasing concentration of putrescine show change in secondary structure with increasing putrescine concentration. (C) Change in secondary structure, with increasing concentration of putrescine (0–200 µM) was monitored by molar ellipticity value at θ₂₂₂, curve showing change in secondary structure with increasing concentration of putrescine upto 50 µM. (D) Limited proteolysis with increasing concentration of putrescine (10–90 µM) also does not show effect on folding pattern of <i>L. donovani</i> ADL. (E) Thermal denaturation profiles of native ADL (black), <i>L. donovani</i> ADL in complex with putrescine (blue), <i>L. donovani</i> ADL in complex with SAM (green) and <i>L. donovani</i> ADL in complex with both SAM and putrescine (red) reveals substrates binding causes decrease in thermal stability of <i>L. donovani</i> ADL.</p

Homology modeling and prediction of SAM binding site.

<p>(A) Cartoon representation of the homology model of <i>L. donovani</i> ADL (cyan), superimposed on to the crystal structure of human AD (pink). Ligands observed in the human structure, SAMe (red) and putrescine (red) are also shown, as are the docked SAM and putrescine (yellow) to <i>L. donovani</i> ADL. Figure prepared with the help of Chimera 1.6.1. (B) Surface representation of the <i>L. donovani</i> ADL monomer showing the five binding pockets of SAM as predicted by Q-site prediction server. The different pockets are colored red, yellow, green, magenta and blue. The active site where SAM docked successfully is shown in inset (red). Figures are generated with the aid of Pymol molecular visualization tool <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065912#pone.0065912-The1" target="_blank">[55]</a>.</p

Over-expression, purification, size exclusion chromatography and trypsin digestion of of <i>L.</i><i>donovani</i> ADL.

<p>(A) 12% SDS PAGE of the purified <i>L. donovani</i> ADL. M-unstained protein marker; lane 1-unduced; lane 2-induced lane 3-load; lane 4-flow through; lane 5-equilibration; lane 6-wash; lane 7-elution of <i>L. donovani</i> ADL showing single band after metal affinity chromatography. (B) Size exclusion chromatography profile of <i>L. donovani</i> ADL showing protein elution at 10.4 ml on a Superdex 75 column, corresponding to 66 kDa molecular weight i.e. dimer. (C) Confirmation of the dimer by Native Polyacrylamide Gel Electrophoresis. (D) Trypsin assisted limited proteolysis analysis of <i>L. donovani</i> ADL at different time intervals showing that trypsin has no effect on <i>L. donovani</i> ADL.</p

Showing interaction of putrescine with residues in ADL and their comparison with interaction involved in putrescine binding in human AD.

<p>Showing interaction of putrescine with residues in ADL and their comparison with interaction involved in putrescine binding in human AD.</p

Binding site of SAM and putrescine obtained by molecular docking.

<p>(A) Cartoon representation of homology model of <i>L. donovani</i> ADL (cyan) with the docked SAM (cyan), superimposed on SAM-bound crystal structure of human AD (pink) illustrating the differences in relative SAM binding positions. LIGPLOT diagram showing the interactions of the docked SAM with the ADL residues are shown in the bottom panel (B)Cartoon representaton of putrescine (cyan) docked to the ADL homology model. For comparison, the corresponding region of the human AD structure is also shown, with its putrescine (pink). The bottom panel shows LIGPLOT representation of the interaction between putrescine and ADL.</p

Showing AD-ADL interaction in trypanosomatids and their respective interaction score.

<p>Showing AD-ADL interaction in trypanosomatids and their respective interaction score.</p