Crystal structure of ZmGLXI mutants in the path to elucidate the functional role of its site I and II (Póster)

CLARISA E. ALVAREZ; JAVIER M. GONZÁLEZ; ROMINA B. AGOSTINI; CARLOS S. ANDREO; SEBASTIÁN KLINKE; VALERIA CAMPOS-BERMUDEZ

Bahía Blanca

Congreso; XIII Reunión Anual de la Asociación Argentina de Cristalografía (AACr); 2017

Asociación Argentina de Cristalografía (AACr)

The glyoxalase system isubiquitous among all forms of life, due to its central role at relieving thecell from the accumulation of methylglyoxal (MG), a toxic metabolic by-product. Glyoxalase I (GLX1) acts byisomerizing the hemithioacetal formed by spontaneous combination of MG and glutathione(GSH) leading to the formation of S-lactoylglutathione, which is then hydrolyzedto D-lactate by glyoxalase II, regenerating GSH in the process [1]. The enzymesof this pathway are usually metalloenzymes that require metal ions foractivation. In higher plants, this pathway is thought to play an important rolein conferring tolerance to multiple abiotic and biotic stresses. We recently solvedthe first Zea mays GLX1 structure (ZmGLX1,PDB: 5D7Z) which crystallized in the P6(3)space group and diffracted to 1.73 A resolution. We located a bound Ni2+ ion, butnot the GSH substrate. ZmGLX1 is a single polypeptide comprising twointer-domain eight-stranded beta-sheets and giving rise to two cavities (sitesI and II). GLX catalytic active sites contain a transition metal ion displayingan octahedral geometry, with four conserved coordinating residues (His/Glu/Gln/Glu),completed by two solvation water molecules. ZmGLX1 site I retains all of these ligandswhile site II exhibits an incomplete metal ligand set, characterized by theabsence of a typical Glu residue. Instead, the presence of Val278 residuereplacing the usual Glu metal-ligand residue in site II suggests that ZmGLX1 isunable to properly bind the metal cofactor, and hence would lack glyoxalaseactivity on site II. The ZmGLX1 structure shows that there are no nearbyresidues in site II whose side chain could compensate for the absence of a Gluresidue in order to allow binding of an octahedrally-coordinated metal ion. Thus, to evaluate thefunctionality of each site we generated two point mutants in each metal site.Biochemical evaluation of the mutant protein on site I (ZmGLX1E144Q) revealedthat the Glu144 residue is essential to perform the enzymatic reaction. On theother hand, the mutant protein in site II where the Val278 residue was replacedby a Glu, aiming to regenerate the typical catalytic site (ZmGLX1V278E), doesnot show an activity enhancement; instead, it showed a small reduction inactivity. In order to understand this behavior, we obtained crystal structuresof both of these mutants in the presence of GSH and Co2+. While V278E crystalsdiffracted to 1.85 A resolution, crystallizing in the same space group as wild-typeZmGLX1, the E144Q mutant diffracted to 1.45 A but crystallized in P4(1)2(1)2. Data reduction and scaling wasperformed with Mosflm and Scala, respectively, while the initial phases wereobtained through Molecular Replacement using Phaser. After densitymodification, the atomic models were built into electron-density maps usingCoot and iteratively refined with Phenix, Buster and Refmac. For the firsttime, we could locate the GSH substrate in the site I of both ZmGLX1 mutants. Theweak density observed in the site II does not allow us to locate any knownligands. Future co-crystallization assays with relevant ligands will provide additionalinformation on the role of this site in GLX1 activity.References: [1] Yadav, SK; Singla-Pareek, SL; and Sopory, S K(2008). Drug Metabol. Drug Interact. 23: 51?68.[2] Turra GL; Agostini RB; Fauguel CM;PreselloDA; Andreo CS; González JM; Campos-Bermudez VA. Acta Crystallogr D BiolCrystallogr. 2015 Oct;71(Pt 10):2009-20..