Deciphering the number and location of active sites in the monomeric glyoxalase I of Zea mays

Abstract

Detoxification of methylglyoxal, a toxic by‐product of central sugar metabolism, is a major issue for all forms of life. The glyoxalase pathway evolved to effectively convert methylglyoxal into d‐lactate via a glutathione hemithioacetal intermediate. Recently, we have shown that the monomeric glyoxalase I from maize exhibits a symmetric fold with two cavities, potentially harboring two active sites, in analogy with homodimeric enzyme surrogates. Here we confirm that only one of the two cavities exhibits glyoxalase I activity and show that it adopts a tunnel‐shaped structure upon substrate binding. Such conformational change gives rise to independent binding sites for glutathione and methylglyoxal in the same active site, with important implications for the molecular reaction mechanism, which has been a matter of debate for several decades. Database Structural data are available in The Protein Data Bank database under the accession numbers https://www.rcsb.org/structure/6bnn, https://www.rcsb.org/structure/6bnx, and https://www.rcsb.org/structure/6bnz.

Two-Substrate Glyoxalase I Mechanism: A Quantum Mechanics/Molecular Mechanics Study

“…For ZmGlxI, we used the 1.55 Å crystal structure of the V278E mutant (PDB ID: 6BNN), 11 which exhibits the same activity as the wild-type enzyme. 11 It contains a single subunit, with 296 residues (the first 14 residues from the N terminal are missing in the crystal structure and were excluded in our model), one Co ion, one H-SG, two formate ions, and 223 crystal waters. ZmGlxI contains two activesite cavities, but only one of them harbors a Co(II) ion.…”

“…The other end of the tunnel is solventaccessible and can allow small molecules of the size of water and MG to reach the active-site metal coordination sphere. 11 As for HuGlxI, the MG was inserted into the active site, by replacing the two crystal water molecules coordinated to the Co ion (HOH-497 and HOH-532; cf. Figure 1b).…”

“…8,9 However, monomeric GlxIs have also been reported in several species, e.g., in Zea mays (corn). 10,11 For the catalytic activity, GlxI requires divalent metal ions (Zn(II), Ni(II), Co(II), or other metal ions, depending on the organism). It has been suggested that the metal specificity is related to the amino-acid sequence and the length of the enzyme.…”

“…In this study, we employ QM/ MM methods to study the proposed two-substrate reaction mechanism for ZmGlxI. 11 We also investigate the possibility of the two-substrate mechanism for HuGlxI, allowing us to compare enzymes from plants or animals with different metal preferences (HuGlxI and ZmGlxI are Zn(II)-active and -inactive, respectively).…”

Preliminary Characterization of a Ni2+-Activated and Mycothiol-Dependent Glyoxalase I Enzyme from Streptomyces coelicolor

“…The molecular structure of the Clostridium acetobutylicum Glo1 (Ni 2+ -activation class) is also homodimeric, but each of the two active sites is formed by protein residues solely from a single subunit [34]. Furthermore, larger single chain Glo1 are also known [35][36][37][38][39][40][41], and the recent report on the X-ray structure of the maize enzyme shows two metal-binding sites formed by the single protein chain with one site being catalytically active [42]. For the homodimeric Glo1, as well as the maize enzyme, detailed studies employing metal activation, NMR and X-ray experiments have provided unambiguous evidence that only a single active site is required for maximal activity [42][43][44].…”