The Crystal Structure of a Homodimeric Pseudomonas Glyoxalase I Enzyme Reveals Asymmetric Metallation Commensurate with Half‐of‐Sites Activity

Abstract

The Zn inactive class of glyoxalase I (Glo1) metalloenzymes are typically homodimeric with two metal-dependent active sites. While the two active sites share identical amino acid composition, this class of enzyme is optimally active with only one metal per homodimer. We have determined the X-ray crystal structure of GloA2, a Zn inactive Glo1 enzyme from Pseudomonas aeruginosa. The presented structures exhibit an unprecedented metal-binding arrangement consistent with half-of-sites activity: one active site contains a single activating Ni(2+) ion, whereas the other contains two inactivating Zn(2+) ions. Enzymological experiments prompted by the binuclear Zn(2+) site identified a novel catalytic property of GloA2. The enzyme can function as a Zn(2+) /Co(2+) -dependent hydrolase, in addition to its previously determined glyoxalase I activity. The presented findings demonstrate that GloA2 can accommodate two distinct metal-binding arrangements simultaneously, each of which catalyzes a different reaction.

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

“…Metal titration studies of PDO with Ni 2+ and Cu 2+ indicated that 1 mole of metal per mole dimeric enzyme could optimize enzymatic activity ( Figure 6B), suggesting a tight binding of the metal, as well as one functional active site (possibly two active sites per dimeric enzyme as PDO exhibits homodimeric quaternary structure as previously mentioned). These data are consistent with previous reports on other Glo1 (such as E. coli Glo1, P. aeruginosa GloA1, GloA2, and many others), which also find that the metal per dimeric enzyme ratio is approximately one [19,24,44,68]. The pH dependency of PDO activity was also determined ( Figure 6C).…”

“…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]. In addition, deletional mutagenesis experiments have recently provided insight into the underlying structural factors involved in metal selectivity among the Glo1 enzymes [45].…”

Structure of the novel monomeric glyoxalase I fromZea mays

“…Even if the protein were able to bind a metal cofactor using the His27, Gln226 and Glu78 triad, an enzyme catalyzing the same reaction with two different active sites seems to be highly unlikely. On the other hand, the observation that site B seems to be able to bind glutathione conjugates opens the possibility of ZmGLX1 activity being noncompetitively affected by such molecules, probably as part of a yet to be discovered regulatory mechanism or a different catalytic activity such as that recently discovered for Glo1 from P. aeruginosa (Bythell-Douglas et al, 2015). These features are not uncommon in this protein family.…”

“…Glyoxalase I enzymes from numerous organisms have been biochemically characterized, including bacteria, plants, yeast, animals and protozoan parasites He et al, 2000;Sukdeo et al, 2004;Deswal & Sopory, 1991;Espartero et al, 1995;Mustafiz et al, 2014;Marmstå l et al, 1979;Gomes et al, 2005;Martins et al, 2001;Aronsson et al, 1978;Cameron et al, 1997;Akoachere et al, 2005;Ariza et al, 2006;Greig et al, 2006). However, only six glyoxalases I have been structurally described thus far, namely the enzymes from Homo sapiens (Aronsson et al, 1978;Cameron et al, 1997), Escherichia coli (He et al, 2000), Leishmania major (Ariza et al, 2006), Mus musculus (Kawatani et al, 2008), Clostridium acetobutylicum and Pseudomonas aeruginosa (Bythell-Douglas et al, 2015). All of them display a characteristic homodimeric quaternary structure, in which each monomer comprises two domains that interact to generate a continuous eightstranded -sheet with another domain present in the opposite monomer (Fig.…”

Glyoxalase biochemistry

“…One of these Glo1 is shorter in length and has higher amino acid sequence homology to the E. coli and the first identified P. aeruginosa Glo1. This Glo1 exhibits the characteristics of the Ni 2+ -activation class enzymes, with no evidence for Zn 2+ activation (the X-ray structure of this enzyme has recently been reported) (70). The third Glo1 has a longer amino acid sequence and a higher amino acid sequence homology to the human and the Pseudomonas putida Glo1, which are known Zn 2+ -activation class enzymes.…”