Trypanosomatid GLO enzymes are monomeric and contain a zinc
Trypanosomatid GLO2 enzymes are monomeric and contain a zinc–iron binuclear metal center (Irsch and Krauth-Siegel, 2004, Silva et al., 2008), similar to all other glyoxalases II. As mentioned, in T. brucei there are two GLO2 genes but only one encodes an enzyme with glyoxalase II activity (Irsch and Krauth-Siegel, 2004, Wendler et al., 2009). So far, there is only one glyoxalase II-solved structure from a trypanosomatid, L. infantum (UniProt Q2PYN0), revealing that the substrate binds through the spermidine moiety of the trypanothione-thioester (Silva et al., 2008). This explains the absolute specificity of glyoxalase II toward the trypanothione-derived substrate (Irsch and Krauth-Siegel, 2004, Silva et al., 2008, Sousa Silva et al., 2005). Two residues in the L. infantum protein, Tyr291 and Cys294, are essential for substrate binding, and their replacement by Arg and Lys from the human enzyme that binds glutathione produced a mutant enzyme able to hydrolyze both the glutathione and trypanothione-derived thioesters (Barata et al., 2011).
The glyoxalase pathway was analyzed during the life histone deacetylase inhibitors of L. infantum (Sousa Silva et al., in press). This parasite has a complex life cycle, alternating between a promastigote form in the vector and an amastigote inside the mammalian host cells. In this parasite, the activity of both glyoxalase I and II is lower in exponentially growing promastigotes, increases during the stationary phase (enriched in infective forms) and further in amastigotes (Sousa Silva et al., in press). Interestingly, these parasites have an aldose reductase (EC 126.96.36.199; UniProt A4I342; Barata et al., 2009) responsible for the NADPH-dependent reduction of methylglyoxal. This enzyme's activity shows a different behavior during L. infantum life cycle stages, being higher in exponential growth phase parasites and lower at the stationary and amastigote stages (Sousa Silva et al., in press). Methylglyoxal catabolism seems to be tightly regulated in Leishmania; in the parasite's infectious forms, glyoxalase I and II increase their activities to compensate for the decrease of aldose reductase activity in order to reduce NADPH consumption, being in dire need of anti-oxidative defenses.
Conclusions The discovery of the enzymatic formation of lactic acid from methylglyoxal dates back from 1913 and was believed to be associated with an enzyme termed ketonaldehyde mutase or glyoxalase, the latter designation having become the accepted one (Dakin and Dudley, 1913, Neuberg, 1913). Only in 1951 was shown that two enzymes were needed for this process to occur and that glutathione was the required catalytic cofactor (Racker, 1951). The concept of a metabolic pathway defined by two enzymes emerged at this time while its association with detoxification (Thornalley, 1990) and anti-glycation defense of the genome and proteome (Gomes et al., 2005, Thornalley, 1996) is its presently accepted role. This functional defense role has been the rationale behind the possible use of the glyoxalase pathway as a therapeutic exploit, since its inhibition might lead to an increased methylglyoxal concentration, hence, cellular damages. However, 100 years after its discovery, research on the glyoxalase pathway in protozoan parasites, namely in P. falciparum and trypanosomatids, reveals a different landscape. In protozoan parasites, glutathione is not always the preferred co-factor, and in some cases the glyoxalase system is either absent or incomplete (Fig. 1). A comparative evolutionary analysis of glyoxalase I and II in human protozoan parasites revealed that both enzymes are very similar among members of the same group: trypanosomatids, apicomplexans, and enteric parasites (Fig. 2A and B). The human GLO1 and GLO2 are more closely related to T. gondii and P. falciparum proteins (44% identity for GLO1 and 40–45% for GLO2), being more divergent from the enteric parasites, lacking glyoxalase I, and possessing metallo-β-lactamase family proteins (Fig. 2). The evolution of protozoan parasites is mirrored in the glyoxalase pathway, which is likely to assume different roles in diverse organisms at different life stages. Also, functionally complementary and alternative pathways to fulfill a defensive role exist in these parasites (specifically the aldose reductase and methylglyoxal reductase), adding robustness to methylglyoxal detoxification and casting serious doubts on its value as a possible therapeutic target.