Methylglyoxal detoxification by an aldo-keto reductase in the cyanobacterium Synechococcus sp. PCC 7002

Abstract

Aldo-keto reductases (AKRs) are a superfamily of enzymes that reduce aldehydes and ketones, and have a broad range of substrates. An AKR gene, sakR1, was identified in the cyanobacterium Synechococcus sp. PCC 7002. A mutant strain with sakR1 inactivated was sensitive to glycerol, a carbon source that can support heterotrophic growth of Synechococcus sp. PCC 7002. It was found that the sakR1 null mutant accumulated more toxic methylglyoxal than the wild-type when glycerol was added to growth medium, suggesting that SakR1 is involved in the detoxification of methylglyoxal, a highly toxic metabolite that can damage cellular macromolecules. Enzymic analysis of recombinant SakR1 protein showed that it can efficiently reduce methylglyoxal with NADPH. Based on immunoblotting, SakR1 was not upregulated at an increased cellular methylglyoxal concentration. A pH-dependent enzyme-activity profile suggested that SakR1 activity could be regulated by cellular pH in Synechococcus sp. PCC 7002. The broad substrate specificity of SakR1 implies that SakR1 could play other roles in cellular metabolism. INTRODUCTIONCyanobacteria are a diverse group of prokaryotes that carry out oxygenic photosynthesis and grow photoautotrophically. Some cyanobacteria can also grow heterotrophically in the presence of organic substrates (Rippka et al., 1979). The substrates that support heterotrophic growth of cyanobacteria include the sugars fructose and glucose. However, in the cyanobacterium Synechococcus sp. PCC 7002, glycerol is the only substrate that supports heterotrophic growth. A high concentration of exogenous glycerol is inhibitory to cyanobacterial growth, and it has been observed that glycerol-dependent growth of Synechococcus sp. PCC 7002 is only established after a period of adaptation to glycerol.Although the pathway of glycerol metabolism has not been studied in cyanobacteria in detail, it is expected that it would be the same as that of other bacteria such as Escherichia coli, since, based on cyanobacterial genome sequences, cyanobacterial cells have all the genes to encode the enzymes required for glycerol metabolism (www.kazusa.or.jp/cyanobase; J. Zhao and D. A. Bryant, unpublished data). The inhibitory effect of glycerol to cyanobacterial growth could largely be due to toxic products of glycerol metabolism. One highly toxic product of cellular glycerol metabolism is methylglyoxal (Freedberg et al., 1971), which is an electrophile and reacts with cellular macromolecules such as proteins and DNA (Ferguson et al., 1998). One of the pathways leading to methylglyoxal production in most cells is the formation of methylglyoxal from dihydroxyacetone phosphate (DHAP) enzymically or non-enzymically (Ferguson et al., 1998;Kalapos, 1999). In most cells, detoxification of methylglyoxal is through glyoxalase I-II systems (Inoue & Kimmura, 1995;Kalapos, 1999). Another important mechanism is reduction of methylglyoxal by aldo-keto reductases (AKRs), which catalyse the formation of acetol from methylglyoxal (Kalapos, 1999).AKRs are a large supe...

A role for trypanosomatid aldo-keto reductases in methylglyoxal, prostaglandin and isoprostane metabolism

“…Subsequent characterisation of glyoxal and methylglyoxal showed them to follow Michaelis–Menten kinetics (Figure 2A,B). Notably, methylglyoxal had a K m of 0.24 mM and k cat / K m (1.95 × 10 4  M s −1 ) which is similar to that of SakRI (1.1 × 10 4  M −1  s −1 ), a cyanobacterial enzyme involved in methylglyoxal detoxification [18].

Figure 2.Testing benznidazole as a substrate or inhibitor of recombinant Tc AKR.( A ) Benznidazole as a substrate of TcAKR, DMSO (open circles), 60 µM benznidazole (closed circles), 60 µM phenylglyoxal (open squares).

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“…All four enzymes across three representative species possess activity with ketoaldehydes with specificity constants ( k cat / K m ) in the order phenylglyoxal > methylglyoxal > glyoxal. Notably, all four enzymes had specificity constants for methylglyoxal (range 1.1–5.7 [×10 4 ] M s −1 ) that are similar to that of SakRI (1.1 × 10 4  M −1  s −1 ), a bacterial enzyme that has been implicated in methylglyoxal metabolism [18]. The specificity constant for Tb PGFS with methylglyoxal (3.8 × 10 4  M −1  s −1 ) is an order of magnitude lower than that reported with PGH 2 (7 × 10 5  M −1  s −1 ) [30], whereas the reverse is observed for Li PGFS2 with methylglyoxal (5.7 × 10 4  M −1  s −1 ) preferred over PGH 2 (8.8 × 10 3  M −1  s −1 ) [20].…”

“…The K m values are thus consistent with a role in defence against carbonyl stress and are at the lower range of K m values reported for broad specificity methylglyoxal reductases from Aspergillus (1.43 and 15.4 mM) [45], Saccharomyces (5.9 mM) [46], Kluyveromyces (0.43 and 1.6 mM) [47] and Synechococcus sp. (0.08 mM) [18]. …”

“…Members of this AKR superfamily, characterised by their ability to reduce aldehydes and/or ketones, have been identified in all domains of life [17]. The activity of these enzymes requires either NADH and/or NADPH to reduce a variety of substrates, including, but not limited to aldoses, steroids, methylglyoxal [18] or prostaglandins [19]. In the trypanosomatid parasites Leishmania spp.…”

The role of rice aldo/keto reductases in the detoxification of reactive carbonyls and their use to create stress resistant transgenic plants

“…12). In cyanobacteria (Synechococcus PCC 7002) an AKR null-mutant strain accumulated more toxic MG than the wild type and was unable to efficiently detoxify exogenous MG compared to the non-mutant strain (Xu et al 2006). Our result is important in the demonstration of the conserved function for these proteins; a rice AKR protein is able to detoxify MG in a bacterial organism by complementing the function of the endogenous proteins.…”