Regulatory Role of CGS in Methionine Biosynthesis

Being the first enzyme specific for methionine biosynthesis, CGS is expected to play an important regulatory role in methionine metabolism. Nevertheless, there is no evidence for the regulation of CGS activity by feedback inhibition loops (Ravanel et al., 1998a, 1998b). Instead, the level of CGS is regulated by either methionine, or its catabolic product(s), through posttranscriptional and posttranslational mechanisms (Amir et al., 2002; Chiba et al., 1999; Hacham et al., 2002; Onouchi et al., 2005). CGS polypeptides (without their plastid transit peptides) in mature plants contain a region of ~100 amino acids at the N-terminus, which is not present in bacterial CGS enzymes and is also not essential for CGS catalytic activity (Hacham et al., 2002). A series of Arabidopsis mto1 mutants, which accumulates up to 40-fold higher methionine in young tissues than in wild-type plants, were shown to be attributed to mutations in the region encoding this N-terminal domain of CGS (Chiba et al., 1999; Inaba et al., 1994). The mto1 mutations are located in a specific subdomain (called the MTO1 region), which is conserved in the CGS genes of all plant species analyzed so far. This region apparently acts to downregulate CGS mRNA level when either the level of methionine or any of its catabolic products rise, via a mechanism that apparently involves specific nascent amino acids translated from this mRNA region (Chiba et al., 1999; Inaba et al., 1994).

Several lines of evidence suggest that the control of methionine synthesis cannot be solely explained by the posttranscriptional regulation through the MTO1 region. No inverse correlation between methionine and CGS mRNA levels were evident in transgenic Arabidopsis plants overexpressing the endogenous CGS, as well as in an Arabidopsis mutant with reduced methionine catabolism (Goto et al., 2002; Kim et al., 2002). Moreover, in contrast to Arabidopsis, no evidence supporting a control of CGS mRNA level by methionine was obtained in potato plants, although the MTO1 region in the potato CGS gene is highly conserved with that of the Arabidopsis counterpart (Kreft et al., 2003). These observations suggest that the regulatory function of the MTO1 region requires interactions with additional factors that are not present in all tissues and/or are not conserved in all plant species. Notably, Arabidopsis and potato also differed in their response to constitutive CGS overexpression. While CGS overexpression in transgenic Arabidopsis plants caused an approximately 4–20-fold increase in methionine (Gakiere et al., 2000; Kim et al., 2002), no increase in methionine was obtained in transgenic potato plants (Kreft et al., 2003). Whether these differences are due to genetic or physiological factors remains to be elucidated.

The regulatory role of the N-terminal region of the mature plant CGS enzyme was also studied by either constitutive expression of a full-length Arabidopsis CGS or its deletion mutant lacking this region, but still containing the plastid transit peptide, in transgenic tobacco plants (Hacham et al., 2002). Expression of the Arabidopsis CGS without its N-terminal region caused significant increases of ethylene and dimethyl sulfide, two catabolic products of methionine, over plants expressing the full-length Arabidopsis CGS (Hacham et al., 2002). However, methionine and SMM levels, although increased over wild-type plants, did not differ significantly between transgenic plants expressing the different CGS constructs. Since the expression levels of the transgenic CGS polypeptides were comparable between the two sets of these transgenic plants, it was suggested that the N-terminal region of CGS might also regulate methionine metabolism by a posttranslational mechanism (Hacham et al., 2002).