Biochemical studies suggest that methionine biosynthesis is regulated by a competition between CGS and TS for their common substrate O-phosphohomoserine (Amir et al., 2002 and references therein). Plant TS enzymes possess approximately 250–500-fold higher affinity for O-phosphohomoserine than the plant CGS enzymes as measured by in vitro studies (Curien et al., 1998; Ravanel et al., 1998b). This indicates that most of the carbon and amino skeleton of aspartate should be channeled toward threonine rather than to methionine. Indeed, when the flux into the threonine/methionine branch of the heaspartate family was increased by overexpressing a bacterial feedback-insensitive AK in transgenic plants, threonine levels were greatly increased but methionine levels hardly changed (Ben Tzvi-Tzchori et al., 1996; Karchi et al., 1993; Shaul and Galili, 1992b). SAM, the immediate catabolic product of methionine, may buffer the competitive fluxes of threonine and methionine biosynthesis because it positively regulates TS activity (Curien et al., 1998).
Studies using transgenic plants support the biochemical studies for a competition between the threonine and methionine branch of the aspartate family pathway (Fig. 3.2). However, they also show that this competition is not simple. Reduction of CGS level by gene silencing or antisense approaches resulted in a 3.3–8.3-fold increase in threonine levels in transgenic Arabidopsis plants, while methionine levels were only slightly reduced (Kim and Leustek, 2000; Kim et al., 2002). In addition, reduction of TS activity due to a mutation in the TS gene (mto2–1 mutant) caused an ~16-fold reduction in threonine as well as a comparable ~22-fold increase in methionine in rosette leaves compared to wildtype Arabidopsis plants (Bartlem et al., 2000). More remarkable results were obtained when the TS levels were reduced by an antisense approach both in transgenic potato and Arabidopsis plants (Avraham and Amir, 2005; Zeh et al., 2001). In the TS antisense transgenic potato plants, threonine levels were only moderately reduced by up to ~45%, whereas methionine levels were dramatically increased by up to ~239-fold compared to nontransformed plants (Zeh et al., 2001). Similarly, in the TS antisense transgenic Arabidopsis plants, threonine levels were only moderately reduced by approximately 1.5–2.5-fold, while the levels of methionine increased by up to ~47-fold than in wild-type plants (Avraham and Amir, 2005). The results imply that the reduction in TS levels, rather than its activity as observed in the Arabidopsis mto2 mutant, causes either an increased flux of the carbon and amino skeleton from aspartate to methionine or a reduced rate of methionine catabolism.
The complex competition between the methionine and threonine branches of the aspartate family pathway was supported by additional studies. In the mto1–1 mutants, the significant increases in methionine were not associated with a significant reduction in threonine (Kim and Leustek, 2000). In addition, constitutive overexpression of CGS in transgenic Arabidopsis, potato, and tobacco plants caused significant increases in methionine levels, but no significant compensatory decreases in threonine levels (Gakiere et al., 2000; Hacham et al., 2002; Kim et al., 2002; Kreft et al., 2003). These results may be explained by a differential ratelimiting effect of O-phosphohomoserine, the common substrate for CGS and TS (Fig. 3.2), for threonine and methionine biosynthesis. The steady-state level of O-phosphohomoserine may be more rate limiting for methionine than for threonine biosynthesis. In addition, increased O-phosphohomoserine utilization by CGS may trigger an increase in the synthesis of this intermediate metabolite, rendering it nonlimiting for threonine biosynthesis. This assumption is supported by the analysis of Arabidopsis and potato plants expressing the antisense form of CGS. The level of O-phosphohomoserine in these plants was increased by ~22-fold in Arabidopsis, and from an undetectable level to 6.5 nmol/g fresh weight in potatoes, while the level of threonine increased only by ~8-fold in Arabidopsis, or was not increased in potato plants (Gakiere et al., 2000; Kreft et al., 2003).
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