A Specialized Aspartokinase Enhances the Biosynthesis of the

The compatible solutes ectoine and hydroxyectoine are widely produced by bacteria as protectants against osmotic and temperature stress. L-Aspartate-beta-semialdehyde is used as the precursor molecule for ectoine/ hydroxyectoine biosynthesis that is catalyzed by the EctABCD enzymes. L-Aspartate-beta-semialdehyde is a central intermediate in different biosynthetic pathways and is produced from L-aspartate by aspartokinase (Ask) and aspartate-semialdehyde-dehydrogenase (Asd). Ask activity is typically stringently regulated by allosteric control to avoid gratuitous synthesis of aspartylphosphate. Many organisms have evolved multiple forms of aspartokinase, and feedback regulation of these specialized Ask enzymes is often adapted to the cognate biochemical pathways. The ectoine/hydroxyectoine biosynthetic genes (ectABCD) are followed in a considerable number of microorganisms by an ask gene (ask_ect), suggesting that Ask_Ect is a specialized enzyme for this osmoadaptive biosynthetic pathway. However, none of these Ask_Ect enzymes have been functionally characterized. Pseudomonas stutzeri A1501 synthesizes both ectoine and hydroxyectoine in response to increased salinity, and it possesses two Ask enzymes: Ask_Lys and Ask_Ect. We purified both Ask enzymes and found significant differences with regard to their allosteric control: Ask_LysC was inhibited by threonine and in a concerted fashion by threonine and lysine, whereas Ask_Ect showed inhibition only by threonine. The ectABCD_ask genes from P. stutzeri A1501 were cloned and functionally expressed in Escherichia coli, and this led to osmostress protection. An E. coli strain carrying the plasmid-based ectABCD_ask gene cluster produced significantly more ectoine/hydroxyectoine than a strain expressing the ectABCD gene cluster alone. This finding suggests a specialized role for Ask_Ect in ectoine/hydroxyectoine biosynthesis. The growth arrest of microbial cells observed under severe high-osmolarity conditions is correlated with the reduction in the amount of free cytoplasmic water (12) and the reduction or collapse of turgor (7, 65). To promote water reentry and retention under high-osmolarity growth conditions and to balance turgor, many microorganisms amass a selected class of organic osmolytes, the compatible solutes (31, 66). Microbial cells accumulate compatible solutes through either synthesis or uptake from environmental resources (7, 65, 69). They can employ them not only as effective osmostress protectants, but also as protectants against heat (8, 10, 13, 17, 26) and cold (2, 25, 33) stress. The tetrahydropyrimidines ectoine and 5-hydroxyectoine (Fig. 1) are an important class of compatible solutes (9, 45, 50). Biosynthesis of ectoine is catalyzed by L-2,4-diaminobutyric acid transaminase (EctB), N-␥-acetyltransferase (EctA), and ectoine synthase (EctC) (40, 44). A subset of the ectoine producers hydroxylate ectoine to 5-hydroxyectoine (9, 17, 48) …