Background:The transglucosidase GBD-CD2 shows a unique ␣-(132) branching specificity among GH70 family members when catalyzing dextran glucosylation from sucrose. Results: The truncated form ⌬N 123 -GBD-CD2 was biochemically studied and structurally characterized at 1.90 Å resolution. Conclusion: Dextran recognition and regiospecificity clearly involves a residue in subsite ϩ1. Significance: This is the first three-dimensional structure of a GH70 enzyme that reveals determinants of ␣-(132) linkage specificity.
2,4-Dihydroxybutyric acid (DHB) is a molecule with considerable potential as a versatile chemical synthon. Notably, it may serve as a precursor for chemical synthesis of the methionine analogue 2-hydroxy-4-(methylthio)butyrate, thus, targeting a considerable market in animal nutrition. However, no natural metabolic pathway exists for the biosynthesis of DHB. Here we have therefore conceived a three-step metabolic pathway for the synthesis of DHB starting from the natural metabolite malate. The pathway employs previously unreported malate kinase, malate semialdehyde dehydrogenase and malate semialdehyde reductase activities. The kinase and semialdehyde dehydrogenase activities were obtained by rational design based on structural and mechanistic knowledge of candidate enzymes acting on sterically cognate substrates. Malate semialdehyde reductase activity was identified from an initial screening of several natural enzymes, and was further improved by rational design. The pathway was expressed in a minimally engineered Escherichia coli strain and produces 1.8 g l−1 DHB with a molar yield of 0.15.
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