2-Keto-4-hydroxybutyrate aldolase. Identification as 2-keto-4-hydroxyglutarate aldolase, catalytic properties, and role in the mammalian metabolism of L-homoserine

An Economical Method for Production of 2H,13CH3-Threonine for Solution NMR Studies of Large Protein Complexes: Application to the 670 kDa Proteasome

“…A simplification to the path taken by nature involves generating homoserine 3 via transamination of the keto acid, 2-keto-4-hydroxybutyrate (KHB, 2 ), as indicated in Figure 2, which summarizes the Thr biosynthesis route used here. The first critical step is condensation of 2 H, pyruvate 1b with 13 C-formaldehyde, catalyzed via the enzyme 2-keto-4-hydroxyglutarate aldolase [52], [53] (KHGA, also known as 2-keto-3-deoxy-6-phosphogluconate aldolase), a member of the pyruvate aldolase family that has been used in biocatalysis previously [54]. The product of this reaction (referred to as reaction 1 in what follows) is KHB 2 , in which the entire carbon chain for Thr is assembled with the correct deuteration and 13 C labeling pattern at C3 and C4.…”

“…Additionally, 2 H-pyruvate, 1b , is readily generated from pyruvate 1a via incubation with catalytic amounts of KHGA in D 2 O based buffer for 1 hour at pH 7.5. The equilibrium constant for the production of 2 from 1b is 250 M −1 [52] so that by adding excess pyruvate the reaction can be made to go to completion; in a mixture starting as 0.1 M formaldehyde and 0.2 M pyruvate 96% of the starting formaldehyde is predicted to be converted to KHB. Indeed, when depolymerized 13 C-paraformaldehyde (0.11 M) is mixed with a two-fold excess of pyruvate in the presence of KHGA the aldehyde signals in 1 H NMR spectra disappear, while those from KHB appear, with greater than 95% conversion (see Supporting Information S1).…”

“…The transamination of KHB 2 to homoserine 3 can reportedly be catalyzed by aspartate and alanine aminotransferases [52]. Aspartate aminotransferase (AAT, see Supporting Information S1) was the most efficient of several enzymes tested with 60% conversion to homoserine in 3 hours using 1.5 equivalents of Glu as the amino donor.…”

Peer Review #1 of "Investigation of microbial community interactions between Lake Washington methanotrophs using ­­­­­­­genome-scale metabolic modeling (v0.1)"

“…Reactions whose fluxes reached the defined lower and upper bounds were determined to be unbounded reactions, group together based on stoichiometry, and systematically corrected. These cycles were eliminated by removing duplicate reactions, turning off lumped reactions, fixing reaction directionality, or selectively turning reactions on or off based on cofactor specificities found from literature (Ishida et al, 1969;Dekker, Lane & Shapley, 1971;Chen, Lee & Chang, 1991;Schomburg & Stephan, 1995;Achterholt, Priefert & Steinbuchel, 1998;Hadfield et al, 1999;Hutter & Singh, 1999;Kai, Matsumura & Izui, 2003;Feist et al, 2007;Dean, 2012;Flamholz et al, 2012;Lin et al, 2014;Christensen et al, 2017). The FVA optimization algorithm is shown below.…”

Biotransformation of 2-keto-4-hydroxybutyrate via aldol condensation using an efficient and thermostable carboligase from Deinococcus radiodurans

“…In recent years, several class II pyruvate aldolases have been reported and engineered to develop powerful biocatalysts that lead to the organic synthesis of enantiomerically pure formulations via aldol condensation of aldehyde with carbonyl donors, which have potential to catalyze the stereochemical formation of C–C bonds (Baker and Seah 2012 ; Dekker et al 1971 ; Royer et al 2010 ; Wang et al 2010a ; Williams et al 2006 ). Specifically, these enzymes have primarily been studied utilizing aldehydes with straight or branched chain consisting of two to five carbons including acetaldehyde, glycolaldehyde, propionaldehyde, and glyceraldehyde (Baker and Seah 2012 ; Laurent et al 2019 ; Wang et al 2010a ).…”