Identity and some properties of the l-threonine aldolase activity manifested by pure 2-amino-3-ketobutyrate ligase of Escherichia coli

Abstract

2-Amino-3-ketobutyrate ligase catalyzes the reversible, pyridoxal 5'-phosphate-dependent condensation of glycine with acetyl CoA forming the unstable intermediate, 2-amino-3-ketobutyrate. Several independent lines of evidence indicate that the pure protein obtained in the purification of this ligase from Escherichia coli also has L-threonine aldolase activity. The evidence includes: (a), a constant ratio of specific activities (aldolase/ligase) at all stages of purifying 2-amino-3-ketobutyrate ligase to homogeneity; (b), the same rate of loss of aldolase and ligase activities during controlled heat inactivation of the pure protein at 60 degrees C in the absence, as well as in the presence of acetyl CoA, a protective substrate; (c), ratios of the two enzymatic activities that are not significantly different during slow inactivation by iodoacetamide, with and without L-threonine added; (d), coincident rates of loss and essentially identical rates of recovery of aldolase activity and ligase activity during resolution of the holoenzyme with hydroxylamine followed by reconstitution with pyridoxal 5'-phosphate. No aldolase activity is observed with D-threonine as substrate and L-allothreonine is about 25% as effective as L-threonine. Whereas ligase activity has a sharp pH optimum at 7.5, the aldolase activity of this pure protein is maximal at pH 9.0. Comparative apparent Km values for glycine (ligase) and L-threonine (aldolase) are 10 mM and 0.9 mM, respectively, whereas corresponding respective Vmax values were found to be 2.5 mumol of CoA released/min per mg vs. 0.014 mumol of acetaldehyde formed (NADH oxidized)/min per mg.

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

“…Second, Thr can be produced via a pathway involving steps catalyzed by 2-amino-3-ketobutyrate ligase and threonine dehydrogenase [44], with 13 C-labeled sodium acetate serving as a source of acetyl-CoA (Figure 1). However, the production of large quantities of Thr is likely to be compromised because (i) the intermediate 2-amino-3-ketobutyrate has a half-life estimated to be less than 1 minute [45] or 10 minutes [46] at pH 7, with spontaneous decarboxylation diverting a large fraction of the isotopically labeled material into aminoacetone [44] and (ii) 2-amino-3-ketobutyrate ligase also possesses some threonine aldolase activity [47] which would destroy the desired product (Thr) in a coupled enzymatic reaction. A third approach is also suggested in Figure 1, starting from 2-ketobutyrate (2KB, 13 CH 3 12 CD 2 COCOONa), a well-established and inexpensive commercially available precursor for labeling of Ile δ1 positions with 1 H and 13 C [11].…”

Role of Operon aaoSo-mutT in Antioxidant Defense in Streptococcus oligofermentans

“…Pronounced aminoacetone oxidase activity was detected for LAAO, although it was first identified as an amino acid oxidase [11]. Cellular aminoacetone can be derived either from threonine or a condensation of glycine and acetyl-CoA [24], [27]. Threonine dehydrogenase, a key enzyme in L-threonine metabolism, catalyzes the conversion of L-threonine to 2-amino-3-ketobutyrate, and the latter is spontaneously decarboxylated to aminoacetone [28].…”

Purification and characterization of l-allo-threonine aldolase from Aeromonas jandaei DK-39

“…This enzyme also acts on both l ‐threonine and l ‐ allo ‐threonine (unpublished results). Marcus and Dekker showed that a homogeneous 2‐amino‐3‐ketobutyrate ligase of E. coli show low activity toward these threonine isomers [13], but the present enzyme, LATA, does not cleave l ‐threonine. To the best of our knowledge, the present reports demonstrates the first instance of the l ‐ allo ‐threonine specific enzyme, LATA, which exhibits neither l ‐threonine aldolase nor SHMT activity.…”