Isolation and Characterization of D‐Threonine Aldolase, A Pyridoxal‐5′‐Phosphate‐Dependentenzyme from Arthrobacter sp. DK‐38

Abstract

-EJB 97 0741/4 D-Threonine aldolase is an enzyme that catalyzes the cleavage of D-threonine into glycine and acetaldehyde. Its activity was found in several genera of bacteria such as Arthrobacter, Alcaligenes, Xanthomonus, and Pseudonzonas, but not in yeasts or fungi. The enzyme was purified to homogeneity from one strain, Arthrobacter sp. DK-38. The enzyme appeared to consist of a single polypeptide chain with an apparent molecular mass of 51 kDa. This enzyme, as we11 as L-threonine aldolase, requires pyridoxal 5'-phosphate (pyridoxal-P) as a coenzyme. Unlike other pyridoxal-P enzymes, D-threonine aldolase also requires a divalent cation such as Co2+, Ni2+, Mnz+, or Mgz+ for its catalytic activity. The enzyme completely lost its activity in the absence of either pyridoxal-P or a divalent cation. A divalent cation was also essential for the thermal stability of the enzyme. The metal-free enzyme tends to become thermally unstable, resulting in the irreversible loss of its catalytic activity. The enzyme is strictly D-specific for the a-position, whereas it cannot distinguish between threo and erythro forms at the /$position. Thus, D-threonine and D-allothreonine act as substrates of the enzyme, but their kinetic parameters are different : the K,,, and V,,;,, values are 3.81 mM and 38.8 pmol . min-l . mg-' toward D-threonine, and 14.0 mM and 102 pmol . min-' . mg-l toward D-allothreonine, respectively. The aldolase reaction is reversible, and the enzyme is therefore able to produce nearly equimolar amounts of D-threonine and D-allothreonine through C-C bond formation between glycine and acetaldehyde. The enzyme also acts, in the same manner, on several other D-P-hydroxy-a-amino acids, including D-P-phenylserine, D-p-hydroxy-a-aminovaleric acid, ~-p-3,4-dihydroxyphenylserine, and D-~-3,4-methylenedioxyphenylserine.Keywords: D-threonine aldolase; threonine; Arthrohacter; pyridoxal %phosphate; divalent cation.Several enzymes are involved in L-threonine metabolism. Some are enzymes concerned in the biosynthesis of L-threonine from aspartic acid via homoserine 11, 21. Others are enzymes responsible for L-threonine degradation, such as L-threonine deaminase, which converts L-threonine into n-ketobutyrate, an intermediate in ~-isoleucine biosynthesis [3-51.L-Threonine aldolase, one of the L-threonine-degrading enzymes, cleaves L-threonine into glycine and acetaldehyde [6-91. Its enzyme activity has been found in several microorganisms, in such genera as Pseudomonas [lo], Clostridium 18,11, 121, Bacillus [13], and Candida [14, 151, as well as in mammalian organs [16, 171. L-Threonine aldolase has been extensively investigated because of its possible role in amino acid metabolism [lo, 111. Studies on the mechanism underlying its catalytic action revealed that L-threonine aldolase belongs to a group of enzymes that require pyridoxal 5'-phosphate (pyridoxal-P) as a coenzyme L-Threonine aldolase can produce the L-forms of threonine stereoisomers via C-C bond formation between glycine and acetaldehyde, though it is highly sele...

A Novel Metal-activated Pyridoxal Enzyme with a Unique Primary Structure, Low Specificity d-Threonine Aldolase fromArthrobacter sp. Strain DK-38

“…The bound metal ions of the enzyme were subsequently analyzed with a mode ICPS-1000III sequential plasma spectrometer (Shimadzu, Kyoto, Japan); the enzyme so treated was determined to contain no detectable divalent cations, such as Mn 2ϩ , Mg 2ϩ , Co 2ϩ , Ni 2ϩ , Fe 2ϩ , and Ca 2ϩ , which were previously shown to be activators of the wild-type low specificity D-TA from Arthrobacter sp. strain DK-38 (16). Kinetic analysis further showed that the gel-filtrated enzyme had the same V max and K m values toward DLthreo-and DL-erythro-phenylserine as those of the metal-free ones treated with EDTA, supporting the finding that the purified enzyme contained no bound metal ions, at least no activating divalent cations.…”

“…strain DK-38 was used as the source of chromosomal DNA (16). E. coli XL1-Blue MRFЈ (recA1 thi endA1 supE44 gyrA46 relA1 hsdR17 lac/FЈ [proAB ϩ lacI q lacZ_M15::Tn10 Tet r ]) (Toyobo, Osaka, Japan) was used as a host for the gene cloning.…”

“…Likewise, Dtype TA, acting on D-threonine and/or D-allo-threonine, might include D-allo-TA, D-TA, and low specificity D-TA. However, only low specificity D-TA activity was found from 3 out of 2,000 strains examined (16). Low specificity D-TA was previously purified from Arthrobacter sp.…”

“…Low specificity D-TA was previously purified from Arthrobacter sp. strain DK-38, and the enzyme was shown to have a monomeric structure and to require unusually both PLP and divalent cations for its catalytic activity (16). Due to little available purified enzyme, the identification of the cofactors was not performed.…”

Gene cloning, biochemical characterization and physiological role of a thermostable low‐specificity L‐threonine aldolase from Escherichia coli

“…Likewise, D-type TA, acting on D-threonine and/or D-hydroxymethyltransferase, as a host, we occasionally observed allo-threonine, might include D-allo-TA, D-TA and low-specificthat E. coli GS245 could grow on the medium containing 1% ity D-TA, although low-specificity D-TA of Athrobacter sp. is glucose as the sole carbon source with a prolonged cultivation the only one identified (Kataoka et al, 1997b). All of the three time (Liu, J.-Q., Dairi, T., Itoh, N., Kataoka, M., Shimizu, S. L-type enzymes have been found to exist in nature.…”