Purification and characterization of an inducible L‐lysine: 2‐oxoglutarate 6‐aminotransferase from Candida utilis

Abstract

L-Lysine:2-oxoglutarate 6-aminotransferase (EC 2.1.6.36) was purified 202-fold from the yeast Candida utilis. The subunit Mr estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 40 kDa. The Mr of the native enzyme was estimated to be 83 kDa by gel filtration, suggesting a dimeric structure. The enzyme exhibits absorption maxima at 280, 340 and 420 nm, and binds 2 mol pyridoxal-5-phosphate/mol of the native enzyme. The aminotransferase has a maximum activity at pH 8.5 and at 4 degrees C. 2-Oxoglutarate is the best amino acceptor with L-lysine as amino donor. Lower activity is observed with oxaloacetate (38%), pyruvate (19%) and 2-oxoadipate (7%) as acceptor or with L-thialysine (13%) as donor. The Km values are 2.5 mM for L-lysine, 3.8 mM for 2-oxoglutarate and 0.015 mM for pyridoxal-5-phosphate. The enzyme activity is induced in cells grown in the presence of L-lysine.

Direct Evidence for a Glutamate Switch Necessary for Substrate Recognition: Crystal Structures of Lysine ε-Aminotransferase (Rv3290c) from Mycobacterium tuberculosis H37Rv

“…The K m values corresponding to L-lysine and α-ketoglutarate were determined to be 1.2(±0.4) and 2.3(±0.3) mM, respectively, largely following procedures reported earlier. 18,19 These values are similar to those reported for LAT from other sources; viz. Flavobacterium lutescens, 17 candida utilis, 18 and Streptomyces lactamdurans, 19 where also the respective K m values are in the low millimolar range.…”

“…18,19 These values are similar to those reported for LAT from other sources; viz. Flavobacterium lutescens, 17 candida utilis, 18 and Streptomyces lactamdurans, 19 where also the respective K m values are in the low millimolar range. The corresponding K cat values for the respective reactions were calculated to be 55(±5) min −1 and 86(±5) min −1 .…”

“…Despite biochemical characterization of the enzyme from other sources for over four decades, 1,11,[16][17][18][19] no crystal structure is available. We undertook the cloning, expression and purification of the M. tuberculosis enzyme (MtLAT) and assayed it for LAT activity in the first instance to demonstrate that Rv3290c is indeed a LAT.…”

Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

“…H. elongata DABA aminotransferase showed a large molecular mass corresponding to a homohexamer by gel filtration, whereas A. baumannii DABA aminotransferase, which was overproduced and purified from Escherichia coli, was in the form of a homotetramer with a molecular mass of 188 kDa (18). Although the sizes of each subunit are in a narrow range of 40 to 50 kDa in most aminotransferases, the native molecules exist largely as homodimers (17,41,47,53) or homotetramers (18,45,49). As a rare example, ornithine aminotransferase from rats was reported to be a homohexamer composed of a dimer of trimers, with a molecular mass of 256 kDa in the solution containing NaCl (27).…”

Lysine biosynthesis and metabolism in fungi

“…This enzyme has been purified from C. utilis and characterized as a 83 kDa dimer possessing two identical 40 kDa subunits and requiring pyridoxal 5A-phosphate. 129 Whereas a-ketoglutarate was the preferred amine acceptor, oxaloacetate, pyruvate and 2-oxoadipate can serve in this role.…”