Purification and characterization of thermostable aspartate aminotransferase from a thermophilic Bacillus species

Abstract

Aspartate aminotransferase (EC 2.6.1.1) was purified to homogeneity from cell extracts of a newly isolated thermophilic bacterium, Bacillus sp. strain YM-2. The enzyme consisted of two subunits identical in molecular weight (Mr, 42,000) and showed microheterogeneity, giving two bands with pls of 4.1 and 4.5 upon isoelectric focusing. The enzyme contained 1 mol of pyridoxal 5'-phosphate per mol of subunit and exhibited maxima at about 360 and 415 nm in absorption and circular dichroism spectra. The intensities of the two bands were dependent on the buffer pH; at neutral or slightly alkaline pH, where the enzyme showed its maximum activity, the absorption peak at 360 nm was prominent. The enzyme was specific for L-aspartate and L-cysteine sulfinate as amino donors and ax-ketoglutarate as an amino acceptor; the Kms were determined to be 3.0 mM for L-aspartate and 2.6 mM for ot-ketoglutarate. The enzyme was most active at 70°C and had a higher thermostability than the enzyme from Escherichia coli. The N-terminal amino acid sequence (24 residues) did not show any similarity with the sequences of mammalian and E. coli enzymes, but several residues were identical with those of the thermoacidophilic archaebacterial enzyme recently reported.

A Thermostable Aspartate Aminotransferase from Aeropyrum pernix K1

“…The enzyme was fairly stable at temperature up to 65 o C. Moreover, ApAAT was protected against heat activation by the addition of pyridoxal phosphate or/and α-ketoglutarate, which significantly increased its heat stability by approximately 5 ~ 10 o C. In contrast, the addition of L-aspartate decreased the heat stability of ApAAT. This thermostability is comparable with those of AATs purified from other thermophilic bacteria (2,4,13).…”

“…Aspartate aminotransferase (AAT, EC 2.6.1.1), the best known pyridoxal 5'-phosphate dependent enzyme, is an essential component of nitrogen and carboxylic cellular metabolism, 1,2 where it catalyzes the reversible transamination between dicarboxylic amino and α-keto acids. Aminotransferases from many species are classified into four subgroups.…”

Evolutionary recruitment of biochemically specialized subdivisions of Family I within the protein superfamily of aminotransferases

“…These enzymes had previously been known to have broad specificities that include aromatic amino acids. In striking contrast, another cluster contains members that are highly specific aspartate aminotransferases, as best documented by Bacillus species AspX (Bsp AspX) (61) Fig. 2).…”

Lactococcus lactis LM0230 contains a single aminotransferase involved in aspartate biosynthesis, which is essential for growth in milk

“…Asp also appears to be the only amino acid substrate for AspC. Many other subfamily Iγ AspC homologues also have relative activities with Asp that are at least 100-fold higher than the activities with other amino acids (Marino et al, 1988 ;Sung et al, 1990 ;Xing & Whitman, 1992 ;Okamoto et al, 1996). However, the inability to detect activity of the lactococcal AspC with amino acids other than Asp may also have been due to the lack of sensitivity of the assay used, which was approximately 6 % relative activity to that observed with Asp.…”