Amino acid sequence of Salmonella typhimurium branched-chain amino acid aminotransferase

Abstract

The complete amino acid sequence of the subunit of branched-chain amino acid aminotransferase (transaminase B, EC 2.6.1.42) of Salmonella typhimurium was determined. An Escherichia coli recombinant containing the ilvGEDAY gene cluster of Salmonella was used as the source of the hexameric enzyme. The peptide fragments used for sequencing were generated by treatment with trypsin, Staphylococcus aureus V8 protease, endoproteinase Lys-C, and cyanogen bromide. The enzyme subunit contains 308 residues and has a molecular weight of 33,920. To determine the coenzyme-binding site, the pyridoxal 5-phosphate containing enzyme was treated with tritiated sodium borohydride prior to trypsin digestion. Peptide map comparisons with an apoenzyme tryptic digest and monitoring radioactivity incorporation allowed identification of the pyridoxylated peptide, which was then isolated and sequenced. The coenzyme-binding site is the lysyl residue at position 159. The amino acid sequence of Salmonella transaminase B is 97.4% identical with that of Escherichia coli, differing in only eight amino acid positions. Sequence comparisons of transaminase B to other known aminotransferase sequences revealed limited sequence similarity (24-33%) when conserved amino acid substitutions are allowed and alignments were forced to occur on the coenzyme-binding site.

Mechanism and stereochemistry of .alpha.,.beta.-dihydroxyacid dehydratase

“…Stereochemical Preferences. Racemic ,/3-dihydroxyisovalerate (20 mg, 120 Mmol) was incubated at 35 °C with 45 units of DHAD in 2.5 mL of Tris buffer. Aliquots (5 mL) were removed periodically and assayed for keto acid production.…”

Characterization of a branched-chain amino-acid aminotransferase fromSchizosaccharomyces pombe

“…In yeast, as well as in man, two ECA39-like genes were identified ; also called BAT1/2 and TWT1/2; Kispal et al, 1996). All ECA39 genes show significant similarity to ilvE genes coding for the BCAT in prokaryotes (Feild et al, 1989;Kuramitsu et al, 1985).…”

Structural characterization of sialic acid synthase by electrospray mass spectrometry—A tetrameric enzyme composed of dimeric dimers

“…Furthermore, the ESI-MS analysis can reflect dramatic change of pH-dependent quaternary structure in association with enzyme activity, suggesting the tetrameric form may be the primary species responsible for the enzyme catalysis. Proteins interact with other biomolecules to form supermolecular assembly via hydrophobic, electrostatic, hydrogen bonding and van der Waals interactions [1]. The protein quaternary structure plays an important role in close association with the activity and physiological function.…”