Truncated aspartate aminotransferase from alkalophilic Bacillus circulans with deletion of N-terminal 32 amino acids is a non-functional monomer in a partially structured state

• Published in: Protein engineering 2001-04, Vol.14 (4), p.279-285
• Main Authors: Kravchuk, Zinaida, Tsybovsky, Yaroslav, Koivulehto, Marianne, Vlasov, Alexander, Chumanevich, Alexander, Battchikova, Natalia, Martsev, Sergey, Korpela, Timo
• Format: Article
• Language: English
• Subjects: alkalophilic enzymes; Amino Acid Sequence; aspartate aminotransferase; Aspartate Aminotransferases - chemistry; Aspartate Aminotransferases - genetics; Aspartate Aminotransferases - isolation & purification; Bacillus - enzymology; Bacillus circulans; Calorimetry, Differential Scanning; differential scanning calorimetry; Drug Stability; Guanidine - pharmacology; Hydrogen-Ion Concentration; Models, Molecular; PLP-dependent enzymes; Protein Denaturation - drug effects; Protein Folding; Protein Structure, Secondary; Sequence Deletion; Spectrum Analysis; Sulfolobus solfataricus
• ISSN: 0269-2139; 1741-0126; 1460-213X; 1741-0134
• DOI: 10.1093/protein/14.4.279

Aspartate aminotransferase (AspAT) from alkalophilic Bacillus circulans contains an additional N-terminal sequence of 32 amino acid residues that are absent in all other AspATs from different sources. Modeling suggested that this sequence forms two α-helical segments which establish a continuous network of interactions on the surface of the molecule. In the present study, we studied the role of the N-terminal sequence in folding and stability of AspAT by applying the scanning calorimetry, and CD and fluorescence spectroscopies to the native and truncated enzymes. Truncated AspAT (Δ2α mutant) devoid of N-terminal residues cannot provide sufficient potential of quaternary intersubunit and subunit-cofactor interactions, which results in a monomeric non-functional conformation. However, the residual tertiary interactions in the Δ2α mutant are sufficient to: i) provide stability of a residual structure over a wide pH range; ii) confer moderate cooperativity of the denaturant-induced transition while only low cooperativity of the thermal transition, and iii) maintain the hydrophobic core of a part of the structure which prevents aromatic fluorophores from quenching by water. Furthermore, the present study provides evidence that AspAT from the alkalophilic bacterium follows unfolding pathway comprising a stable non-functional intermediate, in contrast to a two-state mechanism of the thermophilic AspAT from Sulfolobus solfataricus.