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.
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 alpha-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 (Delta2alpha 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 Delta2alpha 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.
Kravchuk
Z
Z
Institute of Bio-Organic Chemistry, Minsk 220141, Belarus. Joint Biotechnology Laboratory, University of Turku, BioCity 6A,Fin-20520 Turku, Finland.
Tsybovsky
Y
Y
Koivulehto
M
M
Vlasov
A
A
Chumanevich
A
A
Battchikova
N
N
Martsev
S
S
Korpela
T
T
D000595:Amino Acid Sequence; D001219:Aspartate Aminotransferases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D001407:Bacillus / Q000201:enzymology*; D002152:Calorimetry, Differential Scanning; D004355:Drug Stability; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D013057:Spectrum Analysis
D001219:Aspartate Aminotransferases; D019791:Guanidine