Evolutionarily conserved structural motifs in bacterial GST (glutathione S-transferase) are involved in protein folding and stability.
The bacterium Proteus mirabilis expresses a cytosolic class beta glutathione S-transferase (PmGST B1-1) that is part of a family of multifunctional detoxication enzymes. Like other cytosolic GSTs, PmGST B1-1 possesses two local structural motifs, an N-capping box and a hydrophobic staple motif, both of which are located between amino acids 151 and 156. The N-capping box consists of a reciprocal hydrogen bonding interaction of Thr152 with Asp155, whereas the hydrophobic staple motif consists of a hydrophobic interaction between Phe151 and Ala156. By contrast with other GSTs, PmGST B1-1 displays distinct hydrogen bond interactions in the N-capping box. In mammalian GSTs these structural elements are critical for protein folding and stability. To investigate the role played by these two motifs in a distantly related organism on the evolutionary scale, site-directed mutagenesis was used to generate several mutants of both motifs in PmGST B1-1. All mutants were efficiently overexpressed and purified, but they were quite unstable, although at different levels, indicating that protein folding was significantly destabilized. The analysis of the T152A and D155G variants indicated that the N-capping box motif plays an important role in the stability and correct folding of the enzyme. The analysis of F151A and A156G mutants revealed that the hydrophobic staple motif influences the structural maintenance of the protein and is implicated in the folding process of PmGST B1-1. Finally, the replacement of Thr152 and Asp155, as well as Phe151 and Ala156 residues influences the catalytic efficiency of the enzyme.
Allocati
Nerino
N
Dipartimento di Scienze Biomediche, Università G. d'Annunzio, Via dei Vestini 31, I-66013 Chieti, Italy. n.allocati@dsb.unich.it
Masulli
Michele
M
Pietracupa
Marilena
M
Federici
Luca
L
Di Ilio
Carmine
C
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004795:Enzyme Stability; D019143:Evolution, Molecular; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D011513:Proteus mirabilis / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D005982:Glutathione Transferase