Effect of Proline Substitutions on Stability and Kinetic Properties of a Cold Adapted Subtilase

Abstract

A cold adapted subtilisin-like serine proteinase from a Vibrio species is two amino acids shorter at the N-terminus than related enzymes adapted to higher temperatures and has a 15 residues' C-terminal extension relative to the highly homologous thermophilic enzyme aqualysin I from Thermus aquaticus. These enzymes are produced as pro-enzymes with an N-terminal chaperone sequence for correct folding and a C-terminal signal peptide for secretion, which are subsequently cleaved off by autocatalysis to give the mature enzyme. A truncated form of the Vibrio proteinase where the C-terminal extension was removed and two residues near the N-terminus were substituted with proline, to resemble the N- and C-terminal regions in aqualysin I, resulted in increased thermostability and diminished catalytic efficiency. The proline substitutions shift the site of autocatalytic cleavage at the N-terminus by two amino acids, apparently by rigidifying the terminal residues and support the formation of a beta-sheet that fixes the N-terminus to the main body of the protein.

Improved thermostability of creatinase from Alcaligenes Faecalis through non-biased phylogenetic consensus-guided mutagenesis

“…For example, the G58D and T199S mutations mainly contributed to higher stability by increasing the hydrophilicity of the protein surface, as well as the inter-helix hydrophobic interactions. In addition, introduction of proline substitutions has been previously shown to be a trend in the stabilization of proteins [28]. The leucine to proline substitution in the L6P mutation ( Fig.S4C) decreased the conformational entropy, localized to this amino acid position, which resulted in made the protein space structure more rigid.…”

Seven N-terminal Residues of a Thermophilic Xylanase Are Sufficient to Confer Hyperthermostability on Its Mesophilic Counterpart

“…The pyrrolidine ring restricts proline to fewer conformations than are available to the other amino acids [30], [31]. Thus, proline is believed to strengthen the rigidity of proteins [32]–[34]. When the single S33P mutation was introduced into the sequence of SoxB, the Asn32 residue, which is immediately adjacent to Pro33, may have restrained the local configuration of Asn32 and Pro33.…”

Cold Adapted Subtilases

“…The optimization of conformational flexibility of cold active enzymes for maintaining activity at low temperature has a trade off, however, in that it requires that some stabilizing noncovalent intramolecular interactions that are altered or weakened, causing destabilization of the protein structure. reduced number of salt bridges [8,67], prolines in loops [12,68], higher content of polar, uncharged amino acids, in particular Ser [12,69], have been realized. Several structural features have been correlated with cold adaptation, such as reduced numbers of salt bridges, hydrogen bonds, aromatic interactions, and internal hydrophobic interactions, as well as reduced Pro and Arg content [3,63,66].…”