Mutational effect for stability in a conserved region of thermolysin

Abstract

Aims: To investigate the mutational effect for the stability of thermolysin (TLN) in conserved regions. Methods and Results: Mutational effects for stability at autodegradation sites of TLN in conserved region were studied. The bands of mutant TLN (34 kDa) on SDS-PAGE were decreased. However, those of mutant TLN cultivated with CaCl 2 recovered to the same level as WT TLN. Dialysis study shows that these mutant TLN require more calcium ions than WT TLN. Conclusions: From these results, calcium affinity of mutant TLN in the conserved regions seem to become weak, subsequently mutant TLN were easily autodegraded in the case of low concentration of CaCl 2 . Significance and Impact of the Study: The autodegradation sites located in conserved regions of bacilli neutral proteases are important for the tertiary structure formation concerning the stability of the protein.

Effects of Site-Directed Mutagenesis of the Loop Residue of the N-Terminal Domain Gly117 of Thermolysin on Its Catalytic Activity

“…L155A has higher stability than WT. 23,24) N112D, 25) F114H, 13) and Q225A 26) have modified pH-activity profiles. In the N-terminal domains of thermolysin, two parallel polypeptide strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122, are connected by a short loop, Asn116-Gly117, to form an anti-parallel -sheet (Fig.…”

“…Sitedirected mutagenesis studies of thermolysin have been done extensively, especially on its active-site residues. [20][21][22][23][24][25][26][27][28] Of the active-site residue variants, L144S, D150E, and I168A have higher activities than wildtype thermolysin (WT) in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide (FAGLA), a widely used substrate for thermolysin, 14,29) and ZDFM. L155A has higher stability than WT.…”

Further Stabilization of Leu155 Mutant Thermolysins by Mutation of an Autodegradation Site

“…Autodegradation of TLN occurs in several regions at the same time; therefore, identification of the primary autodegradation site is difficult. Mutations were previously introduced at autodegradation sites in TLN (Asp 126 -Gly 127 , Gly 154 -Leu 155 , Ser 204 -Met 205 , and Asp 207 -Pro 208 ); however, the thermostability was improved only in Gly 154 -Leu 155 mutant TLNs [3,17]. Therefore, the autodegradation at Gly 154 -Leu 155 may occur in the early stages of autodegradation.…”

“…Asp 126 -Gly 127 , Gly 154 -Leu 155 , Ser 204 -Met 205 , and Asp 207 -Pro 208 were identified as the autodegradation sites under existing calcium ions [3,17]. Among these sites, Leu 155 was substituted with Ala and Ser by site-directed mutagenesis.…”

Effects of the mutational combinations on the activity and stability of thermolysin

“…In addition, it is not easy presently to predict the effect of mutational combination on enzyme properties (LiCata and Ackers, 1995;De Kreij et al, 2002;Mildvan, 2004). Various mutations which alter enzyme properties have been identified for thermolysin (Mansfeld et al, 1997;Hanzawa and Kidokoro, 1999;Matsumiya et al, 2004Matsumiya et al, , 2005Kusano et al, 2006Kusano et al, , 2009Tatsumi et al, 2007;Takita et al, 2008). Of these mutations, activating mutations which increase the hydrolytic activity for N-[3-(2-furyl)acryloyl]-Gly-l-Leu amide (FAGLA) and N-carbobenzoxy-l-Asp-l-Phe methyl ester (ZDFM) and stabilizing mutations which increase the thermal stability are listed in Table 1 (In this study, the mutation of the residue for example Leu144 to Ser was designated as Leu144 → Ser, and the thermolysin variant bearing the mutation 0168-1656/$ -see front matter © 2010 Elsevier B.V. All rights reserved.…”