A Single Free Cysteine Residue and Disulfide Bond Contribute to the Thermostability ofAspergillus saitoi1,2-α-Mannosidase

Abstract

Aspergillus saitoi 1,2--mannosidase contains three conserved cysteine residues (Cys334, Cys363, and Cys443). We showed that Cys334 and Cys363 are involved in a disulfide bond, and that Cys443 contains a free thiol group. The cysteines were not essential for the activity analyzed by site-directed mutagenesis and kinetics. The substitution at each cysteine residue greatly destabilized the enzyme. The T m values of WT, C443A, C443G, C443S, and C443T were 55.8, 51.9, 50.2, 50.0, and 52.8 C respectively. The specific activity of these mutants was almost equal to that of WT. Introducing Asp, Leu, Met, or Val at position 443 caused partial denaturation, although the enzymes had some activity. C443F, C443I, C443N, and C443Y were not secreted. These results suggest that the hydrophilic and large side chain causes the destabilization. Molecular modelling showed that the Cys443 residue is buried and surrounded by a hydrophobic environment. Cys334 and Cys363 form a disulfide bond, and Cys443 is involved in a hydrophobic interaction to stabilize the enzyme.

Biochemical and structural characterization of a novel halotolerant cellulase from soil metagenome

“…The observed near-complete inhibition of endoglucanase activity by thiol inhibitors led us to investigate the role of free cysteines in the activity of Cel5R. Moreover, it has been shown that the substitution of free thiols with other amino acid residues increased30 the thermal stability of the protein in some cases, while in others it is decreased31. Thus, to understand the role of cysteines in Cel5R, different constructs with single, double and quadruple mutations (cysteine to alanine) were made and their activities were checked by DNS (3,5-Dinitrosalicylic acid) assay.…”

Molecular Cloning and Expression of a cDNA Encoding a Coccidioides posadasii 1,2‐α‐Mannosidase Identified in the Coccidioidal T27K Vaccine by Immunoproteomic Methods

“…saitoi , T. reesei , and P . citrinum class I 1,2‐α‐mannosidases have also been studied extensively 18,19,24,26–28 . The filamentous fungal enzymes belong to a functionally distinct subgroup of the class I enzymes that include the mammalian Golgi 1,2‐α‐mannosidases 20 .…”

“…The deduced C. posadasii sequence 1,2‐α‐mannosidase protein sequence shares about 60% identity with other fungal class I 1,2‐α‐mannosidases and contains a number of conserved regions including a conserved glutamic acid residue at position 127 and two conserved cysteine residues (Cys‐337 and Cys‐366) 18,19 . The N‐terminal region of the deduced amino acid sequence contains a hydrophobic amino acid sequence, a putative transmembrane α‐helix, and a predicted cleavable signal peptide.…”

Disulfide bond ofMycoplasma pneumoniaecommunity‐acquired respiratory distress syndrome toxin is essential to maintain the ADP‐ribosylating and vacuolating activities

“…Although the disulfide bridge between C230 and C247 is important, C247 seems to be more significant than C230 for the stability of CARDS toxin ( Figure 3a). Previous studies have demonstrated that both disulfides and thiol groups have important roles in the stability of proteins (Burova, Choiset, Tran, & Haertle, 1998;Tatara, Yoshida, & Ichishima, 2005). It is possible that irrespective of its role in disulfide bond formation, the thiol form of C247 may also be essential to maintain the stability of the toxin.…”