Study of the Structure-Function-Stability Relationships in Yeast D-amino Acid Oxidase: Hydrophobization of Alpha-Helices

Abstract

Hydrophobization of alpha-helices is one of the general approaches used for improving the thermal stability of enzymes. A total of 11 serine residues located in alpha-helices have been found based on multiple alignments of the amino acid sequences of D-amino acid oxidases from different organisms and the analysis of the 3D-structure of D-amino acid oxidase from yeast Trigonopsis variabilis (TvDAAO, EC 1.4.3.3). As a result of further structural analysis, eight Ser residues in 67, 77, 78, 105, 270, 277, 335, and 336 positions have been selected to be substituted with Ala. S78A and S270A substitutions have resulted in dramatic destabilization of the enzyme. Mutant enzymes were inactivated during isolation from cells. Another six mutant TvDAAOs have been highly purified and their properties have been characterized. The amino acid substitutions S277A and S336A destabilized the protein globule. The thermal stabilities of TvDAAO S77A and TvDAAO S335A mutants were close to that of the wild-type enzyme, while S67A and S105A substitutions resulted in approximately 1.5- and 2.0-fold increases in the TvDAAO mutant thermal stability, respectively. Furthermore, the TvDAAO S105A mutant showed on average a 1.2- to 3.0-fold higher catalytic efficiency with D-Asn, D-Tyr, D-Phe, and D-Leu as compared to the wild-type enzyme.

Effect of Additional Amino Acid Replacements on the Properties of Multi-point Mutant Bacterial Formate Dehyderogenase PseFDH SM4S

“…The most frequent Ser/Ala replacement in α-helices is a universal and effective approach for majority of proteins. For example, using this approach, we increased the thermal stability of D-amino acid oxidase by several fold [ 21 ]. The analysis of PseFDH structure revealed five Ser residues located in α-helices, among which only one was conserved.…”

Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion

“…Mutants were expressed in E. coli cells and purified using standard chromatography techniques [27]. All mutants were expressed in the active soluble form.…”

“…TvDAAO mutants were prepared by subcloning of DNA fragments from previously obtained genes with single and double mutations. New mutants were expressed in E. coli and purified as described previously [27]. Concentration of TvDAAO mutants was determined by using a FAD extinction coefficient 10.8 mM −1 ·cm −1 at 455 nm [13] in a UV-1800PC spectrophotometer (Shimadzu, Dusseldorf, Germany).…”

Assays of D-Amino Acid Oxidase Activity

“…Microbial DAAOs possess properties that render them suitable for industrial biotechnological applications: for example, they are stable enzymes and show broad substrate specificity, high turnover number (Table 1 ), and a tight binding with the FAD cofactor. The production of DAAOs in large amounts as recombinant proteins together with the availability of the 3D-structure (e.g., Mattevi et al, 1996 ; Mizutani et al, 1996 ; Umhau et al, 2000 ; Kawazoe et al, 2006 ), allowed the design and production by protein engineering techniques of enzyme variants with new and evolved properties (Pollegioni et al, 2007b ; Rosini et al, 2008 , 2009 , 2010 ; Wang et al, 2008 ; Wong et al, 2010 ; Pollegioni and Molla, 2011 ; Golubev et al, 2014 ).…”