Mutant d-amino acid oxidase with higher catalytic efficiency toward d-amino acids with bulky side chains

Комарова, Н. В.; Golubev, I. V.; Khoronenkova, Svetlana V.; Тишков, В. И.

doi:10.1007/s11172-012-0193-4

Cited by 8 publications

(6 citation statements)

References 11 publications

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“…Nucleotide substitutions were introduced using twostep PCR as described previously [ 13 , 16 ]. The plasmid obtained based on pET -33b (+) with the tvdaao gene being under the control of a strong promoter of RNA polymerase of T7 phage was used as a template.…”

Section: Methodsmentioning

confidence: 99%

“…We showed [ 11 , 13 , 16 ] that inactivation of wild-type TvDAAO and its various mutants at elevated temperatures proceeds according to the following dissociative mechanism:…”

Section: Mechanism Of Inactivation Of Tvdaao Mutantsmentioning

confidence: 99%

“…[ 18 ]. The time dependence of the residual activity of the enzyme in this mechanism is described by a sum of two exponential functions, and the inactivation rate of the enzyme depends on its concentration [ 11 , 13 , 16 ]. The dissociative mechanism of the wild-type TvDAAO thermoinactivation is only observed within a temperature range of 50-60°C, when the rate constants k 1 and k 2 are comparable to each other.…”

Section: Mechanism Of Inactivation Of Tvdaao Mutantsmentioning

confidence: 99%

“…Very scarce data on increasing the thermal stability of TvDAAO using protein engineering have been published so far. Only two papers report data on the obtained TvDAAO mutants with point amino acid substitutions, which demonstrated a slight increase in thermal stability as compared to the wild-type enzyme [ 13 , 14 ]. The covalent crosslinking of two TvDAAO subunits with Lys-Leu dipeptide [ 15 ] was also reported.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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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.

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Section: Methodsmentioning

confidence: 99%

“…We showed [ 11 , 13 , 16 ] that inactivation of wild-type TvDAAO and its various mutants at elevated temperatures proceeds according to the following dissociative mechanism:…”

Section: Mechanism Of Inactivation Of Tvdaao Mutantsmentioning

confidence: 99%

Section: Mechanism Of Inactivation Of Tvdaao Mutantsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

et al. 2014

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“…d -amino acid oxidase from the yeast Trigonopsis variabilis (TvDAAO) is the most stable and active enzyme towards CPC among all known DAAOs [13]. To improve catalytic properties with CPC, mutant TvDAAOs with amino acid changes Phe54Ser, Phe54Tyr and Phe54Ala were prepared and studied [14,15]. All above substitutions showed increased catalytic properties with CPC and d -amino acids with bulky side-chains.…”

Section: Introductionmentioning

confidence: 99%

Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion

Atroshenko

Shelomov

Zarubina

et al. 2019

IJMS

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d-amino acid oxidase (DAAO, EC 1.4.3.3) is used in many biotechnological processes. The main industrial application of DAAO is biocatalytic production of 7-aminocephalosporanic acid from cephalosporin C with a two enzymes system. DAAO from the yeast Trigonopsis variabilis (TvDAAO) shows the best catalytic parameters with cephalosporin C among all known DAAOs. We prepared and characterized multipoint TvDAAO mutants to improve their activity towards cephalosporin C and increase stability. All TvDAAO mutants showed better properties in comparison with the wild-type enzyme. The best mutant was TvDAAO with amino acid changes E32R/F33D/F54S/C108F/M156L/C298N. Compared to wild-type TvDAAO, the mutant enzyme exhibits a 4 times higher catalytic constant for cephalosporin C oxidation and 8- and 20-fold better stability against hydrogen peroxide inactivation and thermal denaturation, respectively. This makes this mutant promising for use in biotechnology. The paper also presents the comparison of TvDAAO catalytic properties with cephalosporin C reported by others.

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Engineering of substrate specificity of D-amino acid oxidase from the yeast Trigonopsis variabilis: Directed mutagenesis of Phe258 residue

Комарова

Golubev

Khoronenkova

et al. 2012

Biochemistry Moscow

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Natural D-amino acid oxidases (DAAO) are not suitable for selective determination of D-amino acids due to their broad substrate specificity profiles. Analysis of the 3D-structure of the DAAO enzyme from the yeast Trigonopsis variabilis (TvDAAO) revealed the Phe258 residue located at the surface of the protein globule to be in the entrance to the active site. The Phe258 residue was mutated to Ala, Ser, and Tyr residues. The mutant TvDAAOs with amino acid substitutions Phe258Ala, Phe258Ser, and Phe258Tyr were purified to homogeneity and their thermal stability and substrate specificity were studied. These substitutions resulted in either slight stabilization (Phe258Tyr) or destabilization (Phe258Ser) of the enzyme. The change in half-inactivation periods was less than twofold. However, these substitutions caused dramatic changes in substrate specificity. Increasing the side chain size with the Phe258Tyr substitution decreased the kinetic parameters with all the D-amino acids studied. For the two other substitutions, the substrate specificity profiles narrowed. The catalytic efficiency increased only for D-Tyr, D-Phe, and D-Leu, and for all other D-amino acids this parameter dramatically decreased. The improvement of catalytic efficiency with D-Tyr, D-Phe, and D-Leu for TvDAAO Phe258Ala was 3.66-, 11.7-, and 1.5-fold, and for TvDAAO Phe258Ser it was 1.7-, 4.75-, and 6.61-fold, respectively.

show abstract

Mutant d-amino acid oxidase with higher catalytic efficiency toward d-amino acids with bulky side chains

Cited by 8 publications

References 11 publications

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

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

Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion

Engineering of substrate specificity of D-amino acid oxidase from the yeast Trigonopsis variabilis: Directed mutagenesis of Phe258 residue

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