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

Acta Naturae ◽  
2014 ◽  
Vol 6 (3) ◽  
pp. 76-88 ◽  
Author(s):  
I. V. Golubev ◽  
N. V. Komarova ◽  
K. V. Ryzhenkova ◽  
T. A. Chubar ◽  
S. S. Savin ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Amino Acid ◽  
3D Structure ◽  
Amino Acid Sequences ◽  
Acid Oxidase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Amino Acid Oxidase ◽  
Thermal Stabilities ◽  
Alpha Helices

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.

scholarly journals Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion

2019 ◽  
Vol 20 (18) ◽  
pp. 4412
Author(s):  
Denis L. Atroshenko ◽  
Mikhail D. Shelomov ◽  
Sophia A. Zarubina ◽  
Nikita Y. Negru ◽  
Igor V. Golubev ◽  
...  
Keyword(s):  
Amino Acid ◽  
Thermal Denaturation ◽  
Catalytic Properties ◽  
Acid Oxidase ◽  
Cephalosporin C ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Amino Acid Oxidase ◽  
Catalytic Constant ◽  
Biotechnological Processes

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.

scholarly journals Thermostabilization of Bacterial Fructosyl-Amino Acid Oxidase by Directed Evolution

2003 ◽  
Vol 69 (1) ◽  
pp. 139-145 ◽  
Author(s):  
Ryoichi Sakaue ◽  
Naoki Kajiyama
Keyword(s):  
Amino Acid ◽  
Directed Evolution ◽  
Amino Acid Substitutions ◽  
Acid Oxidase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Amino Acid Oxidase ◽  
Fourth Round ◽  
Fructosyl Amino Acid Oxidase

ABSTRACT We succeeded in isolating several thermostable mutant fructosyl-amino acid oxidase (FAOX; EC 1.5.3) without reduction of productivity by directed evolution that combined an in vivo mutagenesis and membrane assay screening system. Five amino acid substitutions (T60A, A188G, M244L, N257S, and L261M) occurred in the most thermostable mutant obtained by a fourth round of directed evolution. This altered enzyme, FAOX-TE, was stable at 45°C, whereas the wild-type enzyme was not stable above 37°C. The Km values of FAOX-TE for d-fructosyl-l-valine and d-fructosyl-glycine were 1.50 and 0.58 mM, respectively, in contrast with corresponding values of 1.61 and 0.74 mM for the wild-type enzyme. This altered FAOX-TE will be useful in the diagnosis of diabetes.

P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency

2020 ◽  
Vol 168 (5) ◽  
pp. 557-567
Author(s):  
Wanitcha Rachadech ◽  
Yusuke Kato ◽  
Rabab M Abou El-Magd ◽  
Yuji Shishido ◽  
Soo Hyeon Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Active Site ◽  
Structural Changes ◽  
Catalytic Efficiency ◽  
Acid Oxidase ◽  
Wild Type ◽  
Amino Acid Oxidase ◽  
Adenine Ring ◽  
The Impact

Abstract Human D-amino acid oxidase (DAO) is a flavoenzyme that is implicated in neurodegenerative diseases. We investigated the impact of replacement of proline with leucine at Position 219 (P219L) in the active site lid of human DAO on the structural and enzymatic properties, because porcine DAO contains leucine at the corresponding position. The turnover numbers (kcat) of P219L were unchanged, but its Km values decreased compared with wild-type, leading to an increase in the catalytic efficiency (kcat/Km). Moreover, benzoate inhibits P219L with lower Ki value (0.7–0.9 µM) compared with wild-type (1.2–2.0 µM). Crystal structure of P219L in complex with flavin adenine dinucleotide (FAD) and benzoate at 2.25 Å resolution displayed conformational changes of the active site and lid. The distances between the H-bond-forming atoms of arginine 283 and benzoate and the relative position between the aromatic rings of tyrosine 224 and benzoate were changed in the P219L complex. Taken together, the P219L substitution leads to an increase in the catalytic efficiency and binding affinity for substrates/inhibitors due to these structural changes. Furthermore, an acetic acid was located near the adenine ring of FAD in the P219L complex. This study provides new insights into the structure–function relationship of human DAO.

MOUSE MUTANT DEFICIENT IN d-AMINO ACID OXIDASE ACTIVITY

Genetics ◽  
1983 ◽  
Vol 103 (2) ◽  
pp. 277-285 ◽  
Author(s):  
Ryuichi Konno ◽  
Yosihiro Yasumura
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Oxidase Activity ◽  
Null Allele ◽  
Gene Dosage ◽  
Autosomal Gene ◽  
Acid Oxidase ◽  
Wild Type ◽  
Amino Acid Oxidase ◽  
Gene Dosage Effect

ABSTRACT d-Amino acid oxidase activity in the kidney homogenates of mice of seven strains was measured to search for a mutant for this enzyme. There was a consistent sex difference in the enzyme activity in these strains: male mice showed higher levels of the enzyme activity than females. In contrast to other strains, some mice of the ddY strain did not possess enzyme activity. This trait was inheritable, and a mouse stock without enzyme activity (DAO-) was established. The allele (Dao-1c) carried by the DAO- mice was recessive and behaved as a single autosomal gene in inheritance. Heterozygous mice for this gene (Dao-1  +/Dao-1c) showed nearly half the enzyme activity of the wild-type homozygotes (Dao-1  +/Dao-1  +), suggesting that Dao-1c is a null allele and that there is a gene dosage effect on the enzyme activity.

scholarly journals Limited proteolysis and site-directed mutagenesis reveal the origin of microheterogeneity in Rhodotorula gracilis D-amino acid oxidase

1998 ◽  
Vol 330 (2) ◽  
pp. 615-621 ◽  
Author(s):  
Stefano CAMPANER ◽  
Loredano POLLEGIONI ◽  
D. Brian ROSS ◽  
S. Mirella PILONE
Keyword(s):  
Amino Acid ◽  
Isoelectric Focusing ◽  
Limited Proteolysis ◽  
Single Band ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Acid Oxidase ◽  
Wild Type ◽  
Amino Acid Oxidase ◽  
Rhodotorula Gracilis

When analysed by isoelectric focusing, D-amino acid oxidase from the yeast Rhodotorula gracilis normally consists of three molecular isoforms (pI 7.8, 7.4 and 7.2, respectively) all with the same N-terminal sequence. However, only a single band of pI 7.8 is detected with the recombinant wild-type protein expressed in E. coli. To determine whether the molecular basis of this heterogeneity is due to proteolysed forms of the protein, we treated R. gracilisd-amino acid oxidase with various proteases. Limited proteolysis by chymotrypsin and thermolysin produced truncated and nicked monomeric holoenzymes containing two polypeptides of ≈ 34 kDa (Met1-Leu312) and one of ≈ 5 kDa (Ala319-Arg364 with chymotrypsin or Ala319-Ala362 with thermolysin). On the other hand, treatment with endoproteinase Glu-C gave a dimeric holoenzyme lacking the C-terminal SKL tripeptide. This cleavage of Glu365-Ser366 peptide bond caused the disappearance of the three isoelectric bands and a single homogeneous band (pI 7.2) appeared. To study this protein form, we used site-directed mutagenesis to produce a mutant form of R. gracilisD-amino acid oxidase lacking the SKL C-terminal tripeptide (which is the targeting sequence PTS1 for peroxisomal proteins). As expected, the SKL-deleted mutant gave a single band (pI 7.2) in isoelectric focusing. The three-band pattern of native yeast enzyme was generated by in vitro experiments using an equimolar mixture of the wild-type (pI 7.8) and the SKL-deleted recombinant (pI 7.2) DAAOs. The microheterogeneity of yeast DAAO thus stems from the association of two polypeptide chains differing in the C-terminal tripeptide, giving three different holoenzyme dimers.

scholarly journals Contribution of the dimeric state to the thermal stability of the flavoprotein D-amino acid oxidase

2003 ◽  
Vol 12 (5) ◽  
pp. 1018-1029 ◽  
Author(s):  
Loredano Pollegioni ◽  
Stefania Iametti ◽  
Dimitrios Fessas ◽  
Laura Caldinelli ◽  
Luciano Piubelli ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Amino Acid ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Thermal Stability Of
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