Single-Site Oxidation, Cysteine 108 to Cysteine Sulfinic Acid, in D-Amino Acid Oxidase from Trigonopsis variabilis and Its Structural and Functional Consequences (original) (raw)
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Stability and stabilization of D-amino acid oxidase from the yeast Trigonopsis variabilis
Biochemical Society Transactions, 2007
The use of DAO (d-amino acid oxidase) for the conversion of cephalosporin C has provided a significant case for the successful implementation of an O 2 -dependent biocatalyst on an industrial scale. Improvement of the operational stability of the immobilized oxidase is, however, an important goal of ongoing process optimization. We have examined DAO from the yeast Trigonopsis variabilis with the aim of developing a rational basis for the stabilization of the enzyme activity at elevated temperature and under conditions of substrate turnover. Loss of activity in the resting enzyme can occur via different paths of denaturation. Partial thermal unfolding and release of the FAD cofactor, kinetically coupled with aggregation, contribute to the overall inactivation rate of the oxidase at 50 • C. Oxidation of Cys 108 into a stable cysteine sulfinic acid causes both decreased activity and stability of the enzyme. Strategies to counteract each of the denaturation steps in DAO are discussed. Fusion to a pull-down domain is a novel approach to produce DAO as protein-based insoluble particles that display high enzymatic activity per unit mass of catalyst.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2010
D-amino acid oxidase Oxidative inactivation Cysteine sulfinic and sulfonic acid Site-directed mutagenesis Mass-spectrometric characterization of chemical oxidation Denaturation pathway Oxidative modification of Trigonopsis variabilis D-amino acid oxidase in vivo is traceable as the conversion of Cys108 into a stable cysteine sulfinic acid, causing substantial loss of activity and thermostability of the enzyme. To simulate native and modified oxidase each as a microheterogeneity-resistant entity, we replaced Cys108 individually by a serine (C108S) and an aspartate (C108D), and characterized the purified variants with regard to their biochemical and kinetic properties, thermostability, and reactivity towards oxidation by hypochlorite. Tandem MS analysis of tryptic peptides derived from a hypochlorite-treated inactive preparation of recombinant wild-type oxidase showed that Cys108 was converted into cysteine sulfonic acid, mimicking the oxidative modification of native enzyme as isolated. Colorimetric titration of protein thiol groups revealed that in the presence of ammonium benzoate (0.12 mM), the two muteins were not oxidized at cysteines whereas in the wild-type enzyme, one thiol group was derivatized. Each site-directed replacement caused a conformational change in D-amino acid oxidase, detected with an assortment of probes, and resulted in a turnover number for the O 2 -dependent reaction with D-Met which in comparison with the corresponding wild-type value was decreased two-and threefold for C108S and C108D, respectively. Kinetic analysis of thermal denaturation at 50°C was used to measure the relative contributions of partial unfolding and cofactor dissociation to the overall inactivation rate in each of the three enzymes. Unlike wild-type, C108S and C108D released the cofactor in a quasi-irreversible manner and were therefore not stabilized by external FAD against loss of activity. The results support a role of the anionic side chain of Cys108 in the fine-tuning of activity and stability of D-amino acid oxidase, explaining why C108S was a surprisingly poor mimic of the native enzyme.
Exploring the molecular nature of alternative oxidase regulation and catalysis
Febs Letters, 2002
Plant mitochondria contain a non-protonmotive alternative oxidase (AOX) that couples the oxidation of ubiquinol to the complete reduction of oxygen to water. In this paper we review theoretical and experimental studies that have contributed to our current structural and mechanistic understanding of the oxidase and to the clarification of the molecular nature of post-translational regulatory phenomena. Furthermore, we suggest a
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. KeyWordS D-amino acid oxidase from yeast Trigonopsis variabilis, protein engineering, hydrophobization of alpha-helices, site-directed mutagenesis, substrate specificity, thermal stability.
Mechanistic Studies of an Amine Oxidase Derived from d-Amino Acid Oxidase
Biochemistry, 2017
The flavoprotein D-amino acid oxidase has long served as a paradigm for understanding the mechanism of oxidation of amino acids by flavoproteins. Recently, a mutant D-amino acid oxidase (Y228L/R283G) that catalyzed the oxidation of amines rather than amino acids was described [Yasukawa, K., et al. (2014) Angew. Chem., Int. Ed. 53, 4428-4431]. We describe here the use of pH and kinetic isotope effects with (R)-α-methylbenzylamine as a substrate to determine whether the mutant enzyme utilizes the same catalytic mechanism as the wild-type enzyme. The effects of pH on the steady-state and rapid-reaction kinetics establish that the neutral amine is the substrate, while an active-site residue, likely Tyr224, must be uncharged for productive binding. There is no solvent isotope effect on the k cat /K m value for the amine, consistent with the neutral amine being the substrate. The deuterium isotope effect on the k cat /K m value is pH-independent, with an average value of 5.3, similar to values found with amino acids as substrates for the wild-type enzyme and establishing that there is no commitment to catalysis with this substrate. The k cat /K O2 value is similar to that seen with amino acids as the substrate, consistent with the oxidative halfreaction being unperturbed by the mutation and with flavin oxidation preceding product release. All of the data are consistent with the mutant enzyme utilizing the same mechanism as the wildtype enzyme, transfer of hydride from the neutral amine to the flavin.
D-amino acid oxidase: structure, catalytic mechanism, and practical application
Biochemistry (Moscow), 2005
Flavoproteins include numerous groups of enzymes distinct in structure and function. They catalyze the majority of key biochemical conversions and repre sent the most widely studied family of proteins. In con trast to dehydrogenases, which catalyze the interconver sion of dinucleotides, NAD + and NADP + , from oxidized form to the reduced one and vice versa, the FAD mole cule plays the role of a prosthetic group and its reduction state, oxidized as a rule, is the same in the end of a cat alytic act as it was in the beginning. Among the most intensely studied and actively used in practice FAD con taining oxidoreductases, we note glucose oxidase [3], lac tate oxidase [4], xanthine oxidase [5], cytochrome P450 reductase [6], and cytochrome b 5 reductase [7], etc.
Biochemistry (Moscow), 2012
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.
D-amino acid oxidase: structure, catalytic mechanism and application
Flavoproteins include numerous groups of enzymes distinct in structure and function. They catalyze the majority of key biochemical conversions [1, 2] and repree sent the most widely studied family of proteins. In conn trast to dehydrogenases, which catalyze the interconverr sion of dinucleotides, NAD + and NADP + , from oxidized form to the reduced one and vice versa, the FAD molee cule plays the role of a prosthetic group and its reduction state, oxidized as a rule, is the same in the end of a catt alytic act as it was in the beginning. Among the most intensely studied and actively used in practice FADDconn taining oxidoreductases, we note glucose oxidase [3], lacc tate oxidase [4], xanthine oxidase [5], cytochrome P450 reductase [6], and cytochrome b 5 reductase [7], etc.
Biotechnology Journal, 2009
D-Tagatose is a sweetener with low caloric and non-glycemic characteristics. It can be 16 produced by an enzymatic oxidation of D-galactose specifically at C2 followed by chemical 17 hydrogenation. Pyranose 2-Oxidase (P2Ox) from Trametes multicolor catalyzes the oxidation 18 of many aldopyranoses to their corresponding 2-keto derivatives. Since D-galactose is not 19 the preferred substrate of P2Ox, semi-rational design was employed to improve the catalytic 20 efficiency with this poor substrate. Saturation mutagenesis was applied on all positions in the 21 active site of the enzyme, resulting in a library of mutants, which were screened for improved 22 activity in a 96-well microtiter plate format. Mutants with higher activity than wild-type P2Ox 23 were chosen for further kinetic investigations. Variant V546C was found to show a 2.5-fold 24 increase of k cat with both D-glucose and D-galactose when oxygen was used as electron 25 acceptor. Because of weak substrate binding, however, k cat /K M is lower for both sugar 26 substrates compared to wild-type TmP2Ox. Furthermore, variants at position T169, i.e.,