D-amino acid oxidase: structure, catalytic mechanism, and practical application (original) (raw)
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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.
Properties and applications of microbial D-amino acid oxidases: current state and perspectives
Applied Microbiology and Biotechnology, 2008
D-Amino acid oxidase (DAAO) is a biotechnologically relevant enzyme that is used in a variety of applications. DAAO is a flavine adenine dinucleotidecontaining flavoenzyme that catalyzes the oxidative deamination of D-isomer of uncharged aliphatic, aromatic, and polar amino acids yielding the corresponding imino acid (which hydrolyzes spontaneously to the α-keto acid and ammonia) and hydrogen peroxide. This enzymatic activity is produced by few bacteria and by most eukaryotic organisms. In the past few years, DAAO from mammals has been the subject of a large number of investigations, becoming a model for the dehydrogenase-oxidase class of flavoproteins. However, DAAO from microorganisms show properties that render them more suitable for the biotechnological applications, such as a high level of protein expression (as native and recombinant protein), a high turnover number, and a tight binding of the coenzyme. Some important DAAO-producing microorganisms include Trigonopsis variabilis, Rhodotorula gracilis, and Fusarium solani. The aim of this paper is to provide an overview of the main biotechnological applications of DAAO (ranging from biocatalysis to convert cephalosporin C into 7-amino cephalosporanic acid to gene therapy for tumor treatment) and to illustrate the advantages of using the microbial DAAOs, employing both the native and the improved DAAO variants obtained by enzyme engineering.
Biotechnology Progress, 2008
Lacking an efficient process to produce 7-aminocephalosporanic acid from cephalosporin C in a single step, D-amino acid oxidase (DAAO) is of foremost importance in the industrial, two-step process used for this purpose. We report a detailed study on the catalytic properties of the three available DAAOs, namely, a mammalian DAAO and two others from yeast (Rhodotorula gracilis and Trigonopsis variabilis). In comparing the kinetic parameters determined for the three DAAOs, with both cephalosporin C and D-alanine as substrate, the catalytic efficiency of the two enzymes from microorganism is at least 2 orders of magnitude higher than that of pig kidney DAAO. Furthermore, the mammalian enzyme is more sensitive to product inhibition (from hydrogen peroxide and glutaryl-7-aminocephalosporanic acid). Therefore, enzymes from microorganisms appear to be by far more suitable catalysts for bioconversion, although some different minor differences are present between them (e.g., a higher activity of the R. gracilis enzyme when the bioconversion is carried out at saturating oxygen concentration). The mammalian DAAO, even being a poor catalyst, is more stable with respect to temperature than the R. gracilis enzyme in the free form. In any case, for industrial purposes DAAO is used only in the immobilized form where a strong enzyme stabilization occurs.
Recombinant d-amino acid oxidase with improved properties
Journal of Biotechnology, 2008
D-amino acid oxidase from Trigonopsis variabilis (TvDAAO) was overproduced in Escherichia coli cells, and properties of the recombinant enzyme were studied. Single point mutants of the enzyme with 2.4-fold higher thermal stability or changed spectra of substrate specificity compared with wild-type enzyme were prepared. It was shown that mutant TvDAAO has higher catalytic efficiency in cephalosporin C oxidation in comparison with wild-type enzyme. One mutant of recombinant TvDAAO was crystallized and its structure was solved with resolution 2.8 Å.
Assays of D-Amino Acid Oxidase Activity
Frontiers in Molecular Biosciences, 2018
D-amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs). Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples.
Engineering the Properties of D-Amino Acid Oxidases by a Rational and a Directed Evolution Approach
Current Protein & Peptide Science, 2007
D-amino acid oxidase (DAAO) is a FAD-containing flavoprotein that dehydrogenates the D-isomer of amino acids to the corresponding imino acids, coupled with the reduction of FAD. The cofactor then reoxidizes on molecular oxygen and the imino acid hydrolyzes spontaneously to the -keto acid and ammonia. In vitro DAAO displays broad substrate specificity, acting on several neutral and basic D-amino acids: the most efficient substrates are amino acids with hydrophobic side chains. D-aspartic acid and D-glutamic acid are not substrates for DAAO. Through the years, it has been the subject of a number of structural, functional and kinetic investigations. The most recent advances are represented by site-directed mutagenesis studies and resolution of the 3D-structure of the enzymes from pig, human and yeast. The two approaches have given us a deeper understanding of the structure-function relationships and promoted a number of investigations aimed at the modulating the protein properties. By a rational and/or a directed evolution approach, DAAO variants with altered substrate specificity (e.g., active on acidic or on all D-amino acids), increased stability (e.g., stable up to 60 °C), modified interaction with the flavin cofactor, and altered oligomeric state were produced. The aim of this paper is to provide an overview of the most recent research on the engineering of DAAOs to illustrate their new intriguing properties, which also have enabled us to pursue new biotechnological applications. Fig. (1). (A) Reaction of oxidation of D-amino acids catalyzed by DAAO. (B) Kinetic mechanism of the catalytic cycle proposed for DAAO (upper branch, ping-pong mechanism; lower branch, sequential mechanism). Substrates and products are represented in open circles. Fl = Flavin cofactor. Protein Engineering of DAAO
Protein Engineering Design and Selection, 2004
Recent research on the flavoenzyme D-amino acid oxidase from Rhodotorula gracilis (RgDAAO) has revealed new, intriguing properties of this catalyst and offers novel biotechnological applications. Among them, the reaction of RgDAAO has been exploited in the analytical determination of the D-amino acid content in biological samples. However, because the enzyme does not oxidize acidic D-amino acids, it cannot be used to detect the total amount of D-amino acids. We now present the results obtained using a random mutagenesis approach to produce RgDAAO mutants with a broader substrate specificity. The libraries of RgDAAO mutants were generated by error-prone PCR, expressed in BL21(DE3)pLysS Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. Five random mutants that have a 'modified' substrate specificity, more useful for the analytical determination of the entire content of D-amino acids than wild-type RgDAAO, have been isolated. With the only exception of Y223 and G199, none of the effective amino acid substitutions lie in segments predicted to interact directly with the bound substrate. The substitutions appear to cluster on the protein surface: it would not have been possible to predict that these substitutions would enhance DAAO activity. We can only conclude that these substitutions synergistically generate small structural changes that affect the dynamics and/or stability of the protein in a way that enhances substrate binding or subsequently catalytic turnover.
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.
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.
Carp D-amino acid oxidase: Structural active site basis of its catalytic mechanisms
ScienceAsia
The three-dimensional structure of the flavoprotein D-amino acid oxidase (DAO, EC 1.4.3.3) from carp hepatopancreas (chDAO) and its active site cavity was modelled using ProModII. The structural features relevant for the overall conformation and for the catalytic activity are described. Secondary structure topology consists of 11 -helices and 17 -strands, which differs slightly from pig kidney, and Rhodotorula gracilis DAOs. chDAO showed a theoretical 'head-to-head' mode of dimerization. The presence of a short 'lid' in chDAO covering the active site, commonly found in mammalian DAO but absent in R. gracilis DAO, is interpreted as the origin of the differences in kinetic mechanism among these enzymes. This lid has been proposed to control the access of the substrate to the active site and to regulate dissociation of products. The conformational change in the large size active site loop determines the overall rate of turnover of DAOs. The shorter active site loop foun...