Engineering the Properties of D-Amino Acid Oxidases by a Rational and a Directed Evolution Approach

Pollegioni, Loredano; Sacchi, Silvia; Caldinelli, Laura; Boselli, Angelo; Pilone, Mirella S.; Piubelli, Luciano; Molla, Gianluca

doi:10.2174/138920307783018677

Cited by 36 publications

(32 citation statements)

References 83 publications

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“…The pH-stability profile of RxDAO appeared similar to the profiles of RgDAO, TvDAO, and ApDAO but not to the profiles of pkDAO and CbDAO, which are stable under alkaline conditions (6,9). The pH-activity profile of RxDAO was similar to the profiles of RgDAO and pkDAO in terms of higher activity at alkaline pHs but not to the profiles of TvDAO, CbDAO, and ApDAO, which show lower activity at pH values above 8.5 (39).…”

Section: Discussionmentioning

confidence: 71%

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A Highly Stable d -Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

Takahashi

Furukawara

Omae

et al. 2014

Appl Environ Microbiol

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D-Amino acid oxidase (DAO) is a biotechnologically attractive enzyme that can be used in a variety of applications, but its utility is limited by its relatively poor stability. A search of a bacterial genome database revealed a gene encoding a protein homologous to DAO in the thermophilic bacterium Rubrobacter xylanophilus (RxDAO). The recombinant protein expressed in Escherichia coli was a monomeric protein containing noncovalently bound flavin adenine dinucleotide as a cofactor. This protein exhibited oxidase activity against neutral and basic D-amino acids and was significantly inhibited by a DAO inhibitor, benzoate, but not by any of the tested D-aspartate oxidase (DDO) inhibitors, thus indicating that the protein is DAO. RxDAO exhibited higher activities and affinities toward branched-chain D-amino acids, with the highest specific activity toward D-valine and catalytic efficiency (k cat /K m ) toward D-leucine. Substrate inhibition was observed in the case of D-tyrosine. The enzyme had an optimum pH range and temperature of pH 7.5 to 10 and 65°C, respectively, and was stable between pH 5.0 and pH 8.0, with a T 50 (the temperature at which 50% of the initial enzymatic activity is lost) of 64°C. No loss of enzyme activity was observed after a 1-week incubation period at 30°C. This enzyme was markedly inactivated by phenylmethylsulfonyl fluoride but not by thiol-modifying reagents and diethyl pyrocarbonate, which are known to inhibit certain DAOs. These results demonstrated that RxDAO is a highly stable DAO and suggested that this enzyme may be valuable for practical applications, such as the determination and quantification of branched-chain D-amino acids, and as a scaffold to generate a novel DAO via protein engineering.T he flavoenzyme D-amino acid oxidase (DAO; EC 1.4.3.3) catalyzes the oxidative deamination of neutral and basic D-amino acids but not of acidic D-amino acids, which are substrates of D-aspartate oxidase (DDO). DAO was first identified in pig kidney (pkDAO) by Krebs in 1935 (1) and subsequently has been found mainly in eukaryotic organisms, ranging from fungi to humans (2). These eukaryotic DAOs have been extensively studied as a model of the oxidase-dehydrogenase class of flavoproteins. In fungi, the enzyme plays a role in the assimilation of D-amino acids for cell growth and also in the detoxification of D-amino acid toxicity (2, 3). In vertebrates, this enzyme is mainly localized in the liver and kidney, and it metabolizes both exogenous and endogenous D-amino acids (2). In addition, the enzyme plays a specific role in the regulation of D-serine, a coagonist for the N-methyl-Daspartate receptor, in the brain (2).In contrast, for a long time, it was thought that DAO did not exist in prokaryotic organisms. However, this enzyme was recently identified in Arthrobacter protophormiae (ApDAO) and Streptomyces coelicolor (ScDAO) (4, 5). In addition to these bacterial species, a search of a prokaryotic genome database revealed a wide distribution of DAO homologous proteins in bacteria, especially a...

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Section: Discussionmentioning

confidence: 71%

“…The enzymatic stability of DAO is related to protein concentration, oligomerization, cofactor binding, and the oxidation of amino acid side chains (9,10). To improve its stability, DAO is usually immobilized on solid supports, such as magnetic, agarose, and epoxy beads (11).…”

mentioning

confidence: 99%

A Highly Stable d -Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

Takahashi

Furukawara

Omae

et al. 2014

Appl Environ Microbiol

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“…The second step iteratively converts the imino acid produced (from the D-amino acid) back into a DL-mixture to obtain the full resolution of the racemic mixture into the L-enantiomer (1). This approach requires stable recombinant DAAOs possessing wide substrate specificity as well as variants engineered to act on synthetic amino acids (3). Amino acid oxidases with reverse stereoselectivity are also well known flavooxidases, mainly produced by snakes or by microorganisms.…”

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confidence: 99%

Structure-Function Relationships in l-Amino Acid Deaminase, a Flavoprotein Belonging to a Novel Class of Biotechnologically Relevant Enzymes

Motta

Molla

Pollegioni

et al. 2016

Journal of Biological Chemistry

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L-Amino acid deaminase from Proteus myxofaciens (Pma-LAAD) is a membrane flavoenzyme that catalyzes the deamination of neutral and aromatic L-amino acids into ␣-keto acids and ammonia. PmaLAAD does not use dioxygen to re-oxidize reduced FADH 2 and thus does not produce hydrogen peroxide; instead, it uses a cytochrome b-like protein as an electron acceptor. Although the overall fold of this enzyme resembles that of known amine or amino acid oxidases, it shows the following specific structural features: an additional novel ␣؉␤ subdomain placed close to the putative transmembrane ␣-helix and to the active-site entrance; an FAD isoalloxazine ring exposed to solvent; and a large and accessible active site suitable to bind large hydrophobic substrates. In addition, PmaLAAD requires substrate-induced conformational changes of part of the active site, particularly in Arg-316 and Phe-318, to achieve the correct geometry for catalysis. These studies are expected to pave the way for rationally improving the versatility of this flavoenzyme, which is critical for biocatalysis of enantiomerically pure amino acids.A variety of enzymes has been used to prepare chiral pharmaceutical and agricultural compounds containing enantiomeric amine or amino acid groups. Among these are aminotransferases (EC 2.6.4.X), lipases (EC 3.1.1.X), amine oxidases (EC 1.4.3.22), amino acid dehydrogenases (EC 1.4.1.X), and amino acid oxidases (EC 1.4.3.X) (1, 2).The deracemization of a racemic amino acid to obtain the L-configuration was achieved by using a stereoselective Damino acid oxidase (DAAO, 5 EC 1.4.3.3) followed by chemical reduction. The second step iteratively converts the imino acid produced (from the D-amino acid) back into a DL-mixture to obtain the full resolution of the racemic mixture into the L-enantiomer (1). This approach requires stable recombinant DAAOs possessing wide substrate specificity as well as variants engineered to act on synthetic amino acids (3). Amino acid oxidases with reverse stereoselectivity are also well known flavooxidases, mainly produced by snakes or by microorganisms. In particular, L-amino acid oxidases (LAAO, EC 1.4.3.2) catalyze the stereoselective oxidative deamination of L-amino acids into the corresponding ␣-keto acids and ammonia; the re-oxidation of FADH 2 by dioxygen then generates H 2 O 2 (4). These flavoenzymes catalyze an irreversible reaction (differently from aminotransferases) and do not require a specific step of cofactor regeneration, as otherwise required by the NAD-dependent dehydrogenases. However, because of the problems associated with overexpression of snake venom LAAOs in recombinant hosts and the limited substrate acceptance of the microbial counterparts, no appropriate LAAOs for biocatalysis are available (4).L-Amino acid deaminases (LAADs) represent a suitable alternative to LAAOs. LAAD (first identified in the genera Proteus, Providencia, and Morganella) catalyzes the deamination of the L-isomer of amino acids, yielding the corresponding ␣-keto acids and ammonia without any evide...

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“…Protein engineering studies have allowed modulation of D -amino acid oxidases ' oligomerization state, stability (which is signifi cantly increased in the immobilized form), FAD binding, and substrate specifi city (for a review see [57] ). The residue Met213 of R. gracilis D -amino acid oxidase has been proposed as the most important residue for determining the substrate specifi city of the enzyme.…”

Section: -Amino Acid Oxidase ( Ec 1433)mentioning

confidence: 99%

“…Recently, R. gracilis D -amino acid oxidase was employed in the resolution of racemic mixtures of D , L -naphthyl amino acids and the M213G mutant showed a higher catalytic effi ciency on D -naphthyl alanine and D -naphthyl glycine [28] . Subsequently, fi ve mutants displaying an altered substrate specifi city were selected by a directed evolution approach of R. gracilis D -amino acid oxidase: these Damino acid oxidase variants have a better catalytic effi ciency for all the substrates used [57] .…”

Section: -Amino Acid Oxidase ( Ec 1433)mentioning

confidence: 99%

Chemo‐Enzymatic Deracemization Methods

Tessaro¹,

Molla²,

Pollegioni³

et al. 2008

Modern Biocatalysis

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13 IntroductionIn recent times, the term deracemization has been used for describing techniques becoming increasingly important for the preparation of chiral compounds as single enantiomers. The term refers to a process in which one single enantiomer is obtained when starting from a racemate [1] .The two more common strategies for achieving such an objective are deracemization by stereoinversion or deracemization by dynamic kinetic resolution DKR (Scheme 13.1 ).Deracemization by stereoinversion is a process in which one form (S f ) of the racemic starting material (R f + S f ) is enantioselectively transformed into an intermediate (S i ) which can in turn react to give the form of opposite confi guration (R f ). An example of this method could be the selective oxidation of one enantiomer of a racemic secondary alcohol and the subsequent reduction with a catalyst of opposite stereopreference [2] .Deracemization by DKR is in principle a kinetic resolution process in which the non -transformed enantiomer is racemized in situ. The conditions are that a chiral catalyst promotes the transformation of one enantiomer (R s ) into the product (R p ) while the other enantiomer is racemized at a comparable rate and the racemic mixture (R s + S s ) is restored. The product (R p ) is not racemized under the same conditions. While a simple kinetic resolution yields a maximum of 50% of the product, with this technique a 100% conversion can be reached. Although the majority of chiral molecules of industrial interest are still prepared by kinetic resolution, the continuous development of industrial enzymes and racemizing processes fosters new chemo -or biocatalytic systems for DKR to appear. A great impulse for deracemization methods based on the one -pot/two -steps resolution racemization process was brought about by B ä ckvall et al . over a 10 -year period [3] .The method is of general applicability in the deracemization of secondary alcohols and amines and consists of a lipase -catalyzed irreversible acylation and in situ racemization of the non -reacted enantiomer catalyzed by a ruthenium catalyst,

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Engineering the Properties of D-Amino Acid Oxidases by a Rational and a Directed Evolution Approach

Cited by 36 publications

References 83 publications

A Highly Stable d -Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

A Highly Stable d -Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

Structure-Function Relationships in l-Amino Acid Deaminase, a Flavoprotein Belonging to a Novel Class of Biotechnologically Relevant Enzymes

Chemo‐Enzymatic Deracemization Methods

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