Porcine Kidney D-amino Acid Oxidase Research Articles

Combination of Enzymatic Oxidation of Amino Acid and Native Chemical Ligation with Hydroxylamine for Amide Formation toward a One-pot Process

The formation of amide bonds is important in many areas of chemistry, and the ligation by condensation of N-hydroxylamine and an α-keto acid has been attracting increasing attention. Porcine kidney d-amino acid oxidase, which catalyzes the oxidative deamination of amino acids to yield α-keto acids, ammonia, and hydrogen peroxide, and native chemical ligation were used to access α-keto acids, and amides were synthesized with N-hydroxylamine. d-Phenylalanine was converted to phenylpyruvic acid obtained by recombinant pig d-amino acid oxidase in high yield (97%). The α-keto acid was ligated with N-benzylhydroxylamine and N-hydroxylphenylalanine in N,N-dimethylformamide, and benzyl-2-phenylacetamide and (2-phenylacetyl)phenylalanine in 82% and 79% yields, respectively. Other amide compounds were also obtained via this synthesis.

Expansion of the Substrate Specificity of Porcine Kidney D-Amino Acid Oxidase for S-Stereoselective Oxidation of 4-Cl-Benzhydrylamine.

Discovery and development of enzymes for the synthesis of chiral amines have been a hot topic for basic and applied aspects of biocatalysts. Based on our X‐ray crystallographic analyses of porcine kidney D‐amino acid oxidase (pkDAO) and its variants, we rationally designed a new variant that catalyzed the oxidation of (S)‐4‐Cl‐benzhydrylamine (CBHA) from pkDAO and obtained it by functional high‐throughput screening with colorimetric assay. The variant I230A/R283G was constructed from the variant R283G which had completely lost the activity for D‐amino acids, further gaining new activity toward (S)‐chiral amines with the bulky substituents. The variant enzyme (I230A/R283G) was characterized to have a catalytic efficiency of 1.85 s−1 for (S)‐CBHA, while that for (R)‐1‐phenylethylamine was diminished 10‐fold as compared with the Y228L/R283G variant. The variant was efficiently used for the synthesis of (R)‐CBHA in 96 % ee from racemic CBHA by the deracemization reaction in the presence of reducing agent such as NaBH4 in water. Furthermore, X‐ray crystallographic analysis of the new variant complexed with (S)‐CBHA, together with modelling study clearly showed the basis of understanding the structure‐activity relationship of pkDAO.

Diversity-Oriented Synthesis of Coumarin-Linked Benzimidazoles via a One-Pot, Three-Step, Intramolecular Knoevenagel Cyclization.

Diversity-oriented synthesis of coumarin-linked benzimidazoles from N-(2-aminophenyl)-2-cyanoacetamide was achieved via a one-pot, three-step sequential reaction in excellent yields. In situ intramolecular cyclization of the cyanoacetamide afforded benzimidazoles which subsequently underwent a Knoevenagel condensation of the 2-cyanomethylbenzimidazoles with salicylaldehydes promoted by triethylamine to reach the target compounds. An important intermediate, 2-(2-imino-2H-chromen-3-yl)-1H-benzimidazole was characterized by X-ray analysis and further hydrolyzed to 2-(coumarin-3-yl)benzimidazole in acidic condition. Among the synthesized compounds, some were found to be promising inhibitors of porcine kidney d-amino acid oxidase (pkDAO).

English

Porcine kidney D-amino acid oxidase was stabilized by covalent immobilization on spherical particles of Eupergit C because of its low stability in soluble form. The main focus of this work was put on evaluation of operational stability of the immobilized enzyme. To evaluate D-amino acid oxidase’s operational stability during process conditions, repetitive batch reactor experiments of D-methionine oxidation reaction were carried out with continuous aeration for oxygen supply at air-flow rates: of 5 and 10 dm3 h-1. Kinetic analysis of the immobilized enzyme was done as well. The mathematical model of D-methionine oxidative deamination catalyzed by the immobilized D-amino acid oxidase was developed and it described the data well. It enabled the estimation of operational stability decay rate constant. It was possible to achieve 100 % substrate conversion in all batch experiments.

Tailoring D‐Amino Acid Oxidase from the Pig Kidney to R‐Stereoselective Amine Oxidase and its Use in the Deracemization of α‐Methylbenzylamine

The deracemization of racemic amines to yield enantioenriched amines using S-stereoselective amine oxidases (AOx) has recently been attracting attention. However, R-stereoselective AOx that are suitable for deracemization have not yet been identified. An R-stereoselective AOx was now evolved from porcine kidney D-amino acid oxidase (pkDAO) and subsequently use for the deracemization of racemic amines. The engineered pkDAO, which was obtained by directed evolution, displayed a markedly changed substrate specificity towards R amines. The mutant enzyme exhibited a high preference towards the substrate α-methylbenzylamine and was used to synthesize the S amine through deracemization. The findings of this study indicate that further investigations on the structure-activity relationship of AOx are warranted and also provide a new method for biotransformations in organic synthesis.

Engineering the Substrate Specificity of Porcine Kidney d-Amino Acid Oxidase by Mutagenesis of the “Active-Site Lid”

Comparison of the primary structures of pig kidney D-amino acid oxidase (DAO) and human brain D-aspartate oxidase (DDO) revealed a notable difference at I215-N225 of DAO and the corresponding region, R216-G220, of DDO. A DAO mutant, in which I215-N225 is substituted by R216-G220 of DDO, showed D-aspartate-oxidizing activity that wild-type DAO does not exhibit, together with a considerable decrease in activity toward D-alanine. These findings indicate that I215-N225 of DAO contributes profoundly to its substrate specificity. Based on these results and the crystal structure of DAO, we systematically mutated the E220-Y224 region within the short stretch in question and obtained five mutants (220D224G, 221D224G, 222D224G, 223D224G, and 224D), in each of which an aspartate residue is mutated to E220-Y224. All of the mutants exhibited decreased apparent K(m) values toward D-arginine, i.e., to one-seventh to one-half that of wild type DAO. The specificity constant, k(cat app)/K(m app), for D-arginine increased by one order of magnitude for the 221D224G or 222D224G mutant, whereas that for D-alanine or D-serine decreased to marginal or nil.

Deracemisation and stereoinversion of alpha-amino acids using D-amino acid oxidase and hydride reducing agents.

The deracemisation and stereoinversion of both cyclic and acyclic DL-alpha-amino acids, using porcine kidney D-amino acid oxidase (DAAO) and a hydride reducing agent (NaCNBH3-NaBH4), has been investigated.

Three-dimensional structure of the purple intermediate of porcine kidney D-amino acid oxidase. Optimization of the oxidative half-reaction through alignment of the product with reduced flavin.

The three-dimensional structure of the purple intermediate of porcine kidney D-amino acid oxidase (DAO) was solved by cryo-X-ray crystallography; the purple intermediate is known to comprise a complex between the dehydrogenated product, an imino acid, and the reduced form of DAO. The crystalline purple intermediate was obtained by anaerobically soaking crystals of oxidized DAO in a buffer containing excess D-proline as the substrate. The dehydrogenated product, delta(1)-pyrrolidine-2-carboxylate (DPC), is found sandwiched between the phenol ring of Tyr 224 and the planar reduced flavin ring. The cationic protonated imino nitrogen is within hydrogen-bonding distance of the backbone carbonyl oxygen of Gly 313. The carboxyl group of DPC is recognized by the Arg 283 guanidino and Tyr 228 hydroxyl groups through ion-pairing and hydrogen-bonding, respectively. The (+)HN=C double bond of DPC overlaps the N(5)-C(4a) bond of reduced flavin. The electrostatic effect of the cationic nitrogen of DPC is suggested to shift the resonance hybridization of anionic reduced flavin toward a canonical form with a negative charge at C(4a), thereby augmenting the electron density at C(4a), from which electrons are transferred to molecular oxygen during reoxidation of reduced flavin. The reactivity of reduced flavin in the purple intermediate, therefore, is enhanced through the alignment of DPC with respect to reduced flavin.

Purification and characterization of a novel glycine oxidase from Bacillus subtilis

The open reading frame yjbR which had been sequenced as a part of the Bacillus subtilis genome project encodes a putative 40.9-kDa protein. The yjbR-coding sequence was slightly similar to those of bacterial sarcosine oxidases and possibly compatible with the tertiary structure of the porcine kidney d-amino acid oxidase. The yjbR gene product was overproduced in Escherichia coli, purified to homogeneity from the recombinant strain, and characterized. This protein effectively catalyzed the oxidation of sarcosine ( N-methylglycine), N-ethylglycine and glycine. Lower activities on d-alanine, d-valine, and d-proline were detected although no activities were shown on l-amino acids and other d-amino acids. Since glycine is a product and not a substrate for sarcosine oxidase, this protein is not a type of demethylating enzymes but a novel deaminating oxidase, named glycine oxidase as a common name. Several enzymatic properties of the B. subtilis glycine oxidase were also investigated.

Three-dimensional structure of porcine kidney D-amino acid oxidase at 3.0 A resolution.

The X-ray crystallographic structure of porcine kidney D-amino acid oxidase, which had been expressed in Escherichia coli transformed with a vector containing DAO cDNA, was determined by the isomorphous replacement method for the complex form with benzoate. The known amino acid sequence, FAD and benzoate were fitted to an electron density map of 3.0 A resolution with an R-factor of 21.0%. The overall dimeric structure exhibits an elongated ellipsoidal framework. The prosthetic group, FAD, was found to be in an extended conformation, the isoalloxazine ring being buried in the protein core. The ADP moiety of FAD was located in the typical beta alpha beta dinucleotide binding motif, with the alpha-helix dipole stabilizing the pyrophosphate negative charge. The substrate analog, benzoate, is located on the re-face of the isoalloxazine ring, while the si-face is blocked by hydrophobic residues. The carboxylate group of benzoate is ion-paired with the Arg283 side chain and is within interacting distance with the hydroxy moiety of Tyr228. The phenol ring of Tyr224 is located just above the benzene ring of benzoate, implying the importance of this residue for catalysis. There is no positive charge or alpha-helix dipole near N(1) of flavin. Hydrogen bonds were observed at C(2) = O, N(3)-H, C(4) = O, and N(5) of the flavin ring.

Crystallization of expressed porcine kidney D-amino acid oxidase and preliminary X-ray crystallographic characterization.

The cDNA for porcine kidney D-amino acid oxidase (DAO) was cloned by means of the reverse transcription-polymerase chain reaction system from porcine kidney RNA and over-expressed in Escherichia coli which had been transformed with a vector containing the DAO cDNA. The expressed DAO was purified to homogeneity by a three-step procedure, i.e., heat-treatment, DEAE Sepharose column chromatography, and hydroxyapatite column chromatography. The purified DAO preparation, rDAO (recombinant DAO), showed an identical UV-visible absorption spectrum and catalytic activity with those of the wild-type enzyme purified from porcine kidney. Crystallization of rDAO was performed by the hanging-drop method and crystals of suitable quality for X-ray crystallography were obtained. The crystals so obtained diffracted to 2.5 A with a conventional X-ray source, and to 2.0 A with synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 110.3, b = 92.9, c = 71.6 A. A Vm value of 2.35 A3/Da indicates that there are two subunits related by a twofold non-crystallographic axis in the asymmetric unit. Two heavy atom derivatives have been identified.

Studies on Phe-228 and Leu-307 Recombinant Mutants of Porcine Kidney D-Amino Acid Oxidase: Expression, Purification, and Characterization1

Two recombinant mutants of porcine kidney D-amino acid oxidase [EC 1.4.3.3, DAO], in which Tyr(228) and His(307) are replaced with Phe and Leu, respectively, have been expressed in Escherichia coli and purified to apparent homogeneity. The molecular size and amino-terminal sequence of the two mutants were the same as those of the native DAO. Kinetic analysis revealed that the Michaelis constants of the Phe-228 and Leu-307 mutants for D-alanine were 71- and 10-fold and the inhibition constants for benzoate, a potent competitive inhibitor, were 1,189- and 18-fold greater than those of the native DAO, respectively. The maximum velocities of the Phe-228 and Leu-307 mutants were 66 and 58% that of the native DAO. The kinetically estimated dissociation constant of the Leu-307 mutant for FAD was 28-fold greater than that of the native DAO, whereas the value of the Phe-228 mutant was comparable to that of the native DAO. The Leu-307 mutant and the recombinant wild-type DAO were inactivated by D-propargylglycine (D-PG), a suicide substrate. However, the Phe-228 mutant was resistant to the inactivation. Absorption peaks of the Phe-228 mutant were blue-shifted about 10 nm from the corresponding peaks of the wild-type DAO, and the oxidized form was fully reduced by D-alanine without appearance of the purple intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-Specific Mutagenesis of Lysine-204, Tyrosine-224, Tyrosine-228, and Histidine-307 of Porcine Kidney D-Amino Acid Oxidase and the Implications as to Its Catalytic Function1

In order to evaluate the possible contributions of Lys-204, Tyr-224, Tyr-228, and His-307 in porcine kidney D-amino acid oxidase [EC 1.4.3.3] (DAO) to its catalytic function, we constructed four point mutant cDNAs encoding enzymes possessing Glu-204, Phe-224, Phe-228, and Leu-307 by oligonucleotide-directed in vitro mutagenesis. The four mutant cDNAs and the wild type cDNA could be expressed in vitro with similar efficiencies and about 200 ng of each enzyme protein was produced from 5 micrograms of the respective capped RNA. The electrophoretic mobilities of the in vitro synthesized mutant enzymes on SDS-polyacrylamide gel were almost identical with that of the wild type DAO, and the molecular weight was calculated to be 38,000. The Glu-204 and Phe-224 mutant DAOs showed comparable enzyme activities to that of the wild type enzyme, and were inhibited strongly by sodium benzoate, a potent competitive inhibitor of DAO. The kinetic parameters of the two mutant DAOs were also comparable to those of the wild type DAO. On the other hand, the Phe-228 and Leu-307 mutant DAOs showed no detectable activity. The results indicate that Tyr-228 and His-307 play important roles as to the constitution of the active site or participate in the reaction directly, while Lys-204 and Tyr-224 are not essential in the enzyme reaction.

19F-NMR Study on the Interaction of Fluorobenzoate with Porcine Kidney D-Amino Acid Oxidase

The interactions of competitive inhibitors, o-, m-, and p-fluorobenzoates, with porcine kidney D-amino acid oxidase (DAO) were studied by 19F-NMR spectroscopy. The 19F-signals of DAO-bound fluorobenzoates were observed as considerably broadened peaks. The chemical shifts, which are referenced to 20 mM NaF in 50 mM sodium phosphate, pH 7.0, were 6.0, 8.2, and 11.9 ppm for free o-, m-, and p-fluorobenzoates, respectively, while those of o-, m-, and p-fluorobenzoates bound to DAO were 12.5, 5.4, and 13.1 ppm, respectively. The 19F-signals of bound o- and p-fluorobenzoates were downfield-shifted relative to those of the free species, whereas the 19F-resonance of m-fluorobenzoate was up-field shifted from that of the free ligand. The magnitude of the chemical shift difference between the free and bound forms decreases in the order of o-, m-, and p-fluorobenzoates. The remarkably large downfield shift of the o-fluorobenzoate when bound to DAO was attributed to the close proximity of the ortho-fluorine atom to the flavin nucleus in comparison with meta- or para-fluorine. The pH-dependences of the 19F-resonances of o-, m-, and p-fluorobenzoates were observed and the pKa values of 3.33, 3.80, and 4.05 were obtained for the carboxyl groups of o-, m-, and p-fluorobenzoates, respectively. It was observed that the 19F-resonances of o- and p-fluorobenzoates are highly sensitive to the ionic state of the carboxyl group, while that of m-fluorobenzoate was moderately sensitive.

Effect of site-specific mutagenesis of tyrosine-55, methionine-110 and histidine-217 in porcine kidney D-amino acid oxidase on its catalytic function

To assess the contributions of Tyr-55, Met-110 and His-217 in porcine kidney D-amino acid oxidase (EC 1.4.3.3, DAO) to its catalytic function, we constructed three mutant cDNAs coding for the enzymes possessing Phe-55, Leu-110 and Leu-217 by site-specific mutagenesis. The mutant and wild type cDNAs could be expressed in vitro with similar efficiency. The three mutant enzymes thus synthesized showed catalytic activities comparable to that of the wild type oxidase. It is concluded that Tyr-55, Met-110 and His-217 are not directly involved in the catalytic function.

Molecular cloning and sequence analysis of cDNAs encoding porcine kidney D-amino acid oxidase.

Complementary DNAs encoding D-amino acid oxidase (EC 1.4.3.3, DAO), one of the principal and characteristic enzymes of the peroxisomes of porcine kidney, have been isolated from the porcine kidney cDNA library by hybridization with synthetic oligonucleotide probes corresponding to the partial amino acid sequences. Analysis of the nucleotide sequences of two clones revealed a complete 3211-nucleotide sequence with a 5'-terminal untranslated region of 198 nucleotides, 1041 nucleotides of an open reading frame that encoded 347 amino acids, and a 3'-terminal untranslated region of 1972 nucleotides. The deduced amino acid sequence was completely identical with the reported sequence of the mature enzyme [Ronchi, S., Minchiotti, L., Galliano, M., Curti, B., Swenson, R. P., Williams, C. H. J., & Massey, V. (1982) J. Biol. Chem. 257, 8824-8834]. These results indicate that the primary translation product does not contain a signal peptide at its amino-terminal region for its translocation into peroxisomes. RNA blot hybridization analysis suggests that porcine kidney D-amino acid oxidase is encoded by three mRNAs that differ in size: 3.3, 2.7, and 1.5 kilobases. Comparison of the sequences of the two cDNA clones revealed that multiple polyadenylation signal sequences (ATTAAA and AACAAA) are present in the 3'-untranslated region, making the different mRNA species. The efficiency of 3' processing of the RNA was quite different between the two signal sequences ATTAAA and AACAAA. Southern blot analysis showed the presence of a unique gene for D-amino acid oxidase in the porcine genome.

13C-NMR studies of porcine kidney D-amino acid oxidase reconstituted with 13C-enriched flavin adenine dinucleotide. Effects of competitive inhibitors.

D-Amino acid oxidase (DAO) from porcine kidney was reconstituted with FAD's which were enriched with 13C at the 2-, 4-, 4a-, and 10a-positions of the isoalloxazine moiety, and 13C-NMR spectra of the reconstituted DAO were measured in the absence and presence of various competitive inhibitors. When compared to the corresponding chemical shifts of FMN at infinite dilution (Moonen, C.T.W. et al. (1984) Biochemistry 23, 4859-4867), the resonances of C(2), C(4), C(4a), and C(10a) of FAD bound to apoDAO were all shifted to higher field. However, when the reconstituted DAO was complexed with o-, m-, p-aminobenzoate, or benzoate, each of the four 13C-signals underwent a different change in chemical shift. In these changes we observed no characteristics which distinguish DAO-aminobenzoate complexes from DAO-benzoate complex, even though o-, m-, and p-aminobenzoates are known to form charge-transfer complexes with DAO. The signals due to 2- and 4-13C were more sensitive to the formation of the inhibitor-DAO complexes than those of the other carbon atoms. These findings suggest the modulation of the hydrogen bonds at the oxygen atoms of C(2) = O and C(4) = O with the protein moiety as the result of the inhibitor-binding.

Biosynthesis of porcine kidney D-amino acid oxidase

The biosynthesis of a porcine kidney peroxisomal enzyme, D-amino acid oxidase (EC 1.4.3.3., DAO), was investigated. Pig kidney mRNA as well as free and membrane-bound polysomes were used to investigate in vitro protein synthesis using a rabbit reticulocyte lysate. mRNA and free polysomes, but not membrane-bound polysomes, directed the synthesis of DAO. To examine the in vivo synthesis of the enzyme, a pig kidney cell line (LLC-PK1) was biosynthetically labelled. Both the in vitro and in vivo synthesized DAO had the same molecular weight, 38,000, as that of the purified enzyme. These results indicate strongly that DAO is synthesized on free ribosomes and transferred to the interior of peroxisomes without any proteolytic modification.