Biochemical characterization of mouse d-aspartate oxidase

D-amino acids research field has recently gained an increased interest since these atypical molecules have been discovered to play a plethora of different roles. In the mammalian central nervous system, d-aspartate (D-Asp) is critically involved in the regulation of glutamatergic neurotransmission by acting as an agonist of NMDA receptor. Accordingly, alterations in its metabolism have been related to different pathologies. D-Asp shows a peculiar temporal pattern of emergence during ontogenesis and soon after birth its brain levels are strictly regulated by the catabolic enzyme d-aspartate oxidase (DASPO), a FAD-dependent oxidase. Rodents have been widely used as in vivo models for deciphering molecular mechanisms and for testing novel therapeutic targets and drugs, but human targets can significantly differ. Based on these considerations, here we investigated the structural and functional properties of the mouse DASPO, in particular kinetic properties, ligand and flavin binding, oligomerization state and protein stability. We compared the obtained findings with those of the human enzyme (80% sequence identity) highlighting a different oligomeric state and a lower activity for the mouse DASPO, which apoprotein species exists in solution in two forms differing in FAD affinity. The features that distinguish mouse and human DASPO suggest that this flavoenzyme might control in a distinct way the brain D-Asp levels in different organisms.     

Site-directed mutagenesis of La protease. A catalytically active serine residue.

Comparative sequence analysis of Escherichia coli ATP-dependent La protease led to the suggestion that Ser679 is the catalytically active enzyme residue. Site-directed mutagenesis Ser679----Ala, investigation of the cells containing the mutant plasmid, and study of the partially purified mutant protein produced results in favour of this suggestion.     

Gender-related differences of mouse liver d-aspartate oxidase in the activity and response to administration of d-aspartate and peroxisome proliferators

• 1.1. The hepatic d-aspartate oxidase activity was found to be higher in female ddY and ICR mice than in their male counterparts. On the contrary, the free d-aspartate content in the liver was lower in female mice than in male mice, suggesting that d-aspartate is actually metabolized by d-aspartate oxidase in vivo.
• 2.2. Oral administration of d-aspartate to the animals increased the hepatic d-aspartate oxidase activity 2–3 fold in both genders without any significant difference in the rate of the increase between the genders.
• 3.3. Several peroxisomal enzyme activities other than d-aspartate oxidase examined were not affected by this treatment.
• 4.4. Experiments in vitro suggested that the increase in the d-aspartate activity might be explained in part by stabilization of the enzyme by d-aspartate.
• 5.5. The administration of clofibrate, a peroxisome proliferator, to male mice, increased the hepatic d-aspartate oxidase activity with a significant simultaneous decrease of d-aspartate content in the liver, in agreement with a possible role of the enzyme n vivo.
• 6.6. On the other hand, the administration of clofibrate or dehydroepiandrosterone to female mice decreased the d-aspartate oxidase activity.
• 7.7. The peroxisome proliferators were suggested to act to eliminate the gender difference of hepatic d-aspartate oxidase activity in mice.

     

On the mechanism of Rhodotorula gracilis D-amino acid oxidase: role of the active site serine 335

Serine 335 at the active site of D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO) is not conserved in other DAAO sequences. To assess its role in catalysis, it was mutated to Gly, the residue present in mammalian DAAO, an enzyme with a 35-fold lower turnover number with D-alanine. The spectral and ligand binding properties of the S335G mutant are similar to those of wild-type enzyme, suggesting an active site with minimally altered electrostatic properties. The S335G mutant is catalytically active, excluding an essential role of S335 in catalysis. However, S335-OH contributes to the high efficiency of the mutant enzyme since the catalytic activity of the latter is lower due to a decreased rate of flavin reduction relative to wild-type RgDAAO. Catalytic rates are pH-dependent and appear to converge to very low, but finite and similar values at low pH for both wild-type and S335G RgDAAO. While this dependence exhibits two apparent pKs with wild-type RgDAAO, with the S335G mutant a single, apparent pK approximately 8 is observed, which is attributed to the ionization of the alphaNH2 group of the bound substrate. Removal of S335-OH thus suppresses an apparent pK approximately 6. Both wild-type RgDAAO and the S335G mutant exhibit a substantial deuterium solvent kinetic isotope effect (> or =4) at pH<7 that disappears with increasing pH and reflects a pKapp=6.9 +/- 0.4. Interestingly, the substitution suppresses the activity towards d-lactate, suggesting a role of the serine 335 in removal of the substrate alpha-OH hydrogen.     

On the catalytic role of the conserved active site residue His466 of choline oxidase

The oxidation of alcohols to aldehydes is catalyzed by a number of flavin-dependent enzymes, which have been grouped in the glucose-methanol-choline oxidoreductase enzyme superfamily. These enzymes exhibit little sequence similarity in their substrates binding domains, but share a highly conserved catalytic site, suggesting a similar activation mechanism for the oxidation of their substrates. In this study, the fully conserved histidine residue at position 466 of choline oxidase was replaced with an alanine residue by site-directed mutagenesis and the biochemical, spectroscopic, and mechanistic properties of the resulting CHO-H466A mutant enzyme were characterized. CHO-H466A showed k(cat) and k(cat)/K(m) values with choline as substrate that were 60- and 1000-fold lower than the values for the wild-type enzyme, while the k(cat)/K(m) value for oxygen was unaffected, suggesting the involvement of His(466) in the oxidation of the alcohol substrate but not in the reduction of oxygen. Replacement of His(466) with alanine significantly affected the microenvironment of the flavin, as indicated by the altered behavior of CHO-H466A with sulfite and dithionite. In agreement with this conclusion, a midpoint reduction potential of +106 mV for the two-electron transfer in the catalytically competent enzyme-product complex was determined at pH 7 for CHO-H466A, which was approximately 25 mV more negative than that of the wild-type enzyme. Enzymatic activity in CHO-H466A could be partially rescued with exogenous imidazolium, but not imidazole, consistent with the protonated form of histidine exerting a catalytic role. pH profiles for glycine betaine inhibition, the deprotonation of the N(3)-flavin locus, and the k(cat)/K(m) value for choline all showed a significant shift upward in their pK(a) values, consistent with a change in the polarity of the active site. Finally, kinetic isotope effects with isotopically labeled substrate and solvent indicated that the histidine to alanine substitution affected the timing of substrate OH and CH bond cleavages, consistent with removal of the hydroxyl proton being concerted with hydride transfer in the mutant enzyme. All taken together, the results presented in this study suggest that in choline oxidase, His(466) modulates the electrophilicity of the enzyme-bound flavin and the polarity of the active site, and contributes to the stabilization of the transition state for the oxidation of choline to betaine aldehyde.     

Site-directed mutagenesis of the active site serine290 in flavanone 3beta-hydroxylase from Petunia hybrida.

Flavanone 3beta-hydroxylase (FHT) catalyzes a pivotal reaction in the formation of flavonoids, catechins, proanthocyanidins and anthocyanidins. In the presence of oxygen and ferrous ions the enzyme couples the oxidative decarboxylation of 2-oxoglutarate, releasing carbon dioxide and succinate, with the oxidation of flavanones to produce dihydroflavonols. The hydroxylase had been cloned from Petunia hybrida and expressed in Escherichia coli, and a rapid isolation method for the highly active, recombinant enzyme had been developed. Sequence alignments of the Petunia hydroxylase with various hydroxylating 2-oxoglutarate-dependent dioxygenases revealed few conserved amino acids, including a strictly conserved serine residue (Ser290). This serine was mutated to threonine, alanine or valine, which represent amino acids found at the corresponding sequence position in other 2-oxoglutarate-dependent enzymes. The mutant enzymes were expressed in E. coli and purified to homogeneity. The catalytic activities of [Thr290]FHT and [Ala290]FHT were still significant, albeit greatly reduced to 20 and 8%, respectively, in comparison to the wild-type enzyme, whereas the activity of [Val290]FHT was negligible (about 1%). Kinetic analyses of purified wild-type and mutant enzymes revealed the functional significance of Ser290 for 2-oxoglutarate-binding. The spatial configurations of the related Fe(II)-dependent isopenicillin N and deacetoxycephalosporin C synthases have been reported recently and provide the lead structures for the conformation of other dioxygenases. Circular dichroism spectroscopy was employed to compare the conformation of pure flavanone 3beta-hydroxylase with that of isopenicillin N synthase. A double minimum in the far ultraviolet region at 222 nm and 208-210 nm and a maximum at 191-193 nm which are characteristic for alpha-helical regions were observed, and the spectra of the two dioxygenases fully matched revealing their close structural relationship. Furthermore, the spectrum remained unchanged after addition of either ferrous ions, 2-oxoglutarate or both of these cofactors, ruling out a significant conformational change of the enzyme on cofactor-binding.     

Identification of the active site serine in pancreatic cholesterol esterase by chemical modification and site-specific mutagenesis

Chemical modification and site-specific mutagenesis approaches were used in this study to identify the active site serine residue of pancreatic cholesterol esterase. In the first approach, purified porcine pancreatic cholesterol esterase was covalently modified by incubation with [3H]diisopropylfluorophosphate (DFP). The radiolabeled cholesterol esterase was digested with CNBr, and the peptides were separated by high performance liquid chromatography. A single 3H-containing peptide was obtained for sequence determination. The results revealed the binding of DFP to a serine residue within the serine esterase homologous domain of the protein. Furthermore, the DFP-labeled serine was shown to correspond to serine residue 194 of rat cholesterol esterase (Kissel, J. A., Fontaine, R. N., Turck, C. W., Brockman, H. L., and Hui, D. Y. (1989) Biochim. Biophys. Acta 1006, 227-236). The codon for serine 194 in rat cholesterol esterase cDNA was then mutagenized to ACT or GCT to yield mutagenized cholesterol esterase with either threonine or alanine, instead of serine, at position 194. Expression of the mutagenized cDNA in COS-1 cells demonstrated that substitution of serine 194 with threonine or alanine abolished enzyme activity in hydrolyzing the water-soluble substrate, p-nitrophenyl butyrate, and the lipid substrates cholesteryl [14C]oleate and [14C] lysophosphatidylcholine. These studies definitively identified serine 194 in the catalytic site of pancreatic cholesterol esterase.     

Histidine as an essential residue in the active site of the copper enzyme galactose oxidase.

The pH dependence of the oxidation of β-methyl-d-galactopyranoside by galactose oxidase at 1.33 mm O₂ has been determined. The k_cat exhibits a bell-shaped dependence on the ionization of at least two groups in the enzyme-substrate complex, pK_b' = 6.3 and pK_a' = 7.1, respectively. The pH-independent value for k_cat at 1.33 mm O₂ (nonsaturating) and saturating glycoside is 1435 s^?; the pH optimum is 6.7. Galactose oxidase is inactivated rapidly by iodoacetamide. Although the reaction is much slower, iodoacetate also inactivates the enzyme. The inactivation by iodoacetamide obeys saturation kinetics; at pH 7.0 k₃ = 2.19 min^?1 and K_i = 5.1 mM; k₃ but not K_i exhibits a bell-shaped pH dependence, with pK_a values of 6.3 and 7.6, respectively. Labeling with [¹⁴C]iodoacetamide establishes that one carboxamidomethyl group is incorporated per enzyme molecule. This incorporation parallels the loss of enzymatic activity. Only N-3-carboxymethylhistidine is detected in chromatograms following hydrolysis of the labeled protein. The protein-bound copper is not lost as a consequence of alkylation. Apogalactose oxidase does not react with iodoacetamide. The alkylation is inhibited by the oxidation of an active center tryptophan residue (s) by N-bromosuccinimide. The fraction of residual enzyme activity remaining after tryptophan oxidation corresponds to the extent of labeling by [¹⁴C]iodoacetamide. Although alkylation causes little change in the spin Hamiltonian parameters of the Cu(II) atom, it nearly abolishes both the optical activity and optical absorbance of the metal. The native tryptophan fluorescence of the enzyme, which is a sensitive probe of its active site, is also markedly affected. Since binding of a substrate, β-methyl-d-galactopyranoside, reduces fluorescence as it does in the active enzyme and binding of CN^? at the Cu(II) site as detected by electron spin resonance appears unaffected by the alkylation, the effect of alkylation is on catalysis, per se. Both a catalytic and a subtle conformational role for the active site histidine are inferred from the results.     

Role of the conserved active site residue tryptophan-24 of human dihydrofolate reductase as revealed by mutagenesis

The active sites of all bacterial and vertebrate dihydrofolate reductases that have been examined have a tryptophan residue near the binding sites for NADPH and dihydrofolate. In cases where the three-dimensional structure has been determined by X-ray crystallography, this conserved tryptophan residue makes hydrophobic and van der Waals interactions with the nicotinamide moiety of bound NADPH, and its indole nitrogen interacts with the C4 oxygen of bound folate through a bridge provided by a bound water molecule. We have addressed the question of why even the very conservative replacement of this tryptophan by phenylalanine does not seem to occur naturally. Human dihydrofolate reductase with this replacement of tryptophan by phenylalanine has increased rate constants for dissociation of substrates and products and a considerably decreased rate of hydride transfer. These cause some changes in steady-state kinetic behavior, including substantial increases in Michaelis constants for NADPH and dihydrofolate, but there is also a 3-fold increase in kcat. The branched mechanistic pathway for this enzyme has been completely defined and is sufficiently different from that of wild-type enzyme to cause changes in some transient-state kinetics. The most critical changes resulting from the amino acid substitution appear to be a 50% decrease in stability and a decrease in efficiency from 69% to 21% under intracellular conditions.     

Rat d-aspartate oxidase is more similar to the human enzyme than the mouse enzyme

d-Aspartate oxidase (DDO) is a degradative enzyme that is stereospecific for the acidic amino acid d-aspartate, an endogenous agonist of the N-methyl-d-aspartate (NMDA) receptor. Dysregulation of NMDA receptor-mediated neurotransmission has been implicated in the onset of various neuropsychiatric disorders including schizophrenia, as well as chronic pain. Thus, appropriate regulation of d-aspartate is believed to be important for maintaining proper neural activity in the nervous system. Accordingly, much attention has been paid to the role(s) of DDO in the metabolism of d-aspartate in vivo, and the physiological functions of DDO have been actively investigated using experimental rats and mice. However, detailed characterisation of rat DDO has not yet been performed, and little is known about species-specific differences in the properties of mammalian DDOs. In this study, the structural and enzymatic properties of purified recombinant rat, mouse and human DDOs were examined and compared. The results showed that rat DDO is more similar to human DDO than to mouse DDO. This work provides useful insight into the use of rats as an experimental model for investigating the biological significance of human DDO and/or d-aspartate.This article is part of a Special Issue entitled: d-Amino acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca.     

Identification of a cysteine residue in the active site of nitroalkane oxidase by modification with N-ethylmaleimide

The flavoprotein nitroalkane oxidase catalyzes the oxidative denitrification of primary or secondary nitroalkanes to the corresponding aldehydes or ketones with production of hydrogen peroxide and nitrite. The enzyme is irreversibly inactivated by treatment with N-ethylmaleimide at pH 7. The inactivation is time-dependent and shows first-order kinetics for three half-lives. The second-order rate constant for inactivation is 3.4 +/- 0.06 m(-)(1) min(-)(1). The competitive inhibitor valerate protects the enzyme from inactivation, indicating an active site-directed modification. Comparison of tryptic maps of enzyme treated with N-[ethyl-1-(14)C]maleimide in the absence and presence of valerate shows a single radioactive peptide differentially labeled in the unprotected enzyme. The sequence of this peptide was determined to be LLNEVMCYPLFDGGNIGLR using Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The cysteine residue was identified as the site of alkylation by ion trap mass spectrometry.     

Escherichia coli UmuC active site mutants: Effects on translesion DNA synthesis, mutagenesis and cell survival

Escherichia coli polymerase V (pol V/UmuD(2)'C) is a low-fidelity DNA polymerase that has recently been shown to avidly incorporate ribonucleotides (rNTPs) into undamaged DNA. The fidelity and sugar selectivity of pol V can be modified by missense mutations around the "steric gate" of UmuC. Here, we analyze the ability of three steric gate mutants of UmuC to facilitate translesion DNA synthesis (TLS) of a cyclobutane pyrimidine dimer (CPD) in vitro, and to promote UV-induced mutagenesis and cell survival in vivo. The pol V (UmuC_F10L) mutant discriminates against rNTP and incorrect dNTP incorporation much better than wild-type pol V and although exhibiting a reduced ability to bypass a CPD in vitro, does so with high-fidelity and consequently produces minimal UV-induced mutagenesis in vivo. In contrast, pol V (UmuC_Y11A) readily misincorporates both rNTPs and dNTPs during efficient TLS of the CPD in vitro. However, cells expressing umuD'C(Y11A) were considerably more UV-sensitive and exhibited lower levels of UV-induced mutagenesis than cells expressing wild-type umuD'C or umuD'C(Y11F). We propose that the increased UV-sensitivity and reduced UV-mutability of umuD'C(Y11A) is due to excessive incorporation of rNTPs during TLS that are subsequently targeted for repair, rather than an inability to traverse UV-induced lesions.     

Site-directed mutagenesis of Escherichia coli succinyl-CoA synthetase. beta-Cys325 is a nonessential active site residue

Chemical modification experiments have shown that sulfhydryl groups play an important role in the mechanism of action of Escherichia coli succinyl-CoA synthetase. One of these sulfhydryl groups has been localized in the beta-subunit of the enzyme using the coenzyme A affinity analog, CoA disulfide-S,S-dioxide (Collier, G. E., and Nishimura, J. S. (1978) J. Biol. Chem. 253, 4938-4943). Recently, it has been shown that the reactive sulfhydryl group resides in Cys325 (Nishimura, J. S., Mitchell, T., Ybarra, J., and Matula, J. M., submitted to Eur. J. Biochem. for publication). In the present study, we have changed Cys325 to a glycine residue using the technique of site-directed mutagenesis and have purified the mutant enzyme to homogeneity. The resulting mutant enzyme is 83% as active as wild type enzyme. In contrast to wild type succinyl-CoA synthetase, the mutant is refractory to chemical modification by CoA disulfide-S,S-dioxide and methyl methanethiolsulfonate. It is also less reactive with N-ethylmaleimide. Thus, beta-Cys325 is a nonessential active site residue.     

Crystal structure determination of cholesterol oxidase from Streptomyces and structural characterization of key active site mutants

Cholesterol oxidase is a monomeric flavoenzyme which catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. The enzyme interacts with lipid bilayers in order to bind its steroid substrate. The X-ray structure of the enzyme from Brevibacterium sterolicum revealed two loops, comprising residues 78-87 and residues 433-436, which act as a lid over the active site and facilitate binding of the substrate [Vrielink et al. (1991) J. Mol. Biol. 219, 533-554; Li et al. (1993) Biochemistry 32, 11507-11515]. It was postulated that these loops must open, forming a hydrophobic channel between the membrane and the active site of the protein and thus sequestering the cholesterol substrate from the aqueous environment. Here we describe the three-dimensional structure of the homologous enzyme from Streptomyces refined to 1.5 A resolution. Structural comparisons to the enzyme from B. sterolicum reveal significant conformational differences in these loop regions; in particular, a region of the loop comprising residues 78-87 adopts a small amphipathic helical turn with hydrophobic residues directed toward the active site cavity and hydrophilic residues directed toward the external surface of the molecule. It seems reasonable that this increased rigidity reduces the entropy loss that occurs upon binding substrate. Consequently, the Streptomyces enzyme is a more efficient catalyst. In addition, we have determined the structures of three active site mutants which have significantly reduced activity for either the oxidation (His447Asn and His447Gln) or the isomerization (Glu361Gln). Our structural and kinetic data indicate that His447 and Glu361 act as general base catalysts in association with conserved water H2O541 and Asn485. The His447, Glu361, H2O541, and Asn485 hydrogen bond network is conserved among other oxidoreductases. This catalytic tetrad appears to be a structural motif that occurs in flavoenzymes that catalyze the oxidation of unactivated alcohols.