Massive accumulation of Man2GlcNAc2-Asn in nonneuronal tissues of glycosylasparaginase-deficient mice and its removal by enzyme replacement therapy

Aspartylglycosaminuria (AGU) is caused by deficient enzymatic activity of glycosylasparaginase (GA). The disease is characterized by accumulation of aspartylglucosamine (GlcNAc-Asn) and other glycoasparagines in tissues and body fluids of AGU patients and in an AGU mouse model. In the current study, we characterized a glycoasparagine carrying the tetrasaccharide moiety of alpha-D-Man-(1-->6)-beta-D-Man-(1-->4)-beta-D-GlcNAc-(1-->4)-beta-D-GlcNAc-(1-->N)-Asn (Man2GlcNAc2-Asn) in urine of an AGU patient and also in the tissues of the AGU mouse model. Quantitative analysis demonstrated a massive accumulation of the compound especially in nonneuronal tissues of the AGU mice, in which the levels of Man2GlcNAc2-Asn were typically 30-87% of those of GlcNAc-Asn. The highest level of Man2GlcNAc2-Asn was found in the liver, spleen, and heart tissues of the AGU mice, the respective amounts being 87%, 76%, and 57% of the GlcNAc-Asn levels. In the brain tissue of AGU mice the Man2GlcNAc2-Asn storage was only 9% of that of GlcNAc-Asn. In contrast to GlcNAc-Asn, the storage of Man2GlcNAc2-Asn markedly increased in the liver and spleen tissues of AGU mice as they grew older. Enzyme replacement therapy with glycosylasparaginase for 3.5 weeks reduced the amount of Man2GlcNAc2-Asn by 66-97% in nonneuronal tissues, but only by 13% in the brain tissue of the AGU mice. In conclusion, there is evidence for a role for storage of glycoasparagines other than aspartylglucosamine in the pathogenesis of AGU, and this possibility should be taken into consideration in the treatment of the disease.     

Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability

Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t _1/2 values of 7.0 and 1.5 min at 55°C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instabiliby due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its M _r value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.     

Induced autoprocessing of the cytopathic Makes caterpillars floppy‐like effector domain of the Vibrio vulnificus MARTX toxin

The multifunctional‐autoprocessing repeats‐in‐toxin (MARTX_Vv) toxin that harbours a varied repertoire of effector domains is the primary virulence factor of Vibrio vulnificus. Although ubiquitously present among Biotype I toxin variants, the ‘Makes caterpillars floppy‐like’ effector domain (MCF_Vv) is previously unstudied. Using transient expression and protein delivery, MCF_Vv and MCF_Ah from the Aeromonas hydrophila MARTX_Ah toxin are shown for the first time to induce cell rounding. Alanine mutagenesis across the C‐terminal subdomain of MCF_Vv identified an Arg‐Cys‐Asp (RCD) tripeptide motif shown to comprise a cysteine protease catalytic site essential for autoprocessing of MCF_Vv. The autoprocessing could be recapitulated in vitro by the addition of host cell lysate to recombinant MCF_Vv, indicating induced autoprocessing by cellular factors. The RCD motif is also essential for cytopathicity, suggesting autoprocessing is essential first to activate the toxin and then to process a cellular target protein resulting in cell rounding. Sequence homology places MCF_Vv within the C58 cysteine protease family that includes the type III secretion effectors YopT from Yersinia spp. and AvrPphB from Pseudomonas syringae. However, the catalytic site RCD motif is unique compared with other C58 peptidases and is here proposed to represent a new subgroup of autopeptidase found within a number of putative large bacterial toxins.     

Two-step dimerization for autoproteolysis to activate glycosylasparaginase

Glycosylasparaginase (GA) is an amidase and belongs to a novel family of N-terminal nucleophile hydrolases that use a similar autoproteolytic processing mechanism to generate a mature/active enzyme from a single chain protein precursor. From bacteria to eukaryotes, GAs are conserved in primary sequences, tertiary structures, and activation of amidase activity by intramolecular autoproteolysis. An evolutionarily conserved His-Asp-Thr sequence is cleaved to generate a newly exposed N-terminal threonine, which plays a central role in both autoproteolysis and in its amidase activity. We have recently determined the crystal structure of the bacterial GA precursor at 1.9-A resolution, which reveals a highly distorted and energetically unfavorable conformation at the scissile peptide bond. A mechanism of autoproteolysis via an N-O acyl shift was proposed to relieve these conformational strains. However, it is not understood how the polypeptide chain distortion was generated and preserved during the folding of GA to trigger autoproteolysis. An obstacle to our understanding of GA autoproteolysis is the uncertainty concerning its quaternary structure in solution. Here we have revisited this question and show that GA forms dimers in solution. Mutants with alterations at the dimer interface cannot form dimers and are impaired in the autoproteolytic activation. This suggests that dimerization of GA plays an essential role in autoproteolysis to activate the amidase activity. Comparison of the melting temperatures of GA dimers before and after autoproteolysis suggests two states of dimerization in the process of enzyme maturation. A two-step dimerization mechanism to trigger autoproteolysis is proposed to accommodate the data presented here as well as those in the literature.     

Intraparticle concentration gradients for substrate and acidic product in immobilized cephalosporin C amidase and their dependencies on carrier characteristics and reaction parameters

Cephalosporin C amidase was covalently attached using a protein loading of 7.0–200 mg protein/g dry carrier on four epoxy‐activated Sepabeads differing in particle size and pore diameter. Initial‐rate kinetic analysis showed that for Sepabeads with small pore diameters (30–40 nm), the apparent K_M of the amidase for hydrolysis of cephalosporin C at 37°C and pH 8.0 increased ～3‐fold in response to increased particle size (～120–400 µm) and increased amount of immobilized enzyme (7.0–70 mg protein/g dry carrier) while maximum specific activity (3.2 U/mg protein; 25% of free amidase) was affected only by particle size. In contrast, for Sepabeads with wide pores (150–250 nm), the K_M was independent of the enzyme loading. Internal effectiveness factors calculated from observable Thiele modulus reflected the dependence of K_M on geometrical parameters of the particles. A new method for determination of the overall intraparticle pH was developed based on luminescence lifetime measurements in the frequency domain. Sepabeads were doubly labeled using a lipophilic variant of the pH‐sensitive dye fluorescein, and Ru(II) tris(4,7‐diphenyl‐1,10‐phenantroline) whose phosphorescence properties are independent of pH. Luminescent lifetime measurements of doubly labeled particle suspensions showed superior signal‐to‐noise ratio compared to fluorescence intensity‐based measurements using singly labeled particles. The difference at apparent steady state (ΔpH) between bulk (external pH) and intraparticle pH (internal pH) was as large as ～0.6 units. The ΔpH was dependent on substrate concentration, particle size, and pore diameter. Therefore, these results characterize the role of carrier characteristics and reaction parameters in the formation of concentration gradients for substrate and acidic product during hydrolysis of cephalosporin C by immobilized amidase. The strong pH dependence of the immobilized amidase underscores the importance of considering intraparticle pH gradients in the design of an efficient carrier‐bound biocatalyst. Biotechnol. Bioeng. 2010;106: 528–540. © 2010 Wiley Periodicals, Inc.     

TcdB from hypervirulent Clostridium difficile exhibits increased efficiency of autoprocessing

TcdB, an intracellular bacterial toxin that inactivates small GTPases, is a major Clostridium difficile virulence factor. Recent studies have found that TcdB produced by emerging/hypervirulent strains of C. difficile is more potent than TcdB from historical strains, and in the current work, studies were performed to investigate the underlying mechanisms for this change in TcdB toxicity. Using a series of biochemical analyses we found that TcdB from a hypervirulent strain (TcdB_HV) was more efficient at autoprocessing than TcdB from a historical strain (TcdB_HIST). TcdB_HV and TcdB_HIST were activated by similar concentrations of IP6; however, the overall efficiency of processing was 20% higher for TcdB_HV. Using an activity‐based fluorescent probe (AWP19) an intermediate, activated but uncleaved, form of TcdB_HIST was identified, while only a processed form of TcdB_HV could be detected under the same conditions. Using a much higher concentration (200 µM) of the probe revealed an activated uncleaved form of TcdB_HV, indicating a preferential and more efficient engagement of intramolecular substrate than TcdB_HIST. Furthermore, a peptide‐based inhibitor (Ac‐GSL‐AOMK) was found to block the cytotoxicity of TcdB_HIST at a lower concentration than required to inhibit TcdB_HV. These findings suggest that TcdB_HV may cause increased cytotoxicity due to more efficient autoprocessing.     

MARTX effector cross kingdom activation by Golgi‐associated ADP‐ribosylation factors

Vibrio vulnificus infects humans and causes lethal septicemia. The primary virulence factor is a multifunctional‐autoprocessing repeats‐in‐toxin (MARTX) toxin consisting of conserved repeats‐containing regions and various effector domains. Recent genomic analyses for the newly emerged V. vulnificus biotype 3 strain revealed that its MARTX toxin has two previously unknown effector domains. Herein, we characterized one of these domains, Domain X (DmX_Vv). A structure‐based homology search revealed that DmX_Vv belongs to the C58B cysteine peptidase subfamily. When ectopically expressed in cells, DmX_Vv was autoprocessed and induced cytopathicity including Golgi dispersion. When the catalytic cysteine or the region flanking the scissile bond was mutated, both autoprocessing and cytopathicity were significantly reduced indicating that DmX_Vv cytopathicity is activated by amino‐terminal autoprocessing. Consistent with this, host cell protein export was affected by Vibrio cells producing a toxin with wild‐type, but not catalytically inactive, DmX_Vv. DmX_Vv was found to localize to Golgi and to directly interact with Golgi‐associated ADP‐ribosylation factors ARF1, ARF3 and ARF4, although ARF binding was not necessary for the subcellular localization. Rather, this interaction was found to induce autoprocessing of DmX_Vv. These data demonstrate that the V. vulnificus hijacks the host ARF proteins to activate the cytopathic DmX_Vv effector domain of MARTX toxin.     

The gibberellin biosynthetic genes AtKAO1 and AtKAO2 have overlapping roles throughout Arabidopsis development

Ent‐kaurenoic acid oxidase (KAO), a class of cytochrome P450 monooxygenases of the subfamily CYP88A, catalyzes the conversion of ent‐kaurenoic acid (KA) to gibberellin (GA) GA₁₂, the precursor of all GAs, thereby playing an important role in determining GA concentration in plants. Past work has demonstrated the importance of KAO activity for growth in various plant species. In Arabidopsis, this enzyme is encoded by two genes designated KAO1 and KAO2. In this study, we used various approaches to determine the physiological roles of KAO1 and KAO2 throughout plant development. Analysis of gene expression pattern reveals that both genes are mainly expressed in germinating seeds and young developing organs, thus suggesting functional redundancy. Consistent with this, kao1 and kao2 single mutants are indistinguishable from wild‐type plants. By contrast, the kao1 kao2 double mutant exhibits typical non‐germinating GA‐dwarf phenotypes, similar to those observed in the severely GA‐deficient ga1‐3 mutant. Phenotypic characterization and quantitative analysis of endogenous GA contents of single and double kao mutants further confirm an overlapping role of KAO1 and KAO2 throughout Arabidopsis development.     

Characterisation of Shigella Spa33 and Thermotoga FliM/N reveals a new model for C‐ring assembly in T3SS

Flagellar type III secretion systems (T3SS) contain an essential cytoplasmic‐ring (C‐ring) largely composed of two proteins FliM and FliN, whereas an analogous substructure for the closely related non‐flagellar (NF) T3SS has not been observed in situ. We show that the spa33 gene encoding the putative NF‐T3SS C‐ring component in Shigella flexneri is alternatively translated to produce both full‐length (Spa33‐FL) and a short variant (Spa33‐C), with both required for secretion. They associate in a 1:2 complex (Spa33‐FL/C₂) that further oligomerises into elongated arrays in vitro. The structure of Spa33‐C₂ and identification of an unexpected intramolecular pseudodimer in Spa33‐FL reveal a molecular model for their higher order assembly within NF‐T3SS. Spa33‐FL and Spa33‐C are identified as functional counterparts of a FliM–FliN fusion and free FliN respectively. Furthermore, we show that Thermotoga maritima FliM and FliN form a 1:3 complex structurally equivalent to Spa33‐FL/C₂, allowing us to propose a unified model for C‐ring assembly by NF‐T3SS and flagellar‐T3SS.     

Characterization of the mutation responsible for aspartylglucosaminuria in three Finnish patients. Amino acid substitution Cys163----Ser abolishes the activity of lysosomal glycosylasparaginase and its conversion into subunits

The mutation that causes a deficiency of the lysosomal amidase, glycosylasparaginase, has been characterized in fibroblasts from three Finnish patients diagnosed with aspartylglucosaminuria (AGU). The polymerase chain reaction was used to amplify the glycosylasparaginase protein coding sequence from the three AGU patients in order to compare them to the normal sequence from a full-length human placenta cDNA clone HPAsn.6 (Fisher, K.J., Tollersrud, O.K., and Aronson, N.N., Jr. (1990) FEBS Lett. 269, 440-444). Two base changes were found to be common to all three Finnish AGU patients, a G482----A transition that results in an Arg161----Gln substitution and a G488----C transversion that causes Cys163----Ser. Detection of both point mutations from PCR-amplified cDNA or genomic DNA was facilitated by their creation of new endonuclease restriction sites. Expression studies in COS-1 cells revealed only the Cys163----Ser mutation caused a deficiency of glycosylasparaginase activity. This same substitution also prevented the normal posttranslational processing of the precursor glycosylasparaginase polypeptide into its alpha and beta subunits. Cell-free expression of the single-chain glycosylasparaginase precusor did not produce an active enzyme, suggesting that post-translational generation of subunits may be required for catalytic activity.     

The dioxygenase GIM2 functions in seed germination by altering gibberellin production in Arabidopsis

The phytohormones gibberellic acid (GA) and abscisic acid (ABA) antagonistically control seed germination. High levels of GA favor seed germination, whereas high levels of ABA hinder this process. The direct relationship between GA biosynthesis and seed germination ability need further investigation. Here, we identified the ABA‐insensitive gain‐of‐function mutant germination insensitive to ABA mutant 2 (gim2) by screening a population of XVE T‐DNA‐tagged mutant lines. Based on two loss‐of‐function gim2‐ko mutant lines, the disruption of GIM2 function caused a delay in seed germination. By contrast, upregulation of GIM2 accelerated seed germination, as observed in transgenic lines overexpressing GIM2 (OE). We detected a reduction in endogenous bioactive GA levels and an increase in endogenous ABA levels in the gim2‐ko mutants compared to wild type. Conversely, the OE lines had increased endogenous bioactive GA levels and decreased endogenous ABA levels. The expression levels of a set of GA‐ and/or ABA‐related genes were altered in both the gim2‐ko mutants and the OE lines. We confirmed that GIM2 has dioxygenase activity using an in vitro enzyme assay, observing that GIM2 can oxidize GA₁₂. Hence, our characterization of GIM2 demonstrates that it plays a role in seed germination by affecting the GA metabolic pathway in Arabidopsis.     

Is the bovine lysosomal phospholipase B‐like protein an amidase?

The main function of lysosomal proteins is to degrade cellular macromolecules. We purified a novel lysosomal protein to homogeneity from bovine kidneys. By gene annotation, this protein is defined as a bovine phospholipase B‐like protein 1 (bPLBD1) and, to better understand its biological function, we solved its structure at 1.9 Å resolution. We showed that bPLBD1 has uniform noncomplex‐type N‐glycosylation and that it localized to the lysosome. The first step in lysosomal protein transport, the initiation of mannose‐6‐phosphorylation by a N‐acetylglucosamine‐1‐phosphotransferase, requires recognition of at least two distinct lysines on the protein surface. We identified candidate lysines by analyzing the structural and sequentially conserved N‐glycosylation sites and lysines in bPLBD1 and in the homologous mouse PLBD2. Our model suggests that N408 is the primarily phosphorylated glycan, and K358 a key residue for N‐acetylglucosamine‐1‐phosphotransferase recognition. Two other lysines, K334 and K342, provide the required second site for N‐acetylglucosamine‐1‐phosphotransferase recognition. bPLBD1 is an N‐terminal nucleophile (Ntn) hydrolase. By comparison with other Ntn‐hydrolases, we conclude that the acyl moiety of PLBD1 substrate must be small to fit the putative binding pocket, whereas the space for the rest of the substrate is a large open cleft. Finally, as all the known substrates of Ntn‐hydrolases have amide bonds, we suggest that bPLBD1 may be an amidase or peptidase instead of lipase, explaining the difficulty in finding a good substrate for any members of the PLBD family. Proteins 2014; 82:300–311. © 2013 Wiley Periodicals, Inc.     

Directed evolution of Aspergillus niger glucoamylase to increase thermostability

Using directed evolution and site‐directed mutagenesis, we have isolated a highly thermostable variant of Aspergillus niger glucoamylase (GA), designated CR2‐1 . CR2‐1 includes the previously described mutations Asn20Cys and Ala27Cys (forming a new disulfide bond), Ser30Pro, Thr62Ala, Ser119Pro, Gly137Ala, Thr290Ala, His391Tyr and Ser436Pro. In addition, CR2‐1 includes several new putative thermostable mutations, Val59Ala, Val88Ile, Ser211Pro, Asp293Ala, Thr390Ser, Tyr402Phe and Glu408Lys, identified by directed evolution. CR2‐1 GA has a catalytic efficiency (k_cat/K_m) at 35°C and a specific activity at 50°C similar to that of wild‐type GA. Irreversible inactivation tests indicated that CR2‐1 increases the free energy of thermoinactivation at 80°C by 10 kJ mol^?1 compared with that of wild‐type GA. Thus, CR2‐1 is more thermostable (by 5 kJ mol^?1 at 80°C) than the most thermostable A. niger GA variant previously described, THS8 . In addition, Val59Ala and Glu408Lys were shown to individually increase the thermostability in GA variants by 1 and 2 kJ mol^?1, respectively, at 80°C.     

Overexpression of a recombinant amidase in a complex auto-inducing culture: purification,biochemical characterization,and regio- and stereoselectivity

Rhodococcus erythropolis AJ270 metabolizes a wide range of nitriles via the two-step nitrile hydratase/amidase pathway. In this study, an amidase gene from R. erythropolis AJ270 was cloned and expressed in Escherichia coli BL21 (DE3). The activity reached the highest level of 22.04 U/ml in a complex auto-inducing medium using a simplified process of fermentation operation. The recombinant amidase was purified to more than 95% from the crude lysate using Ni-NTA affinity chromatography and Superose S10-300 gel filtration. The V _max and K _m values of the purified enzyme with acetamide (50 mM) were 6.89 μmol/min/mg protein and 4.12 mM, respectively, which are similar to those of the enzyme from the wild-type cell. The enzyme converted racemic α-substituted amides, O-benzylated β-hydroxy amides, and N-benzylated β-amino amides to the corresponding (S)-acids with remarkably high enantioselectivity. The ionic liquid [BMIm][PF₆] (1-butyl-3-methylimidazolium hexafluorophosphate) enhanced the activity by 1.5-fold compared with water. The adequate expression of the enzyme and excellent enantioselectivity of the recombinant amidase to a broad spectrum of amides suggest that the enzyme has prospective industrial-scale practical applications in pharmaceutical chemistry.     

Characterization of an enantioselective amidase with potential application to asymmetric hydrolysis of (<Emphasis Type="Italic">R</Emphasis>, <Emphasis Type="Italic">S</Emphasis>)-2, 2-dimethylcyclopropane carboxamide

Amidase is a promising synthesis tool for chiral amides and related derivatives. In the present study, the biochemical properties of the Delftia tsuruhatensis CCTCC M 205114 enantioselective amidase were determined for its potential application in chiral amides synthesis. D. tsuruhatensis CCTCC M 205114 amidase was purified 105.2 fold with total activity recovery of 4.26%. The enzyme is a monomer with a subunit of approximately 50 kDa by analytical gel filtration HPLC and SDS–PAGE. It had a broad substrate spectrum and displayed high enantioselectivity against R-2, 2-dimethylcyclopropane carboxamide and R-mandelic amide. The amidase was applied to enantioselective hydrolysis of R-2, 2-dimethylcyclopropane carboxamide from racemic (R, S)-2, 2-dimethylcyclopropane carboxamide to accumulate S-2, 2-dimethylcyclopropane carboxamide. This enzyme did not require metal ions for the hydrolysis reaction. Its optimal pH and temperature were 8.0 and 35°C, respectively. The K _m and V _max of the amidase for R-2, 2-dimethylcyclopropane carboxamide were 2.54 mM and 8.37 μmol min⁻¹ mg protein⁻¹, respectively. After 60 min of the reaction, R-2, 2-dimethylcyclopropane carboxamide was completely hydrolyzed, generating S-2, 2-dimethylcyclopropane carboxamide with a yield of 45.9% and an e.e. of above 99%. Therefore, this amidase can serve as a promising producer for S-2, 2-dimethylcyclopropane carboxamide and other amides.     

Enhancement of the thermostability of a recombinant β-agarase, AgaB, from Zobellia galactanivorans by random mutagenesis

Random mutagenesis was performed on β-agarase, AgaB, from Zobellia galactanivorans to improve its catalytic activity and thermostability. The activities of three mutants E99K, T307I and E99K–T307I were approx. 140, 190 and 200%, respectively, of wild type β-agarase (661 U/mg) at 40°C. All three mutant enzymes were stable up to 50°C and E99K–T307I had the highest thermostability. The melting temperature (T _m) of E99K–T307I, determined by CD spectra, was increased by 5.2°C over that of the wild-type enzyme (54.6°C). Activities of both the wild-type and E99K–T307I enzymes, as well as their overall thermostabilities, increased in 1 mM CaCl₂. The E99K–T307I enzyme was stable at 55°C with 1 mM CaCl₂, reaching 260% of the activity the wild-type enzyme held at 40°C without CaCl₂.     

Development and characterization of a potent producer of penicillin G amidase by mutagenization

Summary The penicillin G amidase (PGA) activity of a parent strain of E. coli (PCSIR-102) was enhanced by chemical mutagenization with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). After screening and optimization, a penicillinase deficient mutant (MNNG-37) was isolated and found effective for the production of penicillin G amidase as compared to the parent strain of E. coli (PCSIR-102). Penicillin G amidase activity of MNNG-37 appeared during an early stage of growth, whereas PCSIR-102 did not exhibit PGA activity due to the presence of penicillinase enzyme which inhibits the activity of enzyme PGA. However, MNNG-37 gave a three-fold increase in enzyme activity (231 IU mg⁻¹) as compared to PCSIR-102 (77 IU mg⁻¹) in medium containing 0.15 and 0.1% concentrations of phenylacetic acid, respectively which was added after 6 h of cultivation. The difference in K _m values of the enzyme produced by parent strain PCSIR-102 (0.26 mM) and mutant strain MNNG-37 (0.20 mM) is significant (1.3-fold increase in K _m value) which may show the superiority of the latter in terms of better enzyme properties.     

NMR studies of an immunomodulatory benzodiazepine binding to its molecular target on the mitochondrial F1F0‐ATPase

Bz‐423 is an inhibitor of the mitochondrial F₁F₀‐ATPase, with therapeutic properties in murine models of immune diseases. Here, we study the binding of a water‐soluble Bz‐423 analog (5‐(3‐(aminomethyl)phenyl)‐7‐chloro‐ 1‐methyl‐3‐(naphthalen‐2‐ylmethyl)‐1H‐benzo][e][1,4]diazepin‐2(3H)‐one); (1) to its target subunit on the enzyme, the oligomycin sensitivity conferring protein (OSCP), by NMR spectroscopy using chemical shift perturbation and cross‐relaxation experiments. Titration experiments with constructs representing residues 1–120 or 1–145 of the OSCP reveals that (a) 1 binds to a region of the protein, at the minimum, comprising residues M51, L56, K65, V66, K75, K77, and N92, and (b) binding of 1 induces conformational changes in the OSCP. Control experiments employing a variant of 1 in which a key binding element on the small molecule was deleted; it had no perturbational effect on the spectra of the OSCP, which indicates that the observed changes with 1 represent specific binding interactions. Collectively, these data suggest that 1 might inhibit the enzyme through an allosteric mechanism where binding results in conformational changes that perturb the OSCP‐F₁ interface resulting in disrupted communication between the peripheral stalk and the F₁‐domain of the enzyme. © 2009 Wiley Periodicals, Inc. Biopolymers 29: 85–92, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Continuous hydrolysis of 4-cyanopyridine by nitrilases from <Emphasis Type="Italic">Fusarium solani</Emphasis> O1 and <Emphasis Type="Italic">Aspergillus niger</Emphasis> K10

The operational stabilities of nitrilases from Aspergillus niger K10 and Fusarium solani O1 were examined with 4-cyanopyridine as the substrate in continuous-stirred membrane reactors (CSMRs). The former enzyme was fairly stable at 30 °C with a deactivation constant (k _d) and enzyme half-life of 0.014 h⁻¹ and 50 h, respectively, but the latter exhibited an even higher stability characterized by k _d = 0.008 h⁻¹ and half-life of 87 h at 40 °C. Another advantage of this enzyme was its high chemoselectivity, i.e., selective transformation of nitriles into carboxylic acids, while the amide formed a high ratio of A. niger K10 nitrilase product. High conversion rates (>90%) were maintained for about 52 h using the nitrilase from F. solani O1 immobilized in cross-linked enzyme aggregates (CLEAs). The purity of isonicotinic acid was increased from 98% to >99.9% by using two CSMRs connected in series, the first one containing the F. solani O1 nitrilase and the second the amidase from Rhodococcus erythropolis A4 (both enzymes as CLEAs), the amidase hydrolyzing the by-product isonicotinamide.     

Action of Serine Carboxypeptidase from Paecilomyces carneus on Oligopeptides Containing Carboxy-Terminally Amidated Peptides

Paecilomyces carneus carboxypeptidase sequentially liberated amino acids from the carboxy-terminus of neurotensin, angiotensin I, bradykinin, and delta sleep-inducing peptide, indicating that the sequential hydrolysis of peptides was limited by the occurrence of intermediates with the structure of -Gly-X (X = L-amino acid), -Pro-X, -X-Gly, and -X-Pro. The enzyme had carboxyamidase and/or amidase activities for the carboxy-terminally amidated peptides. The enzyme essentially acted as a carboxyamidase for the long carboxy-terminally amidated peptides; an amidase became dominant for the substrates in the presence of bulky amino acids such as Arg, Met, Leu, and Phe in the penultimate (P₁) and P₂ positions, corresponding with the S₁ and S₂ sites of the enzyme, and the P₃ position of carboxy-terminally amidated peptides played a significant role in the action as a carboxyamidase or a amidase. Received: 10 June 1997 / Accepted: 14 July 1997