Identification of the putative tumor suppressor Nit2 as ω-amidase, an enzyme metabolically linked to glutamine and asparagine transamination

The present report identifies the enzymatic substrates of a member of the mammalian nitrilase-like (Nit) family. Nit2, which is widely distributed in nature, has been suggested to be a tumor suppressor protein. The protein was assumed to be an amidase based on sequence homology to other amidases and on the presence of a putative amidase-like active site. This assumption was recently confirmed by the publication of the crystal structure of mouse Nit2. However, the in vivo substrates were not previously identified. Here we report that rat liver Nit2 is ω-amidodicarboxylate amidohydrolase (E.C. 3.5.1.3; abbreviated ω-amidase), a ubiquitously expressed enzyme that catalyzes a variety of amidase, transamidase, esterase and transesterification reactions. The in vivo amidase substrates are α-ketoglutaramate and α-ketosuccinamate, generated by transamination of glutamine and asparagine, respectively. Glutamine transaminases serve to salvage a number of α-keto acids generated through non-specific transamination reactions (particularly those of the essential amino acids). Asparagine transamination appears to be useful in mitochondrial metabolism and in photorespiration. Glutamine transaminases play a particularly important role in transaminating α-keto-γ-methiolbutyrate, a key component of the methionine salvage pathway. Some evidence suggests that excess α-ketoglutaramate may be neurotoxic. Moreover, α-ketosuccinamate is unstable and is readily converted to a number of hetero-aromatic compounds that may be toxic. Thus, an important role of ω-amidase is to remove potentially toxic intermediates by converting α-ketoglutaramate and α-ketosuccinamate to biologically useful α-ketoglutarate and oxaloacetate, respectively. Despite its importance in nitrogen and sulfur metabolism, the biochemical significance of ω-amidase has been largely overlooked. Our report may provide clues regarding the nature of the biological amidase substrate(s) of Nit1 (another member of the Nit family), which is a well-established tumor suppressor protein), and emphasizes a) the crucial role of Nit2 in nitrogen and sulfur metabolism, and b) the possible link of Nit2 to cancer biology.     

Efficient enzymatic synthesis of the sialyl-Lewis tetrasaccharide: A ligand for selectin-type adhesion molecules

Sialyl-Lewis^x (NeuAcα2→3Galβ1→4[Fucαl→3]GlcNAc] has been identified as a ligand for E-selectin, P-selectin and recently also for L-selectin. We have synthesized the sialyl-Lewis^x tetrasaccharide by total enzymatic synthesis from N-acetyllactosamine using a placental α2→3-sialyltransferase specific for type-2 chain acceptors, followed by a cloned human α1→3-fucosyltransferase (FucTV, the ‘plasma-type’ enzyme). This procedure resulted in the tetrasaccharide in a 61% overall yield.     

NMR spectroscopy of α-crystallin. Insights into the structure, interactions and chaperone action of small heat-shock proteins

The subunit molecular mass of α-crystallin, like many small heat-shock proteins (sHsps), is around 20 kDa although the protein exists as a large aggregate of average mass around 800 kDa. Despite this large size, a well-resolved ¹H NMR spectrum is observed for α-crystallin which arises from short, polar, highly-flexible and solvent-exposed C-terminal extensions in each of the subunits, αA- and αB-crystallin. These extensions are not involved in interactions with other proteins (e.g. β- and γ-crystallins) under non-chaperone conditions. As determined by NMR studies on mutants of αA-crystallin with alterations in its C-terminal extension, the extensions have an important role in acting as solubilising agents for the relatively-hydrophobic α-crystallin molecule and the high-molecular-weight (HMW) complex that forms during the chaperone action. The related sHsp, Hsp25, also exhibits a flexible C-terminal extension. Under chaperone conditions, and in the HMW complex isolated from old lenses, the C-terminal extension of the αA-crystallin subunit maintains its flexibility whereas the αB-crystallin subunit loses, at least partially, its flexibility, implying that it is involved in interaction with the ‘substrate’ protein. The conformation of ‘substrate’ proteins when they interact with α-crystallin has been probed by ¹H NMR spectroscopy and it is concluded that α-crystallin interacts with ‘substrate’ proteins that are in a disordered molten globule state, but only when this state is on its way to large-scale aggregation and precipitation. By monitoring the ¹H and ³¹P NMR spectra of α-crystallin in the presence of increasing concentations of urea, it is proposed that α-crystallin adopts a two-domain structure with the larger C-terminal domain unfolding first in the presence of denaturant. All these data have been combined into a model for the quaternary structure of α-crystallin. The model has two layers each of approximately 40 subunits arranged in an annulus or toroid. A large central cavity is present whose entrance is ringed by the flexible C-terminal extensions. A large hydrophobic region in the aggregate is exposed to solution and is available for interaction with ‘substrate’ proteins during the chaperone action.     

A digestive beta-glucosidase from the silkworm, Bombyx mori: cDNA cloning, expression and enzymatic characterization

A digestive β-glucosidase cDNA was cloned from the silkworm, Bombyx mori. The B. mori β-glucosidase cDNA contains an open reading frame of 1473 bp encoding 491 amino acid residues. The B. mori β-glucosidase possesses the amino acid residues involved in catalysis and substrate binding conserved in glycosyl hydrolase family 1. Southern blot analysis of genomic DNA suggested the B. mori β-glucosidase to be a single gene. Northern blot analysis of B. mori β-glucosidase gene confirmed larval midgut-specific expression. The B. mori β-glucosidase mRNA expression in larval midgut was detectable only during feeding period, whereas its expression was downregulated during starvation. The B. mori β-glucosidase cDNA was expressed as a 57-kDa polypeptide in baculovirus-infected insect Sf9 cells, and the recombinant β-glucosidase was active on cellobiose and lactose, but not active on salicin, indicating that the B. mori β-glucosidase possesses the characteristics of the Class 2 enzyme. The enzyme activity of the purified recombinant β-glucosidase expressed in baculovirus-infected insect cells was approximately 665 U per μg of recombinant B. mori β-glucosidase. The purified recombinant B. mori β-glucosidase showed the highest activity at 35 °C and pH 6.0, and were stable at 50 °C at least for 10 min. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that the recombinant B. mori β-glucosidase is N-glycosylated, but the carbohydrate moieties are not essential for enzyme activity.     

Single-step purification of a recombinant thermostable α-amylase after solubilization of the enzyme from insoluble aggregates

The expression of the gene encoding a thermostable α-amylase (EC 3.2.1.1) (optimal activity at 100°C) from the hyperthermophilic archaeon Pyrococcus woesei in the mesophilic hosts Escherichia coli and Halomonas elongata resulted in the formation of insoluble aggregates. More than 85% of the recombinant enzyme was present within the cells as insoluble but catalytically active aggregates. The recombinant α-amylase was purified to homogeneity in a single step by hydrophobic interaction chromatography on a phenyl superose column after solubilization of the enzyme under nondenaturing conditions. The enzyme was purified 258-fold with a final yield of 54%.     

Screening of microorganisms producing α-methylserine hydroxymethyltransferase, purification of the enzyme, gene cloning, and application to the enzymatic synthesis of α-methyl-l-serine

Through the screening of microorganisms capable of utilizing α-methylserine, three representative strains belonging to the bacterial genera Paracoccus, Aminobacter, and Ensifer were selected as potent producers of α-methylserine hydroxymethyltransferase, an enzyme that catalyzes the interconversion between α-methyl-l-serine and d-alanine via tetrahydrofolate. Among these strains, Paracoccus sp. AJ110402 was selected as the strain exhibiting the highest α-methylserine hydroxymethyltransferase activity. The enzyme was purified to homogeneity from a cell-free extract of this strain. The native enzyme is a homodimer with apparent molecular mass of 85 kDa and contains 1 mol of pyridoxal-5′-phosphate per mol of the subunit. The K_m for α-methyl-l-serine and tetrahydrofolate was 0.54 mM and 73 μM, respectively. The gene from Paracoccus sp. AJ110402 encoding α-methylserine hydroxymethyltransferase was cloned and expressed in Escherichia coli. Sequence analysis revealed an open reading frame of 1278 bp, encoding a polypeptide with a calculated molecular mass of 46.0 kDa. Using E. coli cells as whole-cell catalysts, 9.7 mmol of α-methyl-l-serine was stereoselectively obtained from 15 mmol of d-alanine and 13.2 mmol of formaldehyde.     

Overexpression, on-column refolding and isotopic labeling of Hahellin from Hahella chejuensis, a putative member of the betagamma-crystallin superfamily

A gene which encodes a hypothetical protein of 40 kDa has been identified in the genome of a marine bacterium Hahella chejuensis, as a putative member of βγ-crystallin superfamily. This hypothetical protein contains a putative βγ-crystallin-like domain, along with other domains for carbohydrate binding regions. It is named as Hahellin. A PCR amplified stretch of 92-amino acid residue long protein was cloned into pET21a vector and overexpressed in Escherichia coli strain BL21(DE3)pLysS cells. The recombinant Hahellin, produced as inclusion bodies, was estimated to be around 50% of the total cellular protein content which was solubilized in 8 M urea. The protein was purified and refolded using an anion exchange column. The MALDI-TOF mass spectrometry revealed the purity and monomeric nature of the protein. Further, a method to prepare isotopically (¹⁵N/¹³C) labeled protein with high yield for NMR studies is reported. The uniformly ¹⁵N-labeled Hahellin thus produced has been characterized by recording a sensitivity enhanced 2D [¹⁵N]–[¹H] HSQC spectrum. The well, dispersed peaks in the spectrum confirm that the protein is indeed well folded and suitable for further studies by NMR.     

Molecular cloning and expression analysis of phospholipase Cdelta from mud loach, Misgurnus mizolepis

A gene encoding phosphoinositide-specific phospholipase C (PLC), designated ML-PLCδ, was cloned from mud loach (Misgurnus mizolepis) liver. A complete cDNA encoding ML-PLCδ was isolated by screening the cDNA library of mud loach liver and using the 5′-rapid amplification of cDNA ends (RACE) method. The full-length ML-PLCδ gene contains an open reading frame of 2325 base pairs encoding a 774 amino acid protein with a molecular mass of 88,072 Da; this corresponds to the size of the protein expressed in Escherichia coli BL21 (DE3) using pET28a vector. It contains all of the characteristic domains found in mammalian PLCδ isozymes (PH domain, EF-hands, X–Y catalytic region, and a C2 domain). A homology search revealed that ML-PLCδ shares relatively high sequence identity with mammalian PLCδ1 (51–52%) and catfish PLCδ (64%). The recombinant ML-PLCδ protein expressed as a histidine-tagged fusion protein in E. coli was purified to apparent homogeneity by Ni²⁺-NTA affinity chromatography. The recombinant ML-PLCδ showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP₂) and its activity was Ca²⁺-dependent, which was similar to mammalian PLCδ isozymes.     

Evolution and differential expression of the (1→3)-β-glucan endohydrolaseencoding gene family in barley, Hordeum vulgare

The (1→3)-β-d-glucan glucanohydrolases [(1→ 3)-GGH; EC 3.2.1.39] of barley (Hordeum vulgare L., cv Clipper) are encoded by a small gene family. Amino acid sequences deduced from cDNA and genomic clones for six members of the family exhibit overall positional identities ranging from 44% to 78%. Specific DNA and oligodeoxyribonucleotide (oligo) probes have been used to demonstrate that the (1→3)-GGH-encoding genes are differentially transcribed in young roots, young leaves and the aleurone of germinated grain. The high degree of sequence homology, coupled with characteristic patterns of codon usage and insertion of a single intron at a highly conserved position in the signal peptide region, indicate that the genes have shared a common evolutionary history. Similar structural features in genes encoding barley (1→3,1→4)-β-glucan 4-glucanohydrolases [(1→3,1→4)-GGH; EC 3.2.1.73] further indicate that the (l→3)-GGHs and (l→3,1→4)-GGHs are derived from a single ‘super’ gene family, in which genes encoding enzymes with related yet quite distinct substrate specificities have evolved, with an associated specialization of function. The (1→3,1→4)-GGHs mediate in plant cell wall metabolism through their ability to hydrolyse the (1→3,1→4)-β-glucans that are the major constituents in barley walls, while the (1→3)-GGHs, which are unable to degrade the plant (1→3,1→4)-β-glucans, can hydrolyse the (1→3)- and (1→3,1→6)-β-glucans of fungal cell walls.     

Stereoselective anti-Prelog reduction of ketones by whole cells of Comamonas testosteroni in a ‘substrate-coupled’ approach

Lyophilized cells of the open accessible bacterium Comamonas testosteroni DSM 1455 proved to be an excellent catalyst for the asymmetric reduction of different α-azido, α-bromo, and α-nitro ketones at elevated substrate concentrations (16 g/L) in a ‘substrate-coupled’ approach using 20% (v/v) of 2-propanol as hydrogen donor. Excellent anti-Prelog stereoselectivity was obtained, which is less common found in nature.     

Cloning, characterisation and expression analysis of α-glucuronidase from the thermophilic fungus Talaromyces emersonii

The aguA gene encoding α-glucuronidase was isolated from the thermophilic fungus Talaromyces emersonii by degenerate PCR. AguA has no introns and consists of an open reading frame of 2511 bp, encoding a putative protein of 837 amino acids. The N-terminus of the protein contains a putative signal peptide of 17 amino acids yielding a mature protein of 820 amino acids with a predicted molecular mass of 91.6 kDa. Twenty putative N-glycosylation sites and four O-glycosylation were identified. The T. emersonii α-glucuronidase falls into glycosyl hydrolase family 67, showing approximately 63% identity to similar enzymes from other fungi. Analysis of the aguA promoter revealed several possible regulatory motifs including two XlnR and a CreA binding site. Enzyme activity was optimal at 50 °C and pH 5. Enzyme production was investigated on a range of carbon sources and showed induction on beechwood, oat spelt and birchwood xylan, and repression by glucose or glucuronic acid.     

Gene cloning, purification, and characterization of α-methylserine aldolase from Bosea sp. AJ110407 and its applicability for the enzymatic synthesis of α-methyl-l-serine and α-ethyl-l-serine

The gene encoding α-methylserine aldolase was isolated from Bosea sp. AJ110407. Sequence analysis revealed that the predicted amino acid sequence encoded by the 1320-bp open reading frame was 65.0% similar to the corresponding sequence of the enzyme isolated from Ralstonia sp. AJ110405. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. Gel filtration revealed the molecular mass of the purified enzyme to be approximately 78 kDa, suggesting that the enzyme is a homodimer. The enzyme exhibited a specific peak at 429 nm in the spectrum and contained 1 mol pyridoxal 5′-phosphate per mole of the subunit. The V_max value was 1.40 μmol min⁻¹ mg⁻¹, and the K_m value was 1.5 mM for the reaction wherein formaldehyde was released from α-methyl-l-serine. This enzyme could also catalyze the reverse reaction, i.e., the synthesis of α-methyl-l-serine from l-alanine and formaldehyde. This activity was inhibited in the excess of formaldehyde; however, α-methyl-l-serine was efficiently produced from l-alanine in the presence of formaldehyde. This method was also applicable for producing α-ethyl-l-serine from l-2-aminobutyric acid.     

α2-macroglobulin ‘fast’ forms inhibit superoxide production by activated macrophages

Mouse peritoneal macrophages activated by bacillus Calmette-Guerin (BCG) were incubated with human α₂-macroglobulin converted to its ‘fast’ form with either trypsin or methylamine before being stimulated with phorbol myrystate acetate. Both α₂-macroglobulin-trypsin and α₂-macroglobulin-methylamine inhibited macrophage production of superoxide anion (O₂⁻) while native α₂-macroglobulin had little effect except at high concentration. The α₂-macroglobulin ‘fast’ forms, which bind with a K_d of about 8 nM, inhibited 50% generation of O₂⁻(ID₅₀) at a concentration of 7 nM while α₂-macroglobulin inhibited O₂⁻ production with an ID₅₀ of 141 nM. The ‘fast’ forms of α₂-macroglobulin may play a role in the feedback regulation of inflammatory reactions.     

Properties of the recombinant α-glucosidase from Sulfolobus solfataricus in relation to starch processing

An α-glucosidase activity (EC 3.2.1.20) isolated from Sulfolobus solfataricus strain MT-4 was characterised and found of interest at industrial level in the saccharification step of hydrolysis process of starch. The gene encoding for the enzyme was expressed in Escherichia coli BL21 (DE3) with a yield of 87.5 U/g of wet biomass. The recombinant cytosolic enzyme was purified to homogeneity with a rapid purification procedure employing only steps of selective and progressive thermal precipitations with a final yield of 75.4% and a purification of 14.5-fold. The properties of this thermophilic α-glucosidase were compared with those of the α-glucosidase of a commercial preparation from Aspergillus niger used in the starch processing.     

Cloning and expression of the gene encoding <Emphasis Type="BoldItalic">Streptomyces coelicolor</Emphasis> A3(2) α-galactosidase belonging to family 36

The α-galactosidase gene of Streptomyces coelicolor A3(2) was cloned, expressed in Escherichia coli and characterized. It consisted of 1497 nucleotides encoding a protein of 499 amino acids with a predicted molecular weight of 57,385. The observed homology between the deduced amino acid sequences of the enzyme and α-galactosidase from Thermus thermophilus was over 40%. The α-galactosidase gene was assigned to family 36 of the glycosyl hydrolases. The enzyme purified from recombinant E. coli showed optimal activity at 40 °C and pH 7. The enzyme hydrolyzed p-nitrophenyl-α-D-galactopyroside, raffinose, stachyose but not melibiose and galactomanno-oligosaccharides, indicating that this enzyme recognizes not only the galactose moiety but also other substrates.     

Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis

Wolbachia pipientis are obligate endosymbionts that infect a wide range of insect and other arthropod species. They act as reproductive parasites by manipulating the host reproduction machinery to enhance their own transmission. This unusual phenotype is thought to be a consequence of the actions of secreted Wolbachia proteins that are likely to contain disulfide bonds to stabilize the protein structure. In bacteria, the introduction or isomerization of disulfide bonds in proteins is catalyzed by Dsb proteins. The Wolbachia genome encodes two proteins, α-DsbA1 and α-DsbA2, that might catalyze these steps. In this work we focussed on the 234 residue protein α-DsbA1; the gene was cloned and expressed in Escherichia coli, the protein was purified and its identity confirmed by mass spectrometry. The sequence identity of α-DsbA1 for both dithiol oxidants (E. coli DsbA, 12%) and disulfide isomerases (E. coli DsbC, 14%) is similar. We therefore sought to establish whether α-DsbA1 is an oxidant or an isomerase based on functional activity. The purified α-DsbA1 was active in an oxidoreductase assay but had little isomerase activity, indicating that α-DsbA1 is DsbA-like rather than DsbC-like. This work represents the first successful example of the characterization of a recombinant Wolbachia protein. Purified α-DsbA1 will now be used in further functional studies to identify protein substrates that could help explain the molecular basis for the unusual Wolbachia phenotypes, and in structural studies to explore its relationship to other disulfide oxidoreductase proteins.     

Purification and properties of a β-glycosidase purified from midgut cells of Spodoptera frugiperda (Lepidoptera) larvae

Two β-glycosidases (BG) (Mr 47,000 and Mr 50,000) were purified from Spodoptera frugiperda (Lepidoptera: Noctuidae) midguts. These two polypeptides associate or dissociate depending on the medium ionic strength. The Mr 47,000 BG probably has two active sites. One of the putative active sites (cellobiase site) hydrolyses p-nitrophenyl β- -glucoside (NPβGlu) (79% of the total activity in saturated enzyme), cellobiose, amygdalin and probably also cellotriose, cellotetraose and cellopentaose. The cellobiase site has four subsites for glucose residue binding, as can be deduced from cellodextrin cleavage data. The enzymatic activity in this site is abolished after carbodiimide modification at pH 6.0. Since the inactivation is reduced in the presence of cellobiose, the results suggest the presence of a carboxylate as a catalytic group. The other active site of Mr 47,000 BG (galactosidase site) hydrolyses p-nitrophenyl β- -galactoside (NPβGal) better than NPβGlu, cleaves glucosylceramide and lactose and is unable to act on cellobiose, cellodextrins and amygdalin. This active site is not modified by carbodiimide at pH 6.0.The Mr 47,000 BG N-terminal sequence has high identity to plant β-glycosidases and to mammalian lactase–phlorizin hydrolase, and contains the QIEGA motif, characteristic of the family of glycosyl hydrolases. The putative physiological role of this enzyme is the digestion of glycolipids (galactosidase site) and di- and oligosaccharides (cellobiase site) derived from hemicelluloses, thus resembling mammalian lactase–phlorizin hydrolase.     

TGFbeta1 and TGFbeta3 are partially redundant effectors in brain vascular morphogenesis

Gene deletion experiments have shown that the three TGFβ isoforms regulate distinct developmental processes. Recent work by our group and others showed that the integrins αvβ6 and αvβ8 activate latent forms of TGFβ1 and TGFβ3. This raises the possibility that TGFβ1 and TGFβ3 act redundantly in developmental processes where both isoforms are expressed and activation is by integrins. To investigate this issue, we generated mice with defective integrin-mediated TGFβ1 activation (Tgfb1^RGE/RGE) that were also homozygous for a null mutation in the TGFβ3 gene. Tgfb1^RGE/RGE; Tgfb3^−/− mice have severely perturbed development of the brain vasculature that is highly similar to that in mice lacking αvβ8. Some Tgfb1^RGE/RGE; Tgfb3^+/− and Tgfb1^RGE/RGE; Tgfb3^+/+ mice have milder, background-dependent versions of the phenotype. In addition, we found that Tgfb3 gene status influences embryonic lethality due to TGFβ1 deficiency after limited backcrossing to the BALB/c background. Conversely, Tgfb1 gene status modifies the extent of palate fusion in Tgfb3^−/− mice after limited backcrossing to the ICR background. Our results are consistent with a functional connection between TGFβ1 and TGFβ3 during development based on a shared mechanism of activation.