Crystal structure analysis of a bacterial aryl acylamidase belonging to the amidase signature enzyme family.

The atomic structure of a bacterial aryl acylamidase (EC 3.5.1.13; AAA) is reported and structural features are investigated to better understand the catalytic profile of this enzyme. Structures of AAA were determined in its native form and in complex with the analgesic acetanilide, p-acetaminophenol, at 1.70 Å and 1.73 Å resolutions, respectively. The overall structural fold of AAA was identified as an α/ß fold class, exhibiting an open twisted ß-sheet core surrounded by α-helices. The asymmetric unit contains one AAA molecule and the monomeric form is functionally active. The core structure enclosing the signature sequence region, including the canonical Ser-cisSer-Lys catalytic triad, is conserved in all members of the Amidase Signature enzyme family. The structure of AAA in a complex with its ligand reveals a unique organization in the substrate-binding pocket. The binding pocket consists of two loops (loop1 and loop2) in the amidase signature sequence and one helix (α10) in the non-amidase signature sequence. We identified two residues (Tyr(136) and Thr(330)) that interact with the ligand via water molecules, and a hydrogen-bonding network that explains the catalytic affinity over various aryl acyl compounds. The optimum activity of AAA at pH > 10 suggests that the reaction mechanism employs Lys(84) as the catalytic base to polarize the Ser(187) nucleophile in the catalytic triad.

The contest for precursors: channelling L-isoleucine synthesis in Corynebacterium glutamicum without byproduct formation.

L-Isoleucine is an essential amino acid, which is required as a pharma product and feed additive. Its synthesis shares initial steps with that of L-lysine and L-threonine, and four enzymes of L-isoleucine synthesis have an enlarged substrate specificity involved also in L-valine and L-leucine synthesis. As a consequence, constructing a strain specifically overproducing L-isoleucine without byproduct formation is a challenge. Here, we analyze for consequences of plasmid-encoded genes in Corynebacterium glutamicum MH20-22B on L-isoleucine formation, but still obtain substantial accumulation of byproducts. In a different approach, we introduce point mutations into the genome of MH20-22B to remove the feedback control of homoserine dehydrogenase, hom, and threonine dehydratase, ilvA, and we assay sets of genomic promoter mutations to increase hom and ilvA expression as well as to reduce dapA expression, the latter gene encoding the dihydrodipicolinate synthase. The promoter mutations are mirrored in the resulting differential protein levels determined by a targeted LC-MS/MS approach for the three key enzymes. The best combination of genomic mutations was found in strain K2P55, where 53 mM L-isoleucine could be obtained. Whereas in fed-batch fermentations with the plasmid-based strain, 94 mM L-isoleucine with L-lysine as byproduct was formed; with the plasmid-less strain K2P55, 109 mM L-isoleucine accumulated with no substantial byproduct formation. The specific molar yield with the latter strain was 0.188 mol L-isoleucine (mol glucose)(-1) which characterizes it as one of the best L-isoleucine producers available and which does not contain plasmids.

Production of p-acetaminophenol by whole-cell catalysis using Escherichia coli overexpressing bacterial aryl acylamidase.

Aryl acylamidase (EC 3.5.1.13, AAA) acts on the amide bond between aryl and acyl groups. Whole cells of Escherichia coli overexpressing a novel bacterial AAA synthesized p-acetaminophenol (p-AAP) from p-aminophenol (p-AP, aryl compound) and acetate (acyl donor). Optimum conditions were pH 5.5 and 35°C with 100 mM p-AP and 600 mM sodium acetate in 100 mM sodium phosphate buffer including 1% (v/v) Triton X-100 for 60 h. 13.1 g p-AAP l(-1) was produced with a conversion yield of 87%.

Molecular characterization of a novel bacterial aryl acylamidase belonging to the amidase signature enzyme family.

In seeking aryl acylamidase (EC 3.5.1.13) acting on an amide bond in p-acetaminophenol (Tylenol), we identified a novel gene encoding 496 residues of a protein. The gene revealed a conserved amidase signature region with a canonical catalytic triad. The gene was expressed in E. coli and characterized for its biochemical properties. The optimum pH and temperature for the activity on p-acetaminophenol were 10 and 37 degrees C, respectively. The half-life of enzyme activity at 37 degrees C was 192 h and 90% of its activity remained after 3 h incubation at 40 degrees C. Divalent metals was found to inhibit the activity of enzyme. The K (m) values for various aryl acylamides such as 4-nitroacetanilide, p-acetaminophenol, phenacetin, 4-chloroacetanilide and acetanilide were 0.10, 0.32, 0.83, 1.9 and 19 mM, respectively. The reverse reaction activity (amide synthesis) was also examined using various chain lengths (C(1) approximately C(4) and C(10)) of carboxylic donors and aniline as substrates. These kinetic parameters and substrate specificity in forward and reverse reaction indicated that the aryl acylamidase in this study has a preference for aryl substrate having polar functional groups and hydrophobic carboxylic donors.

Overexpression and molecular characterization of Aga50D from Saccharophagus degradans 2-40: an exo-type beta-agarase producing neoagarobiose.

beta-Agarases are mostly categorized into three glycoside hydrolase (GH) families 16, 50, and 86. Recent genomic analysis of Saccharophagus degradans 2-40 revealed the presence of five agarase genes belonging to these GH families. Among the five agarases, Aga50D (a member of GH50) had neither been functionally characterized nor overexpressed. In this report, we present soluble overexpression and molecular characterization of Aga50D. Aga50D was expressed in an active form resulting in a single major product from agarose without intermediates. While known GH50 agarases have both endo-lytic and exo-lytic activities, which produce neoagarobiose as a final product through the intermediate, neoagaro-oligosaccharides, identification and analysis of the reaction product by mass spectrometry and 13C NMR showed that Aga50D had unique exo-lytic activity and was able to produce neoagarobiose directly from agarose. The optimum pH and temperature for the activity were 7.0 and 30 degrees C, respectively. The K (m) and V (max) for agarose were 41.9 mg/ml (4.2 mM) and 17.9 U/mg, respectively.

Functional characterization of a bacterial expansin from Bacillus subtilis for enhanced enzymatic hydrolysis of cellulose.

Expansin is a plant protein family that induces plant cell wall-loosening and cellulose disruption without exerting cellulose-hydrolytic activity. Expansin-like proteins have also been found in other eukaryotes such as nematodes and fungi. While searching for an expansin produced by bacteria, we found that the BsEXLX1 protein from Bacillus subtilis had a structure that was similar to that of a beta-expansin produced by maize. Therefore, we cloned the BsEXLX1 gene and expressed it in Escherichia coli to evaluate its function. When incubated with filter paper as a cellulose substrate, the recombinant protein exhibited both cellulose-binding and cellulose-weakening activities, which are known functions of plant expansins. In addition, evaluation of the enzymatic hydrolysis of filter paper revealed that the recombinant protein also displayed a significant synergism when mixed with cellulase. By comparing the activity of a mixture of cellulase and the bacterial expansin to the additive activity of the individual proteins, the synergistic activity was found to be as high as 240% when filter paper was incubated with cellulase and BsEXLX1, which was 5.7-fold greater than the activity of cellulase alone. However, this synergistic effect was observed when only a low dosage of cellulase was used. This is the first study to characterize the function of an expansin produced by a non-eukaryotic source.

Optimization of ascorbic acid-2-phosphate production from ascorbic acid using resting cell of Brevundimonas diminuta.

With the aim to produce ascorbic acid-2-phosphate (AsA-2-P) from L-ascorbic acid (AsA, Vitamin C), nine bacteria conferring the ability to transform AsA to AsA-2-P were isolated from soil samples alongside known strains from culture collections. Most isolates were classified to the genus Brevundimonas by 16S phylogenetic analysis. Among them, Brevundimonas diminuta KACC 10306 was selected as the experimental strain because of its the highest productivity of AsA-2-P. The optimum set of conditions for the AsA-2-P production from AsA using resting cells as the source of the enzyme was also investigated. The optimum cultivation time was 16 h and the cell concentration was 120 g/l (wet weight). The optimum concentrations of AsA and pyrophosphate were 550 mM and 450 mM, respectively. The most effective buffer was 50 mM sodium formate. The optimum pH was 4.5 and temperature was 40 degrees C. Under the above conditions, 27.5 g/l of AsA-2-P was produced from AsA after 36 h of incubation, which corresponded to a 19.7% conversion efficiency based on the initial concentration of AsA.

Genes encoding ribonucleoside hydrolase 1 and 2 from Corynebacterium ammoniagenes.

Two kinds of nucleoside hydrolases (NHs) encoded by rih1 and rih2 were cloned from Corynebacterium ammoniagenes using deoD- and gsk-defective Escherichia coli. Sequence analysis revealed that NH 1 was a protein of 337 aa with a deduced molecular mass of 35,892 Da, whereas NH 2 consisted of 308 aa with a calculated molecular mass of 32 310 Da. Experiments with crude extracts of IPTG-induced E. coli CGSC 6885(pTNU23) and 6885(pTNI12) indicated that the Rih1 enzyme could catalyse the hydrolysis of uridine and cytidine and showed pyrimidine-specific ribonucleoside hydrolase activity. Rih2 was able to hydrolyse both purine and pyrimidine ribonucleosides with the following order of activity -- inosine>adenosine>uridine>guanosine>xanthosine>cytidine -- and was classified in the non-specific NHs family. rih1 and rih2 deletion mutants displayed a decrease in cell growth on minimal medium supplemented with pyrimidine and purine/pyrimidine nucleosides, respectively, compared with the wild-type strain. Growth of each mutant was substantially complemented by introducing rih1 and rih2, respectively. Furthermore, disruption of both rih1 and rih2 led to the inability of the mutant to utilize purine and pyrimidine nucleosides as sole carbon source on minimal medium. These results indicated that rih1 and rih2 play major roles in the salvage pathways of nucleosides in this micro-organism.

Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5-HT6) and the alpha subunit of GS protein.

The serotonin type 6 (5-HT(6)) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (G(S)). To identify the structural basis for the interaction of the 5-HT(6) receptor with the G(S) protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT(6) receptor and the alpha subunit of G(S) (Galpha(S)). The direct interaction of iL3 and Galpha(S) was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and Galpha(S) were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9 x 10(-6)M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to Galpha(S). The critical residues within the iL3 region for the interaction with Galpha(S) were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT(6) receptor mutants.

Use of plant protein hydrolysates for varicella virus production in serum-free medium.

Serum-free media containing no animal-derived components were assessed for their efficacy to produce live attenuated varicella virus. Serum-free medium containing an ultrafiltrate of soy protein acid hydrolysate and lipid resulted in a viral production yield comparable to media containing fetal bovine serum, indicating that varicella virus can be produced without the risk of contamination associated with the use of bovine serum.

Effect of chelators and reductants on the mobilization of metals from ambient particulate matter.

Ambient urban particulate matter (PM) contains various transition metals. When the PM is inhaled into the lung, not all but some part of metals from the particles might be mobilized to participate in a reaction that can damage various biomolecules, such as DNA and proteins. The dust particle size as well as organic acids may influence the metal mobilization. Thus, the mobilization of the metal from two standard reference materials (SRM; NIST, USA) and urban PM (PM2.5 and PM10) collected in the Seoul area was measured in the presence of artificial or biological chelator with or without reductant. The degree of the mobilization was higher with the artificial or biological chelator than the control with saline. In some cases, a reductant increased the mobilization as much as about 5 times the control without the reductant. Especially, the mobilization of Fe was greatly influenced by the presence of reductants. In general, the degree of the mobilization of the transition metal was higher with PM2.5 than with PM10. Therefore, it is expected that, considering the previously known toxicities of the transition metals, PM2.5 is more damaging to various biomolecules than PM10. The results also suggest that not the total amount but the mobilizable fraction of the metal in the ambient PM should be considered with regard to the toxicity of the urban particulate matter.

Enhanced production of recombinant B-domain deleted factor VIII from Chinese hamster ovary cells by propionic and butyric acids.

Sodium propionate, as well as sodium butyrate, enhanced the production of recombinant B-domain-deleted, factor VIII (rFVIIIdB) by Chinese hamster ovary cells growing in a spinner-flask with a protein-free medium by more than six-fold. The two acids, however, had different cytotoxicities.

Identification of immunodominant Helicobacter pylori proteins with reactivity to H. pylori-specific egg-yolk immunoglobulin.

The importance of hens eggs as a source of specific antibodies (IgY) is well recognized. The protective effect of IgY obtained from hens immunized with Helicobacter pylori whole-cell lysate has been reported for the control of H. pylori infection. However, IgY produced by whole-cell lysates presents the possibility of cross-reactivity with other bacteria, including the normal human flora, and this could decrease the efficiency of IgY. In the present study, the immunodominant proteins of H. pylori with reactivity to H. pylori-specific IgY (IgY-Hp) were identified. IgY obtained from hens immunized with various fractions of H. pylori proteins was isolated and purified, titres of IgY-Hp against H. pylori were determined and cross-reactivity between IgY-Hp and normal human bacteria was examined by Western blot analysis. Finally, immunodominant H. pylori proteins were identified by LC/MS analysis. IgY obtained 2 months after immunization with H. pylori whole-cell lysate showed the highest antibody titre. Five immunodominant proteins were identified that were strongly reactive to IgY-Hp: urease beta-subunit (62 kDa), heat-shock protein 60 (60 kDa), urease alpha-subunit (26 kDa), probable peroxiredoxin (22 kDa) and probable thiol peroxidase (18 kDa). Immunization of hens with the immunodominant proteins identified would produce a more specific IgY against H. pylori.

Use of egg yolk-derived immunoglobulin as an alternative to antibiotic treatment for control of Helicobacter pylori infection.

The present study evaluated the potential use of immunoglobulin prepared from the egg yolk of hens immunized with Helicobacter pylori (immunoglobulin Y [IgY]-Hp) in the treatment of H. pylori infections. The purity of our purified IgY-Hp was 91.3%, with a yield of 9.4 mg of IgY per ml of egg yolk. The titer for IgY-Hp was 16 times higher than that for IgY in egg yolk from nonimmunized hens, and IgY-Hp significantly inhibited the growth and urease activity of H. pylori in vitro. Bacterial adhesion on AGS cells was definitely reduced by preincubation of both H. pylori (10(8) CFU/ml) and 10 mg of IgY-Hp/ml. In Mongolian gerbil models, IgY-Hp decreased H. pylori-induced gastric mucosal injury as determined by the degree of lymphocyte and neutrophil infiltration. Therefore, in this experimental model, H. pylori-associated gastritis could be successfully treated by orally administered IgY-Hp. The immunological activity of IgY-Hp stayed active at 60 degrees C for 10 min, suggesting that pasteurization can be applied to sterilize the product. Fortification of food products with this immunoglobulin would significantly decrease the H. pylori infection. In conclusion, the IgY-Hp obtained from hens immunized by H. pylori could provide a novel alternative approach to treatment of H. pylori infection.