Bioproduction of 4-Vinylphenol and 4-Vinylguaiacol ß-Primeverosides Using Transformed Bamboo Cells Expressing Bacterial Phenolic Acid Decarboxylase.

Phenolic acid decarboxylase (PAD) catalyzes the decarboxylation of hydroxycinnamic acids to produce hydroxystyrenes, which serve as starting materials for the production of polymers. Bamboo (Phyllostachys nigra; Pn) cells, a suitable host for producing phenylpropanoid-derived compounds, were transformed to express PAD of Bacillus amyloliquefaciens (BaPAD). BaPAD-transformed cells accumulated several metabolites that were not detected in wild-type Pn cells or BaPAD-negative transformant. Two major metabolites were isolated from BaPAD-transformed cells, and elucidation of their chemical structures confirmed these as 4-vinylphenol ß-primeveroside (4-VPP) and 4-vinylguaiacol ß-primeveroside (4-VGP). The production titers of 4-VPP and 4-VGP reached 48 and 33 mg/L at the maximum, respectively. Feeding experiments with 4-vinylphenol (4-VP), 4-vinylguaiacol (4-VG), and their glucosides indicated that 4-VPP and 4-VGP are formed by sequential glycosylation of 4-VP and 4-VG via their corresponding glucosides. Our results demonstrate the versatility of Pn cells for producing styrene derivatives, and indicate the presence of a unique glycosylation pathway to produce 4-VPP and 4-VGP in Pn cells.

Evidences for agmatine alterations in Aß1-42induced memory impairment in mice.

Alzheimer's disease is an age related progressive neurodegenerative disorder characterized by decline in cognitive functions, such as memory loss and behavioural abnormalities. The present study sought to assess alterations in agmatine metabolism in the beta-amyloid (Aß1-42) Alzheimer's disease mouse model. Aß1-42 injected mice showed impairment of cognitive functioning as evidenced by increased working and reference memory errors in radial arm maze (RAM). This cognitive impairment was associated with a reduction in the agmatine levels and elevation in its degrading enzyme, agmatinase, whereas reduced immunocontent was observed in its synthesizing enzyme arginine decarboxylase expression within hippocampus and prefrontal cortex. Chronic agmatine treatment and its endogenous modulation by l-arginine, or arcaine or aminoguanidine prevented the learning and memory impairment induced by single intracranial Aß1-42 peptide injection. In conclusion, the present study suggests the importance of the endogenous agmatinergic system in ß-amyloid induced memory impairment in mice.

A three-gene methylation marker panel for the nodal metastatic risk assessment of muscle-invasive bladder cancer.

PURPOSE: In this study, we aimed to identify a DNA methylation pattern suitable for prognosis assessment of muscle-invasive bladder cancer and to investigate metastasis-associated processes regulated by DNA methylation. METHODS: Genome-wide methylation analysis was performed on 23 muscle-invasive bladder tumors by microarray analysis. Validation was performed by the qAMP technique in two different patient cohorts (n = 32 and n = 100). mRNA expression was analyzed in 12 samples. Protein expression was determined using tissue microarrays of 291 patients. Bladder cancer cell lines T24 and 253JB-V were used for functional analyses. RESULTS: Microarray analyses revealed KISS1R, SEPT9 and CSAD as putative biomarkers with hypermethylation in node-positive tumors. The combination of the three genes predicted the metastatic risk with sensitivity of 73% and specificity of 71% in cohort 1, and sensitivity of 82% and specificity of 54% in cohort 2. mRNA expression differences were detected for KISS1R (p = 0.04). Protein expression of KISS1R was significantly reduced (p < 0.001). Knockdown of SEPT9v3 resulted in increased cell migration by 28% (p = 0.04) and increased invasion by 22% (p = 0.004). KISS1R overexpression resulted in decreased cell migration (25%, p = 0.1). CONCLUSIONS: We identified a methylation marker panel suitable to differentiate between patients with positive and negative lymph nodes at time of cystectomy. This enables a risk assessment for patients who potentially benefit from extended lymph node resection as well as from neoadjuvant chemotherapy and could improve the survival rates. Furthermore, we examined the impact of putative markers on tumor behavior. Hence, KISS1R and SEPT9 could represent a starting point for the development of novel therapy approaches.

Knockdown of PAICS inhibits malignant proliferation of human breast cancer cell lines.

BACKGROUND: Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), an enzyme required for de novo purine biosynthesis, is associated with and involved in tumorigenesis. This study aimed to evaluate the role of PAICS in human breast cancer, which remains the most frequently diagnosed cancer and the leading cause of cancer-related death among women in less developed countries. RESULTS: Lentivirus-based short hairpin RNA targeting PAICS specifically depleted its endogenous expression in ZR-75-30 and MDA-MB-231 breast cancer cells. Depletion of PAICS led to a significant decrease in cell viability and proliferation. To ascertain the mechanisms through which PAICS modulates cell proliferation, flow cytometry was performed, and it was confirmed that G1-S transition was blocked in ZR-75-30 cells through PAICS knockdown. This might have occurred partly through the suppression of Cyclin E and the upregulation of Cyclin D1, P21, and CDK4. Moreover, PAICS knockdown obviously promoted cell apoptosis in ZR-75-30 cells through the activation of PARP and caspase 3 and downregulation of Bcl-2 and Bcl-xl expression in ZR-75-30 cells. CONCLUSIONS: These findings demonstrate that PAICS plays an essential role in breast cancer proliferation in vitro, which provides a new opportunity for discovering and identifying novel effective treatment strategies.

Cloning, expression, and functional analysis of lysine decarboxylase in mulberry (Morus alba L.).

1-Deoxynojirimycin (DNJ) is the main bioactive compound of Morus alba L.. DNJ has pharmacological effects, including blood sugar level regulation and antiviral activity. In this study, the mulberry lysine decarboxylase gene (MaLDC), which is involved in the biosynthesis of DNJ alkaloids, was cloned, expressed, and functionally verified. MaLDC was induced and expressed in Escherichia coli BL21 (DE3). The recombinant soluble MaLDC protein had a relative molecular mass of 24.0 kDa. The protein was purified by Ni-NTA separation. The results showed that MaLDC protein could catalyze lysine decarboxylation to produce cadaverine. The Km and Vmax values were 19.2 µM and 3.31 µM/min, respectively. Quantitative real-time reverse transcription polymerase chain reaction revealed that MaLDC expression was positively correlated with DNJ content (P < 0.001), indicating that the MaLDC could encode a functional protein involved in the biosynthesis of DNJ alkaloid in mulberry. Our results provided a foundation for further studies of the enzymatic properties of LDC and established a basis for the analysis of key enzymes involved in the biosynthetic pathway of mulberry DNJ alkaloid.

Characterization and induction of phenolic acid decarboxylase from Aspergillus luchuensis.

Awamori is a traditional distilled liquor in the Ryukyu Islands, made from steamed rice by the action of the black-koji mold Aspergillus luchuensis and awamori yeast Saccharomyces cerevisiae. One of the specific flavors in aged awamori kusu is vanillin, which is derived from ferulic acid (FA) in rice grains. FA is released from the cell wall material in the rice grain by ferulic acid esterase produced by A. luchuensis. Through decarboxylation of FA, 4-vinylguaiacol (4-VG) is produced, which is transferred to the distilled liquor, and converted to vanillin by natural oxidization during the aging process. However, the actual mechanism for conversion of FA to 4-VG in the awamori brewing process is unknown. A genetic sequence having homology to the phenolic acid decarboxylase (PAD)-encoding region from bacteria and the yeast Candida guilliermondii has been identified in A. luchuensis mut. kawachii. In the present study, recombinant PAD from A. luchuensis, designated as AlPAD, expressed as a homodimer, catalyzed the conversion of FA to 4-VG, displayed optimal catalytic activity at pH 5.7 and 40°C, and was stable up to 50°C. Both rice bran and FA could induce the bioconversion of FA to 4-VG and the expression of AlPAD in A. luchuensis. The amount of AlPAD determined using western blotting correlated with the level of FA decarboxylase activity during koji production. In awamori brewing process, AlPAD might be responsible for a part of the conversion of FA to 4-VG.

Characterization of a Whole-Cell Biotransformation Using a Constitutive Lysine Decarboxylase from Escherichia coli for the High-Level Production of Cadaverine from Industrial Grade L-Lysine.

Cadaverine is used for the synthesis of the novel bio-polyamides 54, 56, and 510. Here, we examine the feasibility of using a lysine decarboxylase (LdcC) from Escherichia coli for high-level production of cadaverine. After sequential optimization of whole-cell biotransformation conditions, recombinant E. coli-overexpressing LdcC (EcLdcC) could produce 1.0 M cadaverine from 1.2 M crude L-lysine solution after 9 h. EcLdcC retained a higher cadaverine yield after being reused 10 times at acidic and alkaline pH values than that of a recombinant E. coli strain overexpressing an inducible lysine decarboxylase (CadA), a conventional cadaverine producer (90 vs. 51% at pH 6 and 55 vs. 15% at pH 8). This study reveals that EcLdcC is a promising whole-cell biocatalyst for the bio-based production of cadaverine from industrial grade L-lysine in comparison to EcCadA.

Knockdown of PAICS inhibits malignant proliferation of human breast cancer cell lines

BACKGROUND: Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), an enzyme required for de novo purine biosynthesis, is associated with and involved in tumorigenesis. This study aimed to evaluate the role of PAICS in human breast cancer, which remains the most frequently diagnosed cancer and the leading cause of cancer-related death among women in less developed countries. RESULTS: Lentivirus-based short hairpin RNA targeting PAICS specifically depleted its endogenous expression in ZR-75-30 and MDA-MB-231 breast cancer cells. Depletion of PAICS led to a significant decrease in cell viability and proliferation. To ascertain the mechanisms through which PAICS modulates cell proliferation, flow cytometry was performed, and it was confirmed that G1-S transition was blocked in ZR-75-30 cells through PAICS knockdown. This might have occurred partly through the suppression of Cyclin E and the upregulation of Cyclin D1, P21, and CDK4. Moreover, PAICS knockdown obviously promoted cell apoptosis in ZR-75-30 cells through the activation of PARP and caspase 3 and downregulation of Bcl-2 and Bcl-xl expression in ZR-75-30 cells. CONCLUSIONS: These findings demonstrate that PAICS plays an essential role in breast cancer proliferation in vitro, which provides a new opportunity for discovering and identifying novel effective treatment strategies.

Production of low-expressing recombinant cationic biopolymers with high purity.

The growing complexity of recombinant biopolymers for delivery of bioactive agents requires the ability to control the biomaterial structure with high degree of precision. Genetic engineering techniques have provided this opportunity to synthesize biomaterials in an organism such as E. coli with full control over their lengths and sequences. One class of such biopolymers is recombinant cationic biopolymers with applications in gene delivery, regenerative medicine and variety of other biomedical applications. Unfortunately, due to their highly cationic nature and complex structure, their production in E. coli expression system is marred by low expression yield which in turn complicates the possibility of obtaining pure biopolymer. SlyD and ArnA endogenous E. coli proteins are considered the major culprits that copurify with the low-expressing biopolymers during the metal affinity chromatography. Here, we compared the impact of different parameters such as the choice of expression hosts as well as metal affinity columns in order to identify the most effective approach in obtaining highly pure recombinant cationic biopolymers with acceptable yield. The results of this study showed that by using E. coli BL21(DE3) LOBSTR strain and in combination with our developed stringent expression and Ni-NTA purification protocols highly pure products in one purification step (>99% purity) can be obtained. This approach could be applied to the production of other complex and potentially toxic biopolymers with wide range of applications in biomedicine.

Pgas, a Low-pH-Induced Promoter, as a Tool for Dynamic Control of Gene Expression for Metabolic Engineering of Aspergillus niger.

The dynamic control of gene expression is important for adjusting fluxes in order to obtain desired products and achieve appropriate cell growth, particularly when the synthesis of a desired product drains metabolites required for cell growth. For dynamic gene expression, a promoter responsive to a particular environmental stressor is vital. Here, we report a low-pH-inducible promoter, Pgas, which promotes minimal gene expression at pH values above 5.0 but functions efficiently at low pHs, such as pH 2.0. First, we performed a transcriptional analysis of Aspergillus niger, an excellent platform for the production of organic acids, and we found that the promoter Pgas may act efficiently at low pH. Then, a gene for synthetic green fluorescent protein (sGFP) was successfully expressed by Pgas at pH 2.0, verifying the results of the transcriptional analysis. Next, Pgas was used to express the cis-aconitate decarboxylase (cad) gene of Aspergillus terreus in A. niger, allowing the production of itaconic acid at a titer of 4.92 g/liter. Finally, we found that Pgas strength was independent of acid type and acid ion concentration, showing dependence on pH only.IMPORTANCE The promoter Pgas can be used for the dynamic control of gene expression in A. niger for metabolic engineering to produce organic acids. This promoter may also be a candidate tool for genetic engineering.

Enhanced production of butanol and acetoin by heterologous expression of an acetolactate decarboxylase in Clostridium acetobutylicum.

Butanol is an important industrial chemical and an attractive transportation fuel. However, the deficiency of reducing equivalents NAD(P)H in butanol fermentation results in a large quantity of oxidation products, which is a major problem limiting the atom economy and economic viability of bio-butanol processes. Here, we integrated the butanol fermentation process with a NADH-generating, acetoin biosynthesis process to improve the butanol production. By overexpressing the α-acetolactate decarboxylase gene alsD from Bacillus subtilis in Clostridium acetobutylicum, acetoin yield was significantly increased at the cost of acetone. After optimization of fermentation conditions, butanol (12.9g/L), acetoin (6.5g/L), and ethanol (1.9g/L) were generated by the recombinant strain, with acetone no more than 1.8g/L. Thus, both mass yield and product value were greatly improved. This study demonstrates that reducing power compensation is effective to improve the atom economy of butanol fermentation, and provides a novel approach to improve the economic viability of bio-butanol production.

Calcium-insensitive splice variants of mammalian E1 subunit of 2-oxoglutarate dehydrogenase complex with tissue-specific patterns of expression.

The 2-oxoglutarate dehydrogenase (OGDH) complex is an important control point in vertebrate mitochondrial oxidative metabolism, including in the citrate cycle and catabolism of alternative fuels including glutamine. It is subject to allosteric regulation by NADH and the ATP/ADP ratio, and by Ca(2+) through binding to the E1 subunit. The latter involves a unique Ca(2+)-binding site which includes D(114)ADLD (site 1). Here, we describe three splice variants of E1 in which either the exon expressing this site is replaced with another exon (loss of site 1, LS1) or an additional exon is expressed leading to the insertion of 15 amino acids just downstream of site 1 (Insert), or both changes occur together (LS1/Insert). We show that all three variants are essentially Ca(2+)-insensitive. Comparison of massive parallel sequence (RNA-Seq) databases demonstrates predominant expression of the Ca(2+)-sensitive archetype form in heart and skeletal muscle, but substantial expression of the Ca(2+)-insensitive variants in brain, pancreatic islets and other tissues. Detailed proteomic and activity studies comparing OGDH complexes from rat heart and brain confirmed the substantial difference in expression between these tissues. The evolution of OGDH variants was explored using bioinformatics, and this indicated that Ca(2+)-sensitivity arose with the emergence of chordates. In all species examined, this was associated with the co-emergence of Ca(2+)-insensitive variants suggesting a retained requirement for the latter in some settings. Tissue-specific expression of OGDH splice variants may thus provide a mechanism that tunes the control of the enzyme to the specialized metabolic and signalling needs of individual cell types.

VID22 is required for transcriptional activation of the PSD2 gene in the yeast Saccharomyces cerevisiae.

Phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae is synthesized through decarboxylation of phosphatidylserine (PS), catalysed by PS decarboxylase 1 (Psd1p) and 2 (Psd2p) and the cytidine 5'-diphosphate (CDP)-ethanolamine (CDP-Etn) pathway. PSD1 null (psd1Δ) and PSD2 null (psd2Δ) mutants are viable in a synthetic minimal medium, but a psd1Δ psd2Δ double mutant exhibits Etn auxotrophy, which is incorporated into PE through the CDP-Etn pathway. We have previously shown that psd1Δ is synthetic lethal with deletion of VID22 (vid22Δ) [Kuroda et al. (2011) Mol. Microbiol. 80: , 248-265]. In the present study, we found that vid22Δ mutant exhibits Etn auxotrophy under PSD1-depressed conditions. Deletion of VID22 in wild-type and PSD1-depressed cells caused partial defects in PE formation through decarboxylation of PS. The enzyme activity of PS decarboxylase in an extract of vid22Δ cells was ∼70% of that in wild-type cells and similar to that in psd2Δ cells and the PS decarboxylase activity remaining in the PSD1-depressed cells became almost negligible with deletion of VID22. Thus, the vid22Δ mutation was suggested to cause a defect in the Psd2p activity. Furthermore, vid22Δ cells were shown to be defective in expression of the PSD2 gene tagged with 6×HA, the defect being ameliorated by replacement of the native promoter of the PSD2 gene with a CYC1 promoter. In addition, an α-galactosidase reporter assay revealed that the activity of the promoter of the PSD2 gene in vid22Δ cells was ∼5% of that in wild-type cells. These results showed that VID22 is required for transcriptional activation of the PSD2 gene.

Involvement of microRNA214 and transcriptional regulation in reductions in mevalonate pyrophosphate decarboxylase mRNA levels in stroke-prone spontaneously hypertensive rat livers.

Hypocholesterolemia has been epidemiologically identified as one of the causes of stroke (cerebral hemorrhage). We previously reported that lower protein levels of mevalonate pyrophosphate decarboxylase (MPD), which is responsible for reducing serum cholesterol levels in stroke-prone spontaneously hypertensive rats (SHRSP), in the liver were caused by a reduction in mRNA levels. However, the mechanism responsible for reducing MPD expression levels in the SHRSP liver remains unclear. Thus, we compared microRNA (miR)-214 combined with the 3'-untranslated region of MPD mRNA and heterogeneous nuclear RNA (hnRNA) between SHRSP and normotensive Wistar Kyoto rats (WKY). miR-214 levels in the liver were markedly higher in SHRSP than in WKY, whereas hnRNA levels were significantly lower. These results indicate that the upregulation of miR-214 and downregulation of MPD transcription in the liver both play a role in the development of hypocholesterolemia in SHRSP.

Metabolic engineering of Corynebacterium glutamicum for the production of itaconate.

The capability of Corynebacterium glutamicum for glucose-based synthesis of itaconate was explored, which can serve as building block for production of polymers, chemicals, and fuels. C. glutamicum was highly tolerant to itaconate and did not metabolize it. Expression of the Aspergillus terreus CAD1 gene encoding cis-aconitate decarboxylase (CAD) in strain ATCC13032 led to the production of 1.4mM itaconate in the stationary growth phase. Fusion of CAD with the Escherichia coli maltose-binding protein increased its activity and the itaconate titer more than two-fold. Nitrogen-limited growth conditions boosted CAD activity and itaconate titer about 10-fold to values of 1440 mU mg(-1) and 30 mM. Reduction of isocitrate dehydrogenase activity via exchange of the ATG start codon to GTG or TTG resulted in maximal itaconate titers of 60 mM (7.8 g l(-1)), a molar yield of 0.4 mol mol(-1), and a volumetric productivity of 2.1 mmol l(-1) h(-1).

Cysteine dioxygenase and cysteine sulfinate decarboxylase genes of the deep-sea mussel Bathymodiolus septemdierum: possible involvement in hypotaurine synthesis and adaptation to hydrogen sulfide.

It has been suggested that invertebrates inhabiting deep-sea hydrothermal vent areas use the sulfinic acid hypotaurine, a precursor of taurine, to protect against the toxicity of hydrogen sulfide contained in the seawater from the vent. In this protective system, hypotaurine is accumulated in the gill, the primary site of sulfide exposure. However, the pathway for hypotaurine synthesis in mollusks has not been identified. In this study, we screened for the mRNAs of enzymes involved in hypotaurine synthesis in the deep-sea mussel Bathymodiolus septemdierum and cloned cDNAs encoding cysteine dioxygenase and cysteine sulfinate decarboxylase. As mRNAs encoding cysteamine dioxygenase and cysteine lyase were not detected, the cysteine sulfinate pathway is suggested to be the major pathway of hypotaurine and taurine synthesis. The two genes were found to be expressed in all the tissues examined, but the gill exhibited the highest expression. The mRNA level in the gill was not significantly changed by exposure to sulfides or thiosulfate. These results suggests that the gill of B. septemdierum maintains high levels of expression of the two genes regardless of ambient sulfide level and accumulates hypotaurine continuously to protect against sudden exposure to high level of sulfide.

Simultaneous production of butanol and acetoin by metabolically engineered Clostridium acetobutylicum.

Biobutanol is a potential fuel substitute and has been receiving increased attention in recent years. However, the economics of biobutanol production have been hampered by a number of bottlenecks such as high cost of raw material and low yield of solvent. Co-production of value-added products is a possible way to improve the economics of biobutanol production. Here, we present metabolic engineering strategies to substitute the major by-product acetone for a value-added product acetoin during butanol fermentation. By overexpressing the α-acetolactate decarboxylase gene alsD in Clostridium acetobutylicum B3, the acetoin yield was markedly increased while acetone formation was reduced. Subsequent disruption of adc gene effectively abolished acetone formation and further increased acetoin yield. After optimization of fermentation conditions, the alsD-overexpressing adc mutant generated butanol (13.8g/L), acetoin (4.3g/L), and ethanol (3.9g/L), but no acetone. Thus, acetone was completely substituted for acetoin, and both mass yield and product value were improved. This study provides valuable insights into the regulation of acetoin synthesis and should be highly useful for the development of acetoin-derived products like 2,3-butanediol and 2-butanol in C. acetobutylicum.

[Cloning, prokaryotic expression and characterization of lysine decarboxylase gene from Huperzia serrata].

Huperzine A is a promising drug to treat Alzheimer's disease (AD). To date, its biosynthetic pathway is still unknown. Lysine decarboxylase (LDC) has been proposed to catalyze the first-step of the biosynthesis of huperzine A. To identify and characterize LDCs from Huperzia serrata, we isolated two LDC fragments (LDC1 and LDC2) from leaves of H. serrata by RT-PCR and then cloned them into pMD 19-T vector. Sequence analysis showed that LDC1 and LDC2 genes shared 95.3% identity and encoded the protein of 212 and 202 amino acid residues respectively. Thus, we ligated LDC genes into pET-32a(+) to obtain recombinant expressing vectors pET-32a(+)/LDC1 and pET-32a(+)/LDC2 respectively. We further introduced two expression vectors into Escherichia coli BL21(DE3) and cultured positive colonies of E. coli in liquid LB medium. After inducing for 4 hours with 260 µg/mL IPTG at 30 degrees C, soluble recombinant Trx-LDC1 and Trx-LDC2 were obtained and isolated for purification using a Ni-NTA affinity chromatography. We incubated purified recombinant proteins with L-lysine in the enzyme reaction buffer at 37 degrees C and then derived the reaction products using dansyl chloride. It was found that both Trx-LDC1 and Trx-LDC2 had decarboxylase activity, could convert L-lysine into cadaverine by way of thin layer chromatography assay. Further, bioinformatics analysis indicated that deduced LDC1 and LDC2 had different physicochemical properties, but similar secondary and three-dimensional structures.

Putrescine reduces antibiotic-induced oxidative stress as a mechanism of modulation of antibiotic resistance in Burkholderia cenocepacia.

Communication of antibiotic resistance among bacteria via small molecules is implicated in transient reduction of bacterial susceptibility to antibiotics, which could lead to therapeutic failures aggravating the problem of antibiotic resistance. Released putrescine from the extremely antibiotic-resistant bacterium Burkholderia cenocepacia protects less-resistant cells from different species against the antimicrobial peptide polymyxin B (PmB). Exposure of B. cenocepacia to sublethal concentrations of PmB and other bactericidal antibiotics induces reactive oxygen species (ROS) production and expression of the oxidative stress response regulator OxyR. We evaluated whether putrescine alleviates antibiotic-induced oxidative stress. The accumulation of intracellular ROS, such as superoxide ion and hydrogen peroxide, was assessed fluorometrically with dichlorofluorescein diacetate, while the expression of OxyR and putrescine synthesis enzymes was determined in luciferase assays using chromosomal promoter-lux reporter system fusions. We evaluated wild-type and isogenic deletion mutant strains with defects in putrescine biosynthesis after exposure to sublethal concentrations of PmB and other bactericidal antibiotics. Exogenous putrescine protected against oxidative stress induced by PmB and other antibiotics, whereas reduced putrescine synthesis resulted in increased ROS generation and a parallel increased sensitivity to PmB. Of the 3 B. cenocepacia putrescine-synthesizing enzymes, PmB induced only BCAL2641, an ornithine decarboxylase. This study reveals BCAL2641 as a critical component of the putrescine-mediated communication of antibiotic resistance and as a plausible target for designing inhibitors that would block the communication of such resistance among different bacteria, ultimately reducing the window of therapeutic failure in treating bacterial infections.

Characterization of a pyridoxal-5'-phosphate-dependent l-lysine decarboxylase/oxidase from Burkholderia sp. AIU 395.

A novel enzyme, which catalyzed decarboxylation of l-lysine into cadaverine with release of carbon dioxide and oxidative deamination of l-lysine into l-2-aminoadipic 5-semialdehyde with release of ammonia and hydrogen peroxide, was found from a newly isolated Burkholderia sp. AIU 395. The enzyme was specific to l-lysine and did not exhibit enzyme activities for other l-amino acids, l-lysine derivatives, d-amino acids, and amines. The apparent Km values for l-lysine in the oxidation and decarboxylation reactions were estimated to be 0.44 mM and 0.84 mM, respectively. The molecular mass was estimated to be 150 kDa, which was composed of two identical subunits with molecular mass of 76.5 kDa. The enzyme contained one mol of pyridoxal 5'-phosphate per subunit as a prosthetic group. The enzyme exhibiting decarboxylase and oxidase activities for l-lysine was first reported here, while the deduced amino acid sequence was homologous to that of putative lysine decarboxylases from the genus Burkholderia.