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101.
Purification and characterization of guanosine diphospho-D-mannose dehydrogenase. A key enzyme in the biosynthesis of alginate by Pseudomonas aeruginosa 总被引:10,自引:0,他引:10
S Roychoudhury T B May J F Gill S K Singh D S Feingold A M Chakrabarty 《The Journal of biological chemistry》1989,264(16):9380-9385
Alginate-producing Pseudomonas aeruginosa are usually associated with the cystic fibrosis lung environment and contribute to the high mortality rates observed among these patients. The present paper describes the purification and enzymatic properties of guanosine diphospho-D-mannose dehydrogenase (EC 1.1.1.132), a key enzyme in alginate biosynthesis by mucoid P. aeruginosa. The enzyme was overproduced using a plasmid vector containing algD (the gene encoding this enzyme) under control of the tac promoter. It was purified from cell-free lysates by lowering the pH to 5.0, heating the extract to 57.5 degrees C for 10 min, and discarding the protein pellet. The enzyme was selectively precipitated from the supernatant fraction with 45% acetone, resuspended in a 100 mM triethanolamine acetate buffer, pH 7.6, and ultimately purified by Bio-Sil TSK-400 gel filtration chromatography. The subunit molecular weight (Mr 48,000) as well as the N-terminal amino acid sequence corresponded to those predicted from the DNA sequence of algD. The native protein migrated as a hexamer of 290,000 molecular weight upon Bio-Gel A-1.5m gel filtration chromatography. Kinetic analysis demonstrated an apparent Km of 14.9 microM for the substrate GDP-D-mannose and 185 microM for the cofactor NAD+. GDP-D-mannuronic acid was identified as the enzyme reaction product. Several compounds (including GMP, ATP, GDP-D-glucose, and maltose) were found to inhibit enzymatic activity. GMP, the most potent of these inhibitors, exhibited competitive inhibition with an apparent Ki of 22.7 microM. Enzyme activity was also sensitive to the sulfhydryl group modifying agents iodoacetamide and p-hydroxymercuribenzoate. The addition of excess dithiothreitol restored enzyme activity, suggesting a possible involvement of cysteine residues in enzymatic activity. 相似文献
102.
103.
D. Chakrabarty Y. H. Dewir E. J. Hahn S. K. Datta K. Y. Paek 《Plant Growth Regulation》2007,51(1):11-19
The present study investigated the dynamics of nutrient utilization and various growth and physiological parameters during
in vitro proliferation of apple root stock ‘M9 EMLA’ in two different bioreactor systems, i.e. temporary and continuous immersions.
Individual shoots obtained from temporary immersion system had higher dry mass and were of better quality than those obtained
from continuous immersion. In continuous immersion bioreactor, apple shoots appeared to utilize more nutrients from liquid
culture medium than that from temporary immersion. The shoot growth was limited by the availability of phosphate and nitrogen
in continuous immersion system. The shoots produced in temporary immersion bioreactor showed higher photosynthetic rate, maximum
quantum yield of photosystem-II and slow but steady rate of nutrient absorption, indicating the occurrence of higher photomixotrophic
metabolism. The study also showed that high level of antioxidant scavenging enzymes in shoots grown in continuous immersion
system induced physiological changes to foster adaptation to stresses. 相似文献
104.
105.
Nucleoside diphosphate kinase: role in bacterial growth, virulence, cell signalling and polysaccharide synthesis 总被引:5,自引:0,他引:5
A.M. Chakrabarty 《Molecular microbiology》1998,28(5):875-882
Nucleoside diphosphate kinase (Ndk) is an important enzyme that generates nucleoside triphosphates (NTPs) or their deoxy derivatives by terminal phosphotransfer from an NTP such as ATP or GTP to any nucleoside diphosphate or its deoxy derivative. As NTPs, particularly GTP, are important for cellular macromolecular synthesis and signalling mechanisms, Ndk plays an important role in bacterial growth, signal transduction and pathogenicity. Specific examples of the role of Ndk in regulating growth, NTP formation and cell surface polysaccharide synthesis in two respiratory tract pathogens, Pseudomonas aeruginosa and Mycobacterium tuberculosis , are discussed. 相似文献
106.
We have previously reported that two genes cloned from a cosmid library of Escherichia coli can restore mucoidy to an algR2 mutant of Pseudomonas aeruginosa . AlgR2 is a protein involved in the regulation of nucleoside diphosphate kinase (Ndk) as well as alginate synthesis in P. aeruginosa . One of the E. coli genes, rnk , encodes a 14.9 kDa protein with no homology to any other proteins. The other gene, sspA , encodes the stringent starvation protein, a regulatory protein involved in stationary-phase regulation and the stringent response of E. coli . While both rnk and sspA restored alginate production to the P. aeruginosa algR2 mutant, only rnk restored Ndk activity to the mutant. In this report, we have examined the effect of mutations in rnk and sspA on the levels of Ndk in E. coli . We find that a mutation in rnk drastically reduces the level of Ndk in E. coli . A mutation in sspA , however, affects the level of another nucleoside diphosphate kinase distinct from Ndk. The proteins can be easily distinguished from each other by their different affinities for nucleoside diphosphates (NDPs) and also by the differential effect of anti-Ndk antibodies on the reactions they catalyse. The ability of either of these two proteins to restore alginate synthesis in the algR2 mutant of P. aeruginosa demonstrates the importance of nucleoside triphosphate synthesis and energy metabolism for alginate synthesis. Additionally, a role for the stringent starvation protein (SspA) in the modulation of nucleoside triphosphate (NTP) levels in E. coli is also suggested from these experiments. 相似文献
107.
Dubey Sonali Shri Manju Chakrabarty Debasis 《Journal of plant biochemistry and biotechnology.》2021,30(4):744-755
Journal of Plant Biochemistry and Biotechnology - Plants being sessile organisms are exposed to innumerable abiotic and biotic stresses on a daily basis. Heavy metal toxicity in plants results in... 相似文献
108.
Genetic rearrangements in plasmids specifying total degradation of chlorinated benzoic acids 总被引:23,自引:0,他引:23
Summary Growth in a chemostat of the 3-chlorobenzoatepositive Pseudomonas putida cells harboring the plasmid pAC25, in presence of cells harboring the TOL plasmid, allows emergence of cells that can also utilize 4-chlorobenzoate (4Cba). Isolation of plasmid DNA from such cells demonstrate the deletion of a 11kb (Kilobase pair) EcoR1 fragment from the pAC25 plasmid; a portion of the TOL plasmid (41.5 kb TOL*) is also found to be transposed onto the chromosome of such cells. Further enrichment of the 4-chlorobenzoate-positive cells with 3,5-dichlorobenzoate (3,5-Dcb) as a sole carbon source has produced cells that can also slowly utilize 3,5-dichlorobenzoate. Isolation of plasmid DNA from such cells demonstrates the appearance of a second plasmid (pAC29). Restriction hybridization of pAC29 EcoRI fragments with pAC25 and TOL demonstrates that pAC29 is derived primarily by duplication of a segment of the pAC27 plasmid and a fragment from TOL, with further mutational divergence. Southern hybridization of the EcoRI-digested chromosomal DNA with 32P-labeled TOL, pTS11 and pTS71 plasmid DNAs demonstrates the presence of the TOL* transposon containing xylD, G, E and F genes in both 4Cba+ (pAC27+) and 3,5-DCb+ (pAC27+, pAC29+) cells. Isolation of plasmid DNA from 3,5-Dcb+ faster growing variants, obtained from slow-growing pAC27+ pAC29+ cells, demonstrates the presence of a single type of plasmid, with identical size and EcoRI digestion profile as pAC27. The implications of gene duplications and subsequent homologous recombination with regard to the biochemical pathway of 3,5-dichlorobenzoate degradation have been discussed. 相似文献
109.
Transformation of Pseudomonas putida with chromosomal DNA 总被引:3,自引:0,他引:3
J R Mylroie D A Friello A M Chakrabarty 《Biochemical and biophysical research communications》1978,82(1):281-288
The deiodination of [125I]-4-iodobiphenyl, [125I]-4-iodonitrobenzene and [125I]-4-iodoaniline was investigated. No deiodination was detected in rat thyroid homogenates. However, at least three biodeiodination mechanisms were indicated for substrates in rat liver subcellar fractions. Microsomal dehalogenation occurred to a minor extent with increased dehalogenation taking place in the cytosol fraction. The cytosol deiodination was extensive for 4-iodonitrobenzene and was mediated by glutathione. A second cytosol deiodination mechanism, not mediated by glutathione, was evident when 4-iodobiphenyl was the substrate. This soluble enzyme system could be enhanced by Arochlor 1254 or 4-iodobiphenyl pretreatment. 相似文献
110.
Nitrogen fixation (acetylene reduction) associated with roots of winter wheat and sorghum in Nebraska 总被引:2,自引:0,他引:2
W L Pedersen K Chakrabarty R V Klucas A K Vidaver 《Applied and environmental microbiology》1978,35(1):129-135
Root segments and root-soil cores (6.5-cm diameter) from fields and nurseries of winter wheat and sorghum were tested for N2 fixation by using the acetylene reduction assay. Wheat samples (approximately 1,200) from 109 sites generally had low or no activity (0 to 3.1 nmol of C2H4 produced per h per g [dry weight] of root segments), even after 24 h of incubation. However, a commercial field of Scout 66, located in western Nebraska, exhibited appreciable activity (290 nmol of C2H4 produced per h per g [dry weight] of root segments). Of 400 sorghum lines and crosses, grain sorghums (i.e., CK-60A, Wheatland A, B517, and NP-16) generally exhibited higher nitrogenase activity than forage sorghums or winter wheats. CK-60A, a male sterile grain sorghum, was sampled at four locations and had the most consistent activity of 24 to 1,100 nmol of C2H4 produced per h per core. The maximum rate extrapolated to 2.5 g of N per hectare per day. Numerous N2-fixing bacterial isolates were obtained from wheat and sorghum roots that exhibited high nitrogenase activity. Most isolates were members of the Enterobacteriacae, i.e., Klebsiella pneumoniae, Enterobacter cloacae, and Erwinia herbicola. 相似文献