|
|
|
|
|
|
The acnD Genes of Shewenella oneidensis and Vibrio cholerae Encode a New Fe/S-Dependent 2-Methylcitrate Dehydratase Enzyme That Requires prpF Function In Vivo. Tracey L. Grimek, 2004.The propionate utilization operons of several bacteria differ from each other in the occurrence of two genes, acnD and prpF, in place of or in addition to the prpD gene encoding an Fe/S-independent 2-methylcitrate dehydratase enzyme . We cloned the acnD and prpF genes from two organisms, Shewanella oneidensis and Vibrio cholerae, and found that, together, the AcnD and PrpF proteins restored the ability of a prpD mutant strain of Salmonella enterica to grow on propionate as a source of carbon and energy . However, neither acnD nor prpF alone was able to substitute for prpD . The AcnD and PrpF proteins were isolated and biochemically analyzed . The AcnD protein required reconstitution of an Fe/S cluster for activity . All detectable AcnD activity was lost after incubation with iron-chelating agents, and no AcnD activity was observed after attempted reconstitution without iron . Nuclear magnetic resonance spectroscopy and in vitro activity assay data showed that AcnD dehydrated 2-methylcitrate and citrate to 2-methyl-cis-aconitate and cis-aconitate, respectively; AcnD also hydrated cis-aconitate . However, 2-methylisocitrate and isocitrate were not substrates for AcnD, indicating that AcnD only catalyzes the first half of the aconitase-like dehydration reactions . No aconitase-like activity was found for PrpF . It is hypothesized that, in vivo, PrpF is an accessory protein required to prevent oxidative damage of the Fe/S center of active AcnD enzyme or that it may be involved in synthesis or repair of the Fe/S cluster present in AcnD .
Domain Interactions on the ntr Signal Transduction Pathway: Two-Hybrid Analysis of Mutant and Truncated Derivatives of Histidine Kinase NtrB. Isabel Martínez-Argudo, 2002.We have used the yeast two-hybrid system to analyze protein-protein interactions mediated by domains of regulatory proteins of the ntr signal transduction system, including interactions among NtrB derivatives and their interactions with NtrC and PII from Klebsiella pneumoniae . Interactions took place only between proteins or protein domains belonging to the ntr signal transduction system and not between proteins or domains from noncognate regulators . NtrB and its transmitter domain, but not NtrC, CheA, or the cytoplasmic C terminus of EnvZ, interacted with PII . In addition, interaction of NtrB with NtrC, but not with PII, depended on the histidine phosphotransfer domain . Point mutation A129T, diminishing the NtrC phosphatase activity of NtrB, affected the strength of the signals between NtrC and the transmitter module of NtrB but had no impact on PII signals, suggesting that A129T prevents the conformational change needed by NtrB to function as a phosphatase for NtrC, rather than disturbing binding to PII . Phosphoprotein with Phosphoglycerate Mutase Activity from the Archaeon Sulfolobus solfataricus. M. Ben Potters, 2003.When soluble extracts of the extreme acidothermophilic archaeon Sulfolobus solfataricus were incubated with [  -32P]ATP, several proteins were radiolabeled . One of the more prominent of these, which migrated with a mass of
 46 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was purified by column chromatography and SDS-PAGE and subjected to amino acid sequence analysis via both the Edman technique and mass spectroscopy . The best match to the partial sequence obtained was the potential polypeptide product of open reading frame sso0417, whose DNA-derived amino acid sequence displayed many features reminiscent of the 2,3-diphosphoglycerate-independent phosphoglycerate (PGA) mutases [iPGMs] . Open reading frame sso0417 was therefore cloned, and its protein product was expressed in Escherichia coli . Assays of its catalytic capabilities revealed that the protein was a moderately effective PGA mutase that also exhibited low levels of phosphohydrolase activity . PGA mutase activity was dependent upon the presence of divalent metal ions such as Co2+ or Mn2+ . The recombinant protein underwent autophosphorylation when incubated with either [-32P]ATP or [-32P]GTP . The site of phosphorylation was identified as Ser59, which corresponds to the catalytically essential serine residue in bacterial and eucaryal iPGMs . The phosphoenzyme intermediate behaved in a chemically and kinetically competent manner . Incubation of the 32P-labeled phosphoenzyme with 3-PGA resulted in the disappearance of radioactive phosphate and the concomitant appearance of 32P-labeled PGA at rates comparable to those measured in steady-state assays of PGA mutase activity .
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
