scholarly journals Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium

2003 ◽  
Vol 370 (2) ◽  
pp. 505-516 ◽  
Author(s):  
Evelyne RAUX ◽  
Helen K. LEECH ◽  
Richard BECK ◽  
Heidi L. SCHUBERT ◽  
Patricio J. SANTANDER ◽  
...  
Keyword(s):  
Bacillus Megaterium ◽  
Metal Ion ◽  
Enzyme Assay ◽  
Sequence Alignments ◽  
Rich Region ◽  
In Vitro Synthesis ◽  
Cobalamin Biosynthesis ◽  
High Degree ◽  
Degree Of Similarity

In Bacillus megaterium, the hemAXBCDL genes were isolated and were found to be highly similar to the genes from Bacillus subtilis that are required for the conversion of glutamyl-tRNA into uroporphyrinogen III. Overproduction and purification of HemC (porphobilinogen deaminase) and -D (uroporphyrinogen III synthase) allowed these enzymes to be used for the in vitro synthesis of uroporphyrinogen III from porphobilinogen. A second smaller cluster of three genes (termed sirABC) was also isolated and found to encode the enzymes that catalyse the transformation of uroporphyrinogen III into sirohaem on the basis of their ability to complement a defined Escherichia coli (cysG) mutant. The functions of SirC and -B were investigated by direct enzyme assay, where SirC was found to act as a precorrin-2 dehydrogenase, generating sirohydrochlorin, and SirB was found to act as a ferrochelatase responsible for the final step in sirohaem synthesis. CbiX, a protein found encoded within the main B. megaterium cobalamin biosynthetic operon, shares a high degree of similarity with SirB and acts as the cobaltochelatase associated with cobalamin biosynthesis by inserting cobalt into sirohydrochlorin. CbiX contains an unusual histidine-rich region in the C-terminal portion of the protein, which was not found to be essential in the chelation process. Sequence alignments suggest that SirB and CbiX share a similar active site to the cobaltochelatase, CbiK, from Salmonella enterica.

scholarly journals Human CNS barrier-forming organoids with cerebrospinal fluid production

Science ◽  
2020 ◽  
Vol 369 (6500) ◽  
pp. eaaz5626 ◽  
Author(s):  
Laura Pellegrini ◽  
Claudia Bonfio ◽  
Jessica Chadwick ◽  
Farida Begum ◽  
Mark Skehel ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Fluid Secretion ◽  
Fluid Production ◽  
New Compounds ◽  
High Degree ◽  
The Brain ◽  
Degree Of Similarity ◽  
By Products

Cerebrospinal fluid (CSF) is a vital liquid, providing nutrients and signaling molecules and clearing out toxic by-products from the brain. The CSF is produced by the choroid plexus (ChP), a protective epithelial barrier that also prevents free entry of toxic molecules or drugs from the blood. Here, we establish human ChP organoids with a selective barrier and CSF-like fluid secretion in self-contained compartments. We show that this in vitro barrier exhibits the same selectivity to small molecules as the ChP in vivo and that ChP-CSF organoids can predict central nervous system (CNS) permeability of new compounds. The transcriptomic and proteomic signatures of ChP-CSF organoids reveal a high degree of similarity to the ChP in vivo. Finally, the intersection of single-cell transcriptomics and proteomic analysis uncovers key human CSF components produced by previously unidentified specialized epithelial subtypes.

scholarly journals Direct Physical and Functional Interaction of the NuA4 Complex Components Yaf9p and Swc4p

2004 ◽  
Vol 3 (4) ◽  
pp. 976-983 ◽  
Author(s):  
Claudia B. Bittner ◽  
Deniz T. Zeisig ◽  
Bernd B. Zeisig ◽  
Robert K. Slany
Keyword(s):  
Transcriptional Activation ◽  
Dna Methyltransferase ◽  
Chromatin Modification ◽  
Gel Filtration ◽  
Dominant Negative ◽  
Yeast Homolog ◽  
High Degree ◽  
Degree Of Similarity

ABSTRACT Saccharomyces cerevisiae Yaf9p and the mammalian leukemia-associated protein ENL share a high degree of similarity. To investigate the biological function of Yaf9p, this protein was used to search for interacting proteins in a two-hybrid system. Here, we demonstrate that Yaf9p binds directly to Swc4p, the yeast homolog of the mammalian DNA-methyltransferase-associated protein 1. Yaf9p and Swc4p associate through C-terminal domains, and both proteins coprecipitate in vitro in pull-down experiments and in vivo by immunoprecipitation. In living cells, Swc4p is present in a megadalton protein complex that shows a fractionation behavior in gel filtration similar to that of Esa1p, the histone acetyltransferase of the NuA4 complex. Recruitment of Yaf9p to DNA leads to promoter-specific transcriptional activation that can be inhibited by dominant negative Swc4p lacking the Yaf9p binding domain. Interference with Swc4p function also increases sensitivity to the microtubule toxin benomyl, a trait that corresponds to the known phenotype of a yaf9 − knockout strain. In summary, the results suggest that Yaf9p and Swc4p form a protein pair that has a role in chromatin modification with possible implications also for the function of their mammalian counterparts.

A Comparative Study of Human and Monkey (Macaca irus) Fibrinogen Degradation Products

1974 ◽  
Vol 31 (02) ◽  
pp. 319-327
Author(s):  
Thomas R Poskitt ◽  
H Philip Fortwengler ◽  
Gerald J Roth ◽  
James A Eaton
Keyword(s):  
Comparative Study ◽  
Disseminated Intravascular Coagulation ◽  
Degradation Products ◽  
Gel Filtration ◽  
Fibrinogen Degradation Products ◽  
Pathogenic Mechanisms ◽  
Intravascular Coagulation ◽  
High Degree ◽  
Degree Of Similarity

SummaryImmunochemical, physical, and anticoagulant properties of human and monkey (Macaca irus) fibrinogen degradation products (FDP’s) were compared by Immunoelectrophoresis, Sephadex G-200 gel filtration, and anticoagulant assay. Both species demonstrated a high degree of similarity in the properties and sequence of generation of FDP’s derived from in vitro plasmin digestion of highly purified fibrinogen. The results of this study should permit a closer analogy to be drawn between the pathogenic mechanisms of disseminated intravascular coagulation occurring experimentally in monkeys and clinically in humans.

Production of cobalamin and sirohaem in Bacillus megaterium: an investigation into the role of the branchpoint chelatases sirohydrochlorin ferrochelatase (SirB) and sirohydrochlorin cobalt chelatase (CbiX)

2002 ◽  
Vol 30 (4) ◽  
pp. 610-613 ◽  
Author(s):  
H. K. Leech ◽  
E. Raux-Deery ◽  
P. Heathcote ◽  
M. J. Warren
Keyword(s):  
Sequence Analysis ◽  
Bacillus Megaterium ◽  
Primary Structure ◽  
Comparative Sequence Analysis ◽  
Functional Complementation ◽  
In Vitro Assays ◽  
Cobalamin Biosynthesis ◽  
Redox Centre

One of the four operons required for cobalamin biosynthesis in Bacillus megaterium is also associated with sirohaem synthesis, and contains three genes, sirA, sirB and sirC. By undertaking functional complementation experiments and in vitro assays using recombinantly produced enzymes, we have been able to demonstrate that (1) SirA acts as a uroporphyrinogen III methyltransferase, transforming uroporphyrinogen III into precorrin-2, (2) SirC acts as an NAD+ dehydrogenase, responsible for the oxidation of precorrin-2 into sirohydrochlorin, and (3) SirB acts as a ferrochelatase, responsible for the insertion of a ferrous ion into sirohydrochlorin to give sirohaem. Comparative sequence analysis reveals that the primary structure of SirB is highly similar to that of the cobalt chelatase involved in cobalamin biosynthesis in Bacillus megaterium, CbiX, with the exception that CbiX contains a C-terminal histidine-rich motif. Surprisingly, CbiX has been shown (using EPR) to contain a 4Fe-4S centre, a redox centre that is absent from SirB.

In vitro synthesis of enzymatically active HIV-1 protease for rapid phenotypic resistance profiling

2005 ◽  
Vol 32 (4) ◽  
pp. 294-299 ◽  
Author(s):  
Dieter Hoffmann ◽  
Bernd Buchberger ◽  
Cordula Nemetz
Keyword(s):  
In Vitro Synthesis ◽  
Phenotypic Resistance ◽  
Hiv 1
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