A chromatographic study of the reaction sequence and effect of ligand on the reaction of human hemoglobin with negatively charged isothiocyanates: characterization of an intermediate modified only on the amino termini of the alpha chains

2000 ◽  
Vol 45 (1) ◽  
pp. 87-98 ◽  
Author(s):  
Michael Cross ◽  
Douglas L Currell ◽  
Mario Marini
Keyword(s):  
Chromatographic Study ◽  
Human Hemoglobin ◽  
Reaction Sequence

scholarly journals Engineering P450 TamI as an Iterative Biocatalyst for Selective Late-Stage C-H Functionalization and Epoxidation of Tirandamycin Antibiotics

2021 ◽  
Author(s):  
Rosa V. Espinoza ◽  
Kersti Caddell Haatveit ◽  
S. Wald Grossman ◽  
Jin Yi Tan ◽  
Caylie A. McGlade ◽  
...  
Keyword(s):  
Natural Product ◽  
Late Stage ◽  
Md Simulations ◽  
Cascade Reactions ◽  
Direct Access ◽  
Tetramic Acid ◽  
Reaction Sequence ◽  
Substrate Reactivity ◽  
Continuous Oxidation

<div> <div> <div> <p>Iterative P450 enzymes are powerful biocatalysts for selective late-stage C-H oxidation of complex natural product scaffolds. These enzymes represent new tools for selectivity and cascade reactions, facilitating direct access to core structure diversification. Recently, we reported the structure of the multifunctional bacterial P450 TamI and elucidated the molecular basis of its substrate binding and strict reaction sequence at distinct carbon atoms of the substrate. Here, we report the design and characterization of a toolbox of TamI biocatalysts, generated by mutations at Leu101, Leu244 and/or Leu295, that alter the native selectivity, step sequence and number of reactions catalyzed, including the engineering of a variant capable of catalyzing a four-step oxidative cascade without the assistance of the flavoprotein and oxidative partner TamL. The tuned enzymes override inherent substrate reactivity enabling catalyst- controlled C-H functionalization and alkene epoxidation of the tetramic acid-containing natural product tirandamycin. Five new, bioactive tirandamycin derivatives (6-10) were generated through TamI-mediated enzymatic synthesis. Quantum mechanics calculations and MD simulations provide important insights on the basis of altered selectivity and underlying biocatalytic mechanisms for enhanced continuous oxidation of the iterative P450 TamI. </p> </div> </div> </div>

scholarly journals Functional Characterization of a Na+-Coupled Dicarboxylate Carrier Protein from Staphylococcus aureus

2005 ◽  
Vol 187 (15) ◽  
pp. 5189-5194 ◽  
Author(s):  
Jason A. Hall ◽  
Ana M. Pajor
Keyword(s):  
Staphylococcus Aureus ◽  
Structural Analysis ◽  
Transport Properties ◽  
Sequence Homology ◽  
Cytoplasmic Membrane ◽  
Functional Characterization ◽  
Carrier Protein ◽  
Dependent Manner ◽  
Reaction Sequence

ABSTRACT We have cloned and functionally characterized a Na+-coupled dicarboxylate transporter, SdcS, from Staphylococcus aureus. This carrier protein is a member of the divalent anion/Na+ symporter (DASS) family and shares significant sequence homology with the mammalian Na+/dicarboxylate cotransporters NaDC-1 and NaDC-3. Analysis of SdcS function indicates transport properties consistent with those of its eukaryotic counterparts. Thus, SdcS facilitates the transport of the dicarboxylates fumarate, malate, and succinate across the cytoplasmic membrane in a Na+-dependent manner. Furthermore, kinetic work predicts an ordered reaction sequence with Na+ (K 0.5 of 2.7 mM) binding before dicarboxylate (Km of 4.5 μM). Because this transporter and its mammalian homologs are functionally similar, we suggest that SdcS may serve as a useful model for DASS family structural analysis.

Synthesis of NTA3-DTDA — A Chelator-Lipid that Promotes Stable Binding of His-Tagged Proteins to Membranes

2006 ◽  
Vol 59 (5) ◽  
pp. 302 ◽  
Author(s):  
Joseph G. Altin ◽  
Martin G. Banwell ◽  
Phillip A. Coghlan ◽  
Christopher J. Easton ◽  
Michael R. Nairn ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Targeted Delivery ◽  
Membrane Vesicles ◽  
Spectroscopic Characterization ◽  
Plasma Membrane Vesicles ◽  
Reaction Sequence ◽  
Type Compound

A six-step reaction sequence is described for the preparation of compound 1 (NTA3-DTDA), a membrane-penetrating and potent chelator that can be incorporated into liposomes and plasma membrane vesicles containing antigens and thus allowing targeted delivery of such assemblies to a variety of cells for the purposes of eliciting anti-tumour responses. Full spectroscopic characterization of this dendritic-type compound as well as certain of its precursors is reported.

Chromatofocusing and isoelectric focusing in immobilized pH gradients compared for characterization of human hemoglobin variants.

1989 ◽  
Vol 35 (3) ◽  
pp. 425-430 ◽  
Author(s):  
R Paleari ◽  
C Arcelloni ◽  
R Paroni ◽  
I Fermo ◽  
A Mosca
Keyword(s):  
Los Angeles ◽  
Isoelectric Focusing ◽  
Ph Gradient ◽  
Resolving Power ◽  
Human Hemoglobin ◽  
Ph Gradients ◽  
Hemoglobin Variants

Abstract We compared the performance of two highly resolving methods, chromatofocusing (CRF) and isoelectric focusing in immobilized pH gradients (IPGF), for the separation of human hemoglobin variants. Lysates containing 13 different hemoglobins, including variants of clinical and geographical importance, and four electrophoretically "silent" variants (Hb Brockton, Hb Cheverly, Hb Köln, and Hb Waco) were analyzed. Both techniques showed a good intrarun precision (CV = 0.87% for CRF, 0.27% for IPGF) and high and similar resolving power (0.010 pH units, with the pH gradients used in this work). The use of an ultranarrow IPGF range (pH 7.15-7.35; pH gradient = 0.019 pH/cm) allowed the resolution between Hb Brockton, Hb Köln, and Hb A. In some cases (Hb D-Los Angeles, Hb F, Hb Waco), the variants were separated from Hb A in different orders, depending on which technique was used, probably because of the different analytical principles of the two methods. As a second-level test, both procedures are informative for characterization of human hemoglobin variants.

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