scholarly journals Structure and Reaction Mechanism of YcjR, an Epimerase That Facilitates the Interconversion of d-Gulosides to d-Glucosides in Escherichia coli

Biochemistry ◽  
2020 ◽  
Vol 59 (22) ◽  
pp. 2069-2077
Author(s):  
Mark F. Mabanglo ◽  
Jamison P. Huddleston ◽  
Keya Mukherjee ◽  
Zane W. Taylor ◽  
Frank M. Raushel
Keyword(s):  
Escherichia Coli ◽  
Reaction Mechanism

scholarly journals Insights into the reaction mechanism of Escherichia coli agmatinase by site-directed mutagenesis and molecular modelling

2002 ◽  
Vol 269 (22) ◽  
pp. 5522-5526 ◽  
Author(s):  
Mónica Salas ◽  
Rolando Rodríguez ◽  
Nelia López ◽  
Elena Uribe ◽  
Vasthi López ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Reaction Mechanism ◽  
Molecular Modelling ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Biosynthesis of dermatan sulphate. Defructosylated Escherichia coli K4 capsular polysaccharide as a substrate for the d-glucuronyl C-5 epimerase, and an indication of a two-base reaction mechanism

1996 ◽  
Vol 313 (2) ◽  
pp. 589-596 ◽  
Author(s):  
Helgi H. HANNESSON ◽  
Åsa HAGNER-McWHIRTER ◽  
Kerstin TIEDEMANN ◽  
Ulf LINDAHL ◽  
Anders MALMSTRÖM
Keyword(s):  
Escherichia Coli ◽  
Reaction Mechanism ◽  
Acid Hydrolysis ◽  
Capsular Polysaccharide ◽  
Glucuronic Acid ◽  
Microsomal Enzyme ◽  
Dermatan Sulphate ◽  
Mild Acid Hydrolysis ◽  
Mild Acid ◽  
Escherichia Coli K4

The capsular polysaccharide from Escherichia coli K4 consists of a chondroitin {[GlcA(β1→3)GalNAc(β1→4)]n} backbone, to which β-fructofuranose units are linked to C-3 of D-glucuronic acid (GlcA) residues. Removal of the fructose units by mild acid hydrolysis provided a substrate for the GlcA C-5 epimerase, which is involved in the generation of L-iduronic acid (IdoA) units during dermatan sulphate biosynthesis. Incubation of this substrate with solubilized fibroblast microsomal enzyme in the presence of 3H2O resulted in the incorporation of tritium at C-5 of hexuronyl units. A Km of 67×10-6 M hexuronic acid (equivalent to disaccharide units) was determined, which is similar to that (80×10-6 M) obtained for dermatan (desulphated dermatan sulphate). Vmax. was about 4 times higher with dermatan than with the K4 substrate. A defructosylated K4 polysaccharide isolated after incubation of bacteria with D-[5-3H]glucose released 3H2O on reaction with the epimerase, and thus could be used to assay the enzyme. Incubation of a K4 substrate with solubilized microsomal epimerase for 6 h in the presence of 3H2O resulted in the formation of about 5% IdoA and approximately equal amounts of 3H in GlcA and IdoA. A corresponding incubation of dermatan yielded approx. 22% GlcA, which contained virtually all the 3H label. These results are tentatively explained in terms of a two-base reaction mechanism, involving a monoprotic L-ido-specific base and a polyprotic D-gluco-specific base. Most of the IdoA residues generated by the enzyme occurred singly, although some formation of two or three consecutive IdoA-containing disaccharide units was observed.

Study of the Reaction Mechanism of the d-Glutamic Acid-Adding Enzyme from Escherichia coli

1996 ◽  
Vol 2 (1) ◽  
pp. 51-54 ◽  
Author(s):  
SABINE VAGANAY ◽  
MARTIN E. TANNER ◽  
JEAN van HEIJENOORT ◽  
DIDIER BLANOT
Keyword(s):  
Escherichia Coli ◽  
Reaction Mechanism ◽  
Glutamic Acid

Structure and mechanism of the chalcogen-detoxifying protein TehB from Escherichia coli

2011 ◽  
Vol 435 (1) ◽  
pp. 85-91 ◽  
Author(s):  
Hassanul G. Choudhury ◽  
Alexander D. Cameron ◽  
So Iwata ◽  
Konstantinos Beis
Keyword(s):  
Escherichia Coli ◽  
Reaction Mechanism ◽  
Active Site ◽  
Kinetic Data ◽  
Protein Analysis ◽  
Nucleophilic Attack ◽  
Conserved Residues ◽  
Low Levels ◽  
Living Organisms ◽  
Derivatives Of

The oxyanion derivatives of the chalcogens tellurium and selenium are toxic to living organisms even at very low levels. Bacteria have developed mechanisms to overcome their toxicity by methylating them. The structure of TehB from Escherichia coli has been determined in the presence of the cofactor analogues SAH (S-adenosylhomocysteine) and sinefungin (a non-hydrolysable form of S-adenosyl-L-methionine) at 1.48 Å (1 Å=0.1 nm) and 1.9 Å respectively. Interestingly, our kinetic data show that TehB does not discriminate between selenium or tellurite oxyanions, making it a very powerful detoxifying protein. Analysis of the active site has identified three conserved residues that are capable of binding and orientating the metals for nucleophilic attack: His176, Arg177 and Arg184. Mutagenesis studies revealed that the H176A and R184A mutants retained most of their activity, whereas the R177A mutant had 65% of its activity abolished. Based on the structure and kinetic data we propose an SN2 nucleophilic attack reaction mechanism. These data provide the first molecular understanding of the detoxification of chalcogens by bacteria.

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