scholarly journals Substrate specificity and characterization of rat liver p-nitrophenol, 3 α-hydroxysteroid and 17 β-hydroxysteroid UDP-glucuronosyltransferases

1986 ◽  
Vol 238 (1) ◽  
pp. 65-73 ◽  
Author(s):  
C N Falany ◽  
M D Green ◽  
E Swain ◽  
T R Tephly
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Limited Proteolysis ◽  
Enzymic Activity ◽  
Substrate Specificities ◽  
Endogenous Substrate ◽  
Udp Glucuronosyltransferase ◽  
Endogenous Substrates ◽  
Peptide Maps

Purified preparations of rat liver 17-hydroxysteroid, 3-hydroxyandrogen and p-nitrophenol (3-methylcholanthrene-inducible) UDP-glucuronosyltransferases were further characterized as to their substrate specificities, phospholipid-dependency and physical properties. The two steroid UDP-glucuronosyltransferases were shown to exhibit strict stereospecificity with respect to the conjugation of steroids and bile acids. These enzymes have been renamed 17 beta-hydroxysteroid and 3 alpha-hydroxysteroid UDP-glucuronosyltransferase to reflect this specificity for important endogenous substrates. An endogenous substrate has not yet been identified for the p-nitrophenol (3-methylcholanthrene-inducible) UDP-glucuronosyltransferase. The steroid UDP-glucuronosyltransferase activities were dependent on phospholipid for maximal catalytic activity. Complete delipidation rendered the UDP-glucuronosyltransferases inactive, and enzymic activity was not restored when phospholipid was added to the reaction mixture. After partial delipidation, phosphatidylcholine was the most efficient phospholipid for restoration of enzymic activity. Partial delipidation also altered the kinetic parameters of the 3 alpha-hydroxysteroid UDP-glucuronosyltransferase. The three purified UDP-glucuronosyltransferases are separate and distinct proteins, with different amino acid compositions and peptide maps generated by limited proteolysis with Staphylococcus aureus V8 proteinase. Some similarity was observed between the amino acid composition and limited proteolytic maps of the steroid UDP-glucuronosyltransferases, suggesting they are more closely related to each other than to the p-nitrophenol UDP-glucuronosyltransferase.

scholarly journals Regiodivergent Biocatalytic Hydroxylation of L-Glutamine Facilitated by Characterization of Non-Heme Dioxygenases from Non-Ribosomal Peptide Biosyntheses

2021 ◽  
Author(s):  
Hans Renata ◽  
Emily Shimizu ◽  
Christian Zwick
Keyword(s):  
Amino Acid ◽  
Building Block ◽  
Solid Phase ◽  
Functional Characterization ◽  
Free Standing ◽  
Substrate Specificities ◽  
Total Turnover

We report the functional characterization of two iron- and a-ketoglutarate-dependent dioxygenases that are capable of hydroxylating free-standing glutamine at its C3 and C4 position respectively. In particular, the C4 hydroxylase, Q4Ox, catalyzes the reaction with approximately 4,300 total turnover numbers, facilitating synthesis of a solid-phase compatible building block and stereochemical elucidation at the C4 position of the hydroxylated product. This work will enable the development of novel synthetic strategies to prepare useful glutamine derivatives and stimulate further discoveries of new amino acid hydroxylases with distinct substrate specificities.<br>

scholarly journals Regiodivergent Biocatalytic Hydroxylation of L-Glutamine Facilitated by Characterization of Non-Heme Dioxygenases from Non-Ribosomal Peptide Biosyntheses

2021 ◽  
Author(s):  
Hans Renata ◽  
Emily Shimizu ◽  
Christian Zwick
Keyword(s):  
Amino Acid ◽  
Building Block ◽  
Solid Phase ◽  
Functional Characterization ◽  
Free Standing ◽  
Substrate Specificities ◽  
Total Turnover

We report the functional characterization of two iron- and a-ketoglutarate-dependent dioxygenases that are capable of hydroxylating free-standing glutamine at its C3 and C4 position respectively. In particular, the C4 hydroxylase, Q4Ox, catalyzes the reaction with approximately 4,300 total turnover numbers, facilitating synthesis of a solid-phase compatible building block and stereochemical elucidation at the C4 position of the hydroxylated product. This work will enable the development of novel synthetic strategies to prepare useful glutamine derivatives and stimulate further discoveries of new amino acid hydroxylases with distinct substrate specificities.<br>

Isolation and partial characterization of amphibian tyrosine oxidase polysomes

Development ◽  
1970 ◽  
Vol 24 (1) ◽  
pp. 109-118
Author(s):  
E. L. Triplett ◽  
R. Herzog ◽  
L. P. Russell
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Enzymic Activity ◽  
Antigenic Determinants ◽  
Free System ◽  
A Cell ◽  
Generating System ◽  
Soluble Components

A population of polysomes isolated from frogskinis capable of supporting protein synthesis in a cell-free system containing an energy generating system, ‘soluble components’, and amino acids. These polysomes catalyse the oxidation of DOPA after gentle trypsinization, and they also have antigenic determinants attributable to tyrosine oxidase. Skin polysomes sedimented in 10–30 % sucrose gradients contain tyrosine oxidase peaks of enzymic activity at the bottom and top of the tube and in the 250 S regions. A peak of tyrosine oxidase antigenic acitvity is found in the 250–350S region of the gradient. Polysomes resolved on the gradient retain the ability to support protein synthesis in a cellfree system. All 250–350S particles capable of supporting the incorporation of [14C]amino acid into tyrosine oxidase are precipitable with tyrosine oxidase antibodies. It is probable that 250–350S tyrosine oxidase antibody precipitates contain only polysomes for this protein.

scholarly journals Purification and characterization of three forms of glutathione transferase from Proteus mirabilis

1988 ◽  
Vol 255 (3) ◽  
pp. 971-975 ◽  
Author(s):  
C Di Ilio ◽  
A Aceto ◽  
R Piccolomini ◽  
N Allocati ◽  
A Faraone ◽  
...  
Keyword(s):  
Amino Acid ◽  
Isoelectric Focusing ◽  
Proteus Mirabilis ◽  
Gel Electrophoresis ◽  
Glutathione Transferase ◽  
Total Activity ◽  
Purification And Characterization ◽  
Substrate Specificities ◽  
Structural Differences

Three forms of glutathione transferase (GST) with pI values of 6.0, 6.4 and 7.3 were isolated from Proteus mirabilis AF 2924 by glutathione-affinity chromatography followed by isoelectric focusing, and their structural, kinetic and immunological properties were investigated. Upon SDS/polyacrylamide-slab-gel electrophoresis, all forms proved to be composed of two subunits of identical (22,500) Mr. GST-6.0 and GST-6.4 together account for about 95% of the total activity, whereas GST-7.3 is present only in trace amounts. Extensive similarities have been found between GST-6.0 and GST-6.4. These include subunit molecular mass, amino acid composition, substrate specificities and immunological characteristics. GST-7.3 also cross-reacted (non-identity) with antisera raised against bacterial GST-6.0. None of the antisera raised against a number of human, rat and mouse GSTs cross-reacted with the bacterial enzymes, indicating major structural differences between them and the mammalian GSTs. This conclusion is further supported by c.d. spectra.

scholarly journals A critical amino acid residue, Asp446, in UDP-glucuronosyltransferase

1997 ◽  
Vol 325 (3) ◽  
pp. 587-591 ◽  
Author(s):  
Hidetomo IWANO ◽  
Hiroshi YOKOTA ◽  
Satoru OHGIYA ◽  
Naomi YOTUMOTO ◽  
Akira YUASA
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Amino Acid Substitution ◽  
Critical Role ◽  
Enzymic Activity ◽  
Male Rat ◽  
Transferase Activity ◽  
Rt Pcr ◽  
Taq Polymerase ◽  
Udp Glucuronosyltransferase

An amino acid residue, Asp446, was found to be essential for the enzymic activity of UDP-glucuronosyltransferase (UGT). We obtained a rat phenol UGT (UGT1*06) cDNA (named Ysh) from male rat liver by reverse-transcription (RT)-PCR using pfu polymerase. A mutant Ysh having two different bases, A1337G and G1384A (named Ysh A1337GC1384A), that result in two amino acid substitutions, D446G and V462M, was obtained by RT-PCR using Taq polymerase. Ysh was expressed functionally in microsomes of Saccharomyces cerevisiae strain AH22. However, the expressed protein from Ysh A1337GG1384A had no transferase activity. Two other mutant cDNAs with Ysh A1337G having one changed base, A1337G, resulting in one amino acid substitution, D446G, and Ysh G1384A having a changed base, G1384A, resulting in an amino acid substitution, V462M, were constructed and expressed in the yeast. The expressed protein from Ysh G1384A (named Ysh V462M) exhibited enzymic activity, but the one from Ysh A1337G (named Ysh D446G) did not show any activity at all. Asp446 was conserved in all UGTs and UDP-galactose:ceramide galactosyltransferases reported, suggesting that Asp446 plays a critical role in each enzyme.

scholarly journals Soluble forms of alpha-d-mannosidases from rat liver. Separation and characterization of two enzymic forms with different substrate specificities

1994 ◽  
Vol 223 (1) ◽  
pp. 99-106 ◽  
Author(s):  
Thierry GRARD ◽  
Agnes SAINT-POL ◽  
Jean-Francois HAEUW ◽  
Catherine ALONSO ◽  
Jean-Michel WIERUSZESKI ◽  
...  
Keyword(s):  
Rat Liver ◽  
Substrate Specificities
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