Evidence for participation of aspartate-84 as a catalytic group at the active site of porphobilinogen deaminase obtained by site-directed mutagenesis of the hemC gene from Escherichia coli

Biochemistry ◽  
1994 ◽  
Vol 33 (9) ◽  
pp. 2688-2695 ◽  
Author(s):  
Sarah C. Woodcock ◽  
Peter M. Jordan
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Porphobilinogen Deaminase

scholarly journals Role of αArg145 and βArg263 in the active site of penicillin acylase of Escherichia coli

2002 ◽  
Vol 365 (1) ◽  
pp. 303-309 ◽  
Author(s):  
Wynand B.L. ALKEMA ◽  
Antoon K. PRINS ◽  
Erik de VRIES ◽  
Dick B. JANSSEN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Penicillin Acylase ◽  
Precursor Protein ◽  
Site Directed Mutagenesis ◽  
Penicillin G ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Arginine Residues

The active site of penicillin acylase of Escherichia coli contains two conserved arginine residues. The function of these arginines, αArg145 and βArg263, was studied by site-directed mutagenesis and kinetic analysis of the mutant enzymes. The mutants αArg145→Leu (αArg145Leu), αArg145Cys and αArg145Lys were normally processed and exported to the periplasm, whereas expression of the mutants βArg263Leu, βArg263Asn and βArg263Lys yielded large amounts of precursor protein in the periplasm, indicating that βArg263 is crucial for efficient processing of the enzyme. Either modification of both arginine residues by 2,3-butanedione or replacement by site-directed mutagenesis yielded enzymes with a decreased specificity (kcat/Km) for 2-nitro-5-[(phenylacetyl)amino]benzoic acid, indicating that both residues are important in catalysis. Compared with the wild type, the αArg145 mutants exhibited a 3–6-fold-increased preference for 6-aminopenicillanic acid as the deacylating nucleophile compared with water. Analysis of the steady-state parameters of these mutants for the hydrolysis of penicillin G and phenylacetamide indicated that destabilization of the Michaelis—Menten complex accounts for the improved activity with β-lactam substrates. Analysis of pH—activity profiles of wild-type enzyme and the βArg263Lys mutant showed that βArg263 has to be positively charged for catalysis, but is not involved in substrate binding. The results provide an insight into the catalytic mechanism of penicillin acylase, in which αArg145 is involved in binding of β-lactam substrates and βArg263 is important both for stabilizing the transition state in the reaction and for correct processing of the precursor protein.

SITE-DIRECTED MUTAGENESIS OF THE ACTIVE SITE OF Escherichia coli FUMARATE REDUCTASE

1991 ◽  
pp. 723-726
Author(s):  
Brian A. C. Ackrell ◽  
Bruce Cochran ◽  
Gary Cecchini ◽  
Imke Schröder ◽  
Robert F. Gunsalus
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Fumarate Reductase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Tailoring a Soluble Diiron Monooxygenase for Synthesis of Aromatic N-oxides

Catalysts ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 356 ◽  
Author(s):  
Vytautas Petkevičius ◽  
Justas Vaitekūnas ◽  
Dovydas Vaitkus ◽  
Narimantas Čėnas ◽  
Rolandas Meškys
Keyword(s):  
Organic Chemistry ◽  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Properties ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Work Site ◽  
Whole Cells ◽  
New Variant

The aromatic N-oxides have received increased attention over the last few years due to their potential application in medicine, agriculture and organic chemistry. As a green alternative in their synthesis, the biocatalytic method employing whole cells of Escherichia coli bearing phenol monooxygenase like protein PmlABCDEF (from here on – PML monooxygenase) has been introduced. In this work, site-directed mutagenesis was used to study the contributions of active site neighboring residues I106, A113, G109, F181, F200, F209 to the regiospecificity of N-oxidation. Based on chromogenic indole oxidation screening, a collection of PML mutants with altered catalytic properties was created. Among the tested mutants, the A113G variant acquired the most distinguishable N-oxidations capacity. This new variant of PML was able to produce dioxides (quinoxaline-1,4-dioxide, 2,5-dimethylpyrazine-1,4-dioxide) and specific mono-N-oxides (2,3,5-trimethylpyrazine-1-oxide) that were unachievable using the wild type PML. This mutant also featured reshaped regioselectivity as N-oxidation shifted towards quinazoline-1-oxide compared to quinazoline-3-oxide that is produced by the wild type PML.

scholarly journals Identification of the amino acid residues involved in an active site of Escherichia coli ribonuclease H by site-directed mutagenesis.

1990 ◽  
Vol 265 (8) ◽  
pp. 4615-4621
Author(s):  
S Kanaya ◽  
A Kohara ◽  
Y Miura ◽  
A Sekiguchi ◽  
S Iwai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Ribonuclease H ◽  
Directed Mutagenesis ◽  
Amino Acid Residues

Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

Biochemistry ◽  
1988 ◽  
Vol 27 (21) ◽  
pp. 7984-7990 ◽  
Author(s):  
A. I. Scott ◽  
C. A. Roessner ◽  
N. J. Stolowich ◽  
P. Karuso ◽  
H. J. Williams ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
High Resolution ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Porphobilinogen Deaminase ◽  
High Resolution Nmr

Site-directed mutagenesis identifies catalytic residues in the active site of Escherichia coli phosphofructokinase

Biochemistry ◽  
1992 ◽  
Vol 31 (38) ◽  
pp. 9237-9242 ◽  
Author(s):  
Stuart A. Berger ◽  
Philip R. Evans
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Catalytic Residues
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