Probing the Catalytic Mechanism of Prephenate Dehydratase by Site-Directed Mutagenesis of theEscherichia coliP-Protein Dehydratase Domain†

Biochemistry ◽  
2000 ◽  
Vol 39 (16) ◽  
pp. 4722-4728 ◽  
Author(s):  
Sheng Zhang ◽  
David B. Wilson ◽  
Bruce Ganem
Keyword(s):  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Prephenate Dehydratase

β-Amyrin biosynthesis: catalytic mechanism and substrate recognition

2017 ◽  
Vol 15 (14) ◽  
pp. 2869-2891 ◽  
Author(s):  
Tsutomu Hoshino
Keyword(s):  
Catalytic Mechanism ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
The Past ◽  
Substrate Analog ◽  
Analog Experiments

In the past five years, there have been remarkable advances in the study of β-amyrin synthase. This review outlines the catalytic mechanism and substrate recognition in β-amyrin biosynthesis, which have been attained by the site-directed mutagenesis and substrate analog experiments.

Catalytic Mechanism of theStreptomycesK15dd-Transpeptidase/Penicillin-Binding Protein Probed by Site-Directed Mutagenesis and Structural Analysis†,‡

Biochemistry ◽  
2003 ◽  
Vol 42 (10) ◽  
pp. 2895-2906 ◽  
Author(s):  
Noureddine Rhazi ◽  
Paulette Charlier ◽  
Dominique Dehareng ◽  
Danièle Engher ◽  
Marcel Vermeire ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Catalytic Mechanism ◽  
Binding Protein ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Penicillin Binding Protein

scholarly journals Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli

1994 ◽  
Vol 303 (2) ◽  
pp. 357-362 ◽  
Author(s):  
M P G van der Linden ◽  
L de Haan ◽  
O Dideberg ◽  
W Keck
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Binding Capacity ◽  
Binding Protein ◽  
Complete Loss ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Active Site Residues

Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis.

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.

Catalytic mechanism of chloramphenicol acetyltransferase investigated by site-directed mutagenesis

1986 ◽  
Vol 14 (6) ◽  
pp. 1227-1228 ◽  
Author(s):  
IAIN A. MURRAY ◽  
ANN LEWENDON ◽  
COLIN KLEANTHOUS ◽  
WILLIAM V. SHAW
Keyword(s):  
Catalytic Mechanism ◽  
Chloramphenicol Acetyltransferase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Abbreviation used: CAT, chloramphenicol acetyltransferase.

scholarly journals Expansion of Substrate Specificity and Catalytic Mechanism of Azoreductase by X-ray Crystallography and Site-directed Mutagenesis

2008 ◽  
Vol 283 (20) ◽  
pp. 13889-13896 ◽  
Author(s):  
Kosuke Ito ◽  
Masayuki Nakanishi ◽  
Woo-Cheol Lee ◽  
Yuehua Zhi ◽  
Hiroshi Sasaki ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
X Ray ◽  
X Ray Crystallography

scholarly journals Structural insights into SETD3-mediated histidine methylation on β-actin

2018 ◽  
Author(s):  
Qiong Guo ◽  
Shanhui Liao ◽  
Sebastian Kwiatkowski ◽  
Weronika Tomaka ◽  
Huijuan Yu ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Enzyme Activity ◽  
Small Molecule ◽  
Conformational Changes ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Domain Protein ◽  
First Time ◽  
Structural Insights

SETD3 is a member of SET (Su(var)3-9, Enhancer of zeste, and Trithorax) domain protein superfamily and plays important roles in hypoxic pulmonary hypertension, muscle differentiation, and carcinogenesis. Recently, we have identified SETD3 as the actin-specific methyltransferase that methylates the N3 of His73 on β-actin. Here we present two structures of S-adenosyl-L-homocysteine-bound SETD3 in complex with either an unmodified β-actin peptide or its His-methylated variant. Structural analyses supported by the site-directed mutagenesis experiments and the enzyme activity assays indicated that the recognition and methylation of β-actin by SETD3 is highly sequence specific, and both SETD3 and β-actin adopt pronounce conformational changes upon binding to each other. In conclusion, the data show for the first time a catalytic mechanism of SETD3-mediated histidine methylation in β-actin, which not only throws light on protein histidine methylation phenomenon, but also facilitates the design of small molecule inhibitors of SETD3.

Dissecting the Catalytic Mechanism of Betaine−HomocysteineS-Methyltransferase by Use of Intrinsic Tryptophan Fluorescence and Site-Directed Mutagenesis†

Biochemistry ◽  
2004 ◽  
Vol 43 (18) ◽  
pp. 5341-5351 ◽  
Author(s):  
Carmen Castro ◽  
Alejandra A. Gratson ◽  
John C. Evans ◽  
Jiri Jiracek ◽  
Michaela Collinsová ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Tryptophan Fluorescence ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Intrinsic Tryptophan Fluorescence
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