NDLI: Structural and Biochemical Evidence That a TEM-1 β-Lactamase N170G Active Site Mutant Acts via Substrate-assisted Catalysis*

Content Provider	PubMed Central
Author	Brown, Nicholas G. Shanker, Sreejesh Prasad, B. V. Venkataram Timothy, Palzkill
Abstract	TEM-1 β-lactamase is the most common plasmid-encoded β-lactamase in Gram-negative bacteria and is a model class A enzyme. The active site of class A β-lactamases share several conserved residues including Ser70, Glu166, and Asn170 that coordinate a hydrolytic water involved in deacylation. Unlike Ser70 and Glu166, the functional significance of residue Asn170 is not well understood even though it forms hydrogen bonds with both Glu166 and the hydrolytic water. The goal of this study was to examine the importance of Asn170 for catalysis and substrate specificity of β-lactam antibiotic hydrolysis. The codon for position 170 was randomized to create a library containing all 20 possible amino acids. The random library was introduced into Escherichia coli, and functional clones were selected on agar plates containing ampicillin. DNA sequencing of the functional clones revealed that only asparagine (wild type) and glycine at this position are consistent with wild-type function. The determination of kinetic parameters for several substrates revealed that the N170G mutant is very efficient at hydrolyzing substrates that contain a primary amine in the antibiotic R-group that would be close to the Asn170 side chain in the acyl-intermediate. In addition, the x-ray structure of the N170G enzyme indicated that the position of an active site water important for deacylation is altered compared with the wild-type enzyme. Taken together, the results suggest the N170G TEM-1 enzyme hydrolyzes ampicillin efficiently because of substrate-assisted catalysis where the primary amine of the ampicillin R-group positions the hydrolytic water and allows for efficient deacylation.
Related Links	http://dx.doi.org/10.1074/jbc.m109.053819
Ending Page	33712
Page Count	10
Starting Page	33703
File Format	PDF
ISSN	00219258
e-ISSN	1083351X
Journal	The Journal of Biological Chemistry
Issue Number	48
Volume Number	284
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2009-11-27
Access Restriction	Open
Rights Holder	American Society for Biochemistry and Molecular Biology
Subject Keyword	Cell Biology Biochemistry Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Structural and Biochemical Evidence That a TEM-1 β-Lactamase N170G Active Site Mutant Acts via Substrate-assisted Catalysis

Structural and biochemical evidence that a TEM-1 beta-lactamase N170G active site mutant acts via substrate-assisted catalysis.

A Triple Mutant in the Ω-loop of TEM-1 β-Lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis*

Site-saturation Mutagenesis of Tyr-105 Reveals Its Importance in Substrate Stabilization and Discrimination in TEM-1 β-Lactamase

A Triple Mutant in the Ω-loop of TEM-1 β-Lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis

Structural Insight into the Kinetics and ΔCp of Interactions between TEM-1 β-Lactamase and β-Lactamase Inhibitory Protein (BLIP)*

High tolerance to simultaneous active-site mutations in TEM-1 β-lactamase: Distinct mutational paths provide more generalized β-lactam recognition

Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM-1 β-lactamase

The Structural Bases of Antibiotic Resistance in the Clinically Derived Mutant β-Lactamases TEM-30, TEM-32, and TEM-34

Structural and Biochemical Evidence That a TEM-1 β-Lactamase N170G Active Site Mutant Acts via Substrate-assisted Catalysis*

Similar Documents

Structural and Biochemical Evidence That a TEM-1 β-Lactamase N170G Active Site Mutant Acts via Substrate-assisted Catalysis

Structural and biochemical evidence that a TEM-1 beta-lactamase N170G active site mutant acts via substrate-assisted catalysis.

A Triple Mutant in the Ω-loop of TEM-1 β-Lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis*

Site-saturation Mutagenesis of Tyr-105 Reveals Its Importance in Substrate Stabilization and Discrimination in TEM-1 β-Lactamase

A Triple Mutant in the Ω-loop of TEM-1 β-Lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis

Structural Insight into the Kinetics and ΔCp of Interactions between TEM-1 β-Lactamase and β-Lactamase Inhibitory Protein (BLIP)*

High tolerance to simultaneous active-site mutations in TEM-1 β-lactamase: Distinct mutational paths provide more generalized β-lactam recognition

Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM-1 β-lactamase

The Structural Bases of Antibiotic Resistance in the Clinically Derived Mutant β-Lactamases TEM-30, TEM-32, and TEM-34

Structural and Biochemical Evidence That a TEM-1 β-Lactamase N170G Active Site Mutant Acts via Substrate-assisted Catalysis*