Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase: Crystallization and preliminary X-ray diffraction analysis of the enzyme

Sebastien Farnaud, Renée Tata, Maninder K. Sohi, Tommy Wan, Paul R. Brown, Brian J. Sutton

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Wild-type and site-specific mutants C166S and C166A (Cys-166→Ser and Cys-166→Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3. Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding. Cys-166 is therefore implicated as the active-site nucleophile. Titration of free thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue. Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a = b = c = 84 Å; α = β = γ = 75°) and space group (R3 or R32). These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility.

Original languageEnglish
Pages (from-to)711-714
Number of pages4
JournalBiochemical Journal
Volume340
Issue number3
DOIs
Publication statusPublished - 15 Jun 1999
Externally publishedYes

Fingerprint

amidase
Amidohydrolases
Nucleophiles
Crystallization
X-Ray Diffraction
Pseudomonas aeruginosa
X ray diffraction analysis
Cysteine
Catalytic Domain
Dithionitrobenzoic Acid
Electrophoretic mobility
Molecular mass
Mutant Proteins
Enzymes
Titration
Sulfhydryl Compounds
Escherichia coli
Gel Chromatography
Immune Sera
Gels

Keywords

  • Quaternary structure
  • Site-directed mutagenesis
  • Thiol groups

ASJC Scopus subject areas

  • Biochemistry

Cite this

Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase : Crystallization and preliminary X-ray diffraction analysis of the enzyme. / Farnaud, Sebastien; Tata, Renée; Sohi, Maninder K.; Wan, Tommy; Brown, Paul R.; Sutton, Brian J.

In: Biochemical Journal, Vol. 340, No. 3, 15.06.1999, p. 711-714.

Research output: Contribution to journalArticle

@article{1ea50812e340424e963f972124102d27,
title = "Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase: Crystallization and preliminary X-ray diffraction analysis of the enzyme",
abstract = "Wild-type and site-specific mutants C166S and C166A (Cys-166→Ser and Cys-166→Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3. Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding. Cys-166 is therefore implicated as the active-site nucleophile. Titration of free thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue. Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a = b = c = 84 {\AA}; α = β = γ = 75°) and space group (R3 or R32). These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility.",
keywords = "Quaternary structure, Site-directed mutagenesis, Thiol groups",
author = "Sebastien Farnaud and Ren{\'e}e Tata and Sohi, {Maninder K.} and Tommy Wan and Brown, {Paul R.} and Sutton, {Brian J.}",
year = "1999",
month = "6",
day = "15",
doi = "10.1042/0264-6021:3400711",
language = "English",
volume = "340",
pages = "711--714",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press",
number = "3",

}

TY - JOUR

T1 - Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase

T2 - Crystallization and preliminary X-ray diffraction analysis of the enzyme

AU - Farnaud, Sebastien

AU - Tata, Renée

AU - Sohi, Maninder K.

AU - Wan, Tommy

AU - Brown, Paul R.

AU - Sutton, Brian J.

PY - 1999/6/15

Y1 - 1999/6/15

N2 - Wild-type and site-specific mutants C166S and C166A (Cys-166→Ser and Cys-166→Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3. Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding. Cys-166 is therefore implicated as the active-site nucleophile. Titration of free thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue. Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a = b = c = 84 Å; α = β = γ = 75°) and space group (R3 or R32). These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility.

AB - Wild-type and site-specific mutants C166S and C166A (Cys-166→Ser and Cys-166→Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3. Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding. Cys-166 is therefore implicated as the active-site nucleophile. Titration of free thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue. Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a = b = c = 84 Å; α = β = γ = 75°) and space group (R3 or R32). These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility.

KW - Quaternary structure

KW - Site-directed mutagenesis

KW - Thiol groups

UR - http://www.scopus.com/inward/record.url?scp=0033564895&partnerID=8YFLogxK

U2 - 10.1042/0264-6021:3400711

DO - 10.1042/0264-6021:3400711

M3 - Article

VL - 340

SP - 711

EP - 714

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 3

ER -