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

Keywords

  • Quaternary structure
  • Site-directed mutagenesis
  • Thiol groups

ASJC Scopus subject areas

  • Biochemistry

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