Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase: crystallization and preliminary X-ray diffraction analysis of the enzyme.
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 A; alpha=beta=gamma=75 degrees) 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.
Farnaud
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S
Molecular Biology and Biophysics Section, Division of Biomolecular Sciences, King's College London, Strand, London WC2R 2LS, UK.
Tata
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Sohi
M K
MK
Wan
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Brown
P R
PR
Sutton
B J
BJ
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites; D002942:Circular Dichroism; D003429:Cross Reactions; D003460:Crystallization; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005779:Immunodiffusion; D008970:Molecular Weight; D011487:Protein Conformation; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism
D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D000581:Amidohydrolases; C019665:amidase; D003545:Cysteine