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Purification and crystallization of benzoylformate decarboxylase
Author(s) -
Hasson Miriam S.,
Petsko Gregory A.,
Ringe Dagmar,
Muscate Angelika,
Henehan Gary T.M.,
Guidinger Peter F.,
Ken Yon George L.
Publication year - 1995
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560040515
Subject(s) - tetramer , pseudomonas putida , chemistry , protein quaternary structure , thiamine pyrophosphate , crystallization , pyrophosphate , crystallography , enzyme , size exclusion chromatography , polyethylene glycol , cofactor , chromatography , stereochemistry , protein subunit , biochemistry , organic chemistry , gene
A new large‐scale purification method for benzoylformate decarboxylase from Pseudomonas putida has allowed us to undertake an X‐ray crystallographic study of the enzyme. The previously observed instability of the enzyme was overcome by addition of 100 μM thiamine pyrophosphate to buffers used in the purification. The final enzyme preparation was more than 97% pure, as determined by denaturing gel electrophoresis and densitometry. The mobility of the enzyme on a gel filtration column indicates that it is a tetramer of 57‐kDa subunits. Large, single crystals of benzoylformate decarboxylase were grown from solutions of buffered polyethylene glycol 400, pH 8.5. The crystals diffract to beyond 1.6 Å resolution and are stable for days to X‐ray radiation. Analysis of X‐ray data from the crystals, along with the newly determined quaternary structure, identifies the space group as 1222. The unit cell dimensions are a = 82 Å, b = 97 Å, c = 138 Å. An average V m value for the crystals is consistent with one subunit per asymmetric unit. The subunits of the tetramer must be arranged with tetrahedral 222 symmetry.