Expression, purification and preliminary crystallographic analysis of 2,4-dihydroxy-hepta-2-ene-1,7-dioate aldolase (HpcH) from Escherichia coli C.
The gene encoding 2,4-dihydroxy-hepta-2-ene-1,7-dioate (HHED) aldolase (HpcH; EC 4.1.2) from Escherichia coli C was cloned into the high-expression plasmid pProEx-HTa and overexpressed in E. coli BL21 (DE3). The 28 kDa enzyme was purified using immobilized metal-affinity and size-exclusion chromatography prior to crystallization. Crystals were obtained by the hanging-drop vapour-diffusion method at 277 K from a number of screening conditions. Type I crystals grown in a solution containing 0.4 M ammonium dihydrogen phosphate belong to space group R32, with unit-cell parameters a = b = 128.92, c = 175.30 A. Type II crystals grown in a solution containing 0.5 M sodium chloride, 0.1 M sodium citrate pH 5.5 belong to space group I222, with unit-cell parameters a = 133.39, b = 155.39, c = 168.80 A. Complete data sets were collected to 1.6 and 2.0 A from type I and type II crystals, respectively, using synchrotron radiation.
Rea
Dean
D
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, England.
Fülöp
Vilmos
V
Bugg
Timothy D H
TD
Roper
David I
DI
D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; C097246:2,4-dihydroxyhept-2-ene-1,7-dioic acid aldolase, E coli; D000446:Aldehyde-Lyases; C413570:2-dehydro-3-deoxygalactarate aldolase