Expression, purification and preliminary crystallographic analysis of 2,4-dihydroxy-hepta-2-ene-1,7-dioate aldolase (HpcH) from Escherichia coli C

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2005 Sep 1;61(Pt 9):821-4. doi: 10.1107/S1744309105023079. Epub 2005 Aug 31.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldehyde-Lyases / chemistry*
  • Aldehyde-Lyases / genetics
  • Aldehyde-Lyases / isolation & purification*
  • Crystallography, X-Ray
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / isolation & purification*
  • Gene Expression*
  • Substrate Specificity

Substances

  • Escherichia coli Proteins
  • 2,4-dihydroxyhept-2-ene-1,7-dioic acid aldolase, E coli
  • Aldehyde-Lyases
  • 2-dehydro-3-deoxygalactarate aldolase