Crystallization and preliminary X-ray crystallographic analysis of EstE1, a new and thermostable esterase cloned from a metagenomic library.
EstE1, a new thermostable esterase, was isolated by functional screening of a metagenomic DNA library from thermal environment samples. This enzyme showed activity towards short-chain acyl derivatives of length C4-C6 at a temperature of 303-363 K and displayed a high thermostability above 353 K. EstE1 has 64 and 57% amino-acid sequence similarity to est(pc)-encoded carboxylesterase from Pyrobaculum calidifontis and AFEST from Archaeoglobus fulgidus, respectively. The recombinant protein with a histidine tag at the C-terminus was overexpressed in Escherichia coli strain BL21(DE3) and then purified by affinity chromatography. The protein was crystallized at 290 K by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.3 A resolution from an EstE1 crystal; the crystal belongs to space group P4(1)2(1)2, with unit-cell parameters a = b = 73.71, c = 234.23 A. Assuming the presence of four molecules in the asymmetric unit, the Matthews coefficient VM is calculated to be 2.2 A3 Da(-1) and the solvent content is 44.1%.
Byun
Jung-Sue
JS
Department of Biology, Yonsei University, Seoul 120-749, South Korea.
Rhee
Jin-Kyu
JK
Kim
Dong-Uk
DU
Oh
Jong-Won
JW
Cho
Hyun-Soo
HS
D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics; D019611:Archaeoglobus fulgidus / Q000201:enzymology; D043182:Carboxylesterase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D015698:Genomic Library; D041543:Pyrobaculum / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D043182:Carboxylesterase