Cloning, expression, purification, crystallization and preliminary X-ray characterization of allantoinase from Bacillus licheniformis ATCC 14580.
Allantoinase, a member of the amidohydrolase superfamily, exists in a wide variety of organisms, including bacteria, fungi, plants and a few animals, such as fishes and amphibians. Allantoinase catalyzes the reversible hydrolysis of allantoin into allantoate by hydrolytic cleavage of the N1-C2 amide bond of the five-membered hydantoin ring. Allantoinase from Bacillus licheniformis (AllBali) presents an inverted enantioselectivity towards allantoin (R-enantioselective), which is a distinguishable feature that is not observed for other allantoinases. In this work, B. licheniformis ATCC 14580 allantoinase (AllBali) containing a C-terminal His6 tag was overproduced in Escherichia coli and purified to homogeneity. Crystals of AllBali were obtained by the vapour-diffusion method using 0.1 M potassium thiocyanate, 20%(w/v) polyethylene glycol 3350 as a crystallization solution. X-ray diffraction data were collected to a resolution of 3.5 Å with an Rmerge of 29.2% from a crystal belonging to space group P12₁1, with unit-cell parameters a=54.93, b=164.74, c=106.89 Å, β=98.49°. There are four molecules in the asymmetric unit with a solvent content of 47% as estimated from the Matthews coefficient (VM=2.34 Å3 Da(-1)).
Laboratorio de Estudios Cristalográficos, IACT, CSIC-Universidad de Granada, Avenida de las Palmeras 4, 18100 Armilla, Spain.
Mayte
M
Conejero-Muriel
Departamento de Química-Física, Universidad de Almería, 04120 Almería, Spain.
Ana Isabel
AI
Martínez-Gómez
Departamento de Química-Física, Universidad de Granada, 18071 Granada, Spain.
Sergio
S
Martínez-Rodríguez
Laboratorio de Estudios Cristalográficos, IACT, CSIC-Universidad de Granada, Avenida de las Palmeras 4, 18100 Armilla, Spain.
Jose A
JA
Gavira
D000581:Amidohydrolases / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D001407:Bacillus / Q000201:enzymology*; D003001:Cloning, Molecular / Q000379:methods; D015964:Gene Expression Regulation, Bacterial; D014961:X-Ray Diffraction
D000581:Amidohydrolases; C019659:allantoinase
Bacillus licheniformis; allantoin; allantoinase; amidohydrolase