Cloning, expression, purification, crystallization and preliminary X-ray characterization of allantoinase fromBacillus licheniformisATCC 14580

Abstract

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—C2amide bond of the five-membered hydantoin ring. Allantoinase fromBacillus 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. licheniformisATCC 14580 allantoinase (AllBali) containing a C-terminal His6tag was overproduced inEscherichia coliand purified to homogeneity. Crystals of AllBali were obtained by the vapour-diffusion method using 0.1 Mpotassium 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 anRmergeof 29.2% from a crystal belonging to space groupP1211, with unit-cell parametersa= 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).