The Hydantoin Transport Protein from Microbacterium liquefaciens

The gene hyuP from Microbacterium liquefaciens AJ 3912 with an added His 6 tag was cloned into the expression plasmid pTTQ18 in an Escherichia coli host strain. The transformed E. coli showed transport of radioisotope-labeled 5-substituted hydantoins with apparent K m values in the micromolar range. This activity exhibited a pH optimum of 6.6 and was inhibited by dinitrophenol, indicating the requirement of energy for the transport system. 5-Indolyl methyl hydantoin and 5-benzyl hydantoin were the preferred substrates, with selectivity for a hydrophobic substituent in position 5 of hydantoin and for the l isomer over the d isomer. Hydantoins with less hydrophobic substituents, cytosine, thiamine, uracil, allantoin, adenine, and guanine, were not effective ligands. The His-tagged hydantoin transport protein was located in the inner membrane fraction, from which it was solubilized and purified and its identity was authenticated.