The Hydantoin Transport Protein from Microbacterium liquefaciens

Abstract

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.

Crystallization and preliminary crystallographic studies of an active-site mutant hydantoin racemase fromSinorhizobium melilotiCECT4114

“…Some hydantoin racemase-encoding genes have been found together with a carbamoylase and a hydantoinase/ dihydropyrimidinase including a potential hydantoin transporter enzyme (Hils et al, 2001;Suzuki, Takenaka et al, 2005;Watabe et al, 1992b;Wiese et al, 2001); this activity has recently been suggested for the corresponding gene from Microbacterium liquefaciens (Suzuki & Henderson, 2006). However, the most common application of these enzymes is the use of the 'hydantoinase process', a cheap and environmentally friendly enzymatic method for the potential production of any optically pure natural or unnatural amino acid from a wide spectrum of d,l-5-monosubstituted hydantoins Martinez-Rodriguez et al, 2002).…”

Crystallization of the hydantoin transporter Mhp1 fromMicrobacterium liquefaciens

“…coli culture conditions and protein expression have been described previously (Suzuki & Henderson, 2006). Cell pellets were resuspended in 50 mM Tris-HCl pH 8.0 with protease inhibitors (Roche) and sonicated.…”

“…Mhp1 transports 5-substituted hydantoin compounds, which are converted to amino acids as part of a metabolic salvage pathway. The transporter activity is proposed to be proton-dependent (Suzuki & Henderson, 2006) because the uptake of a hydantoin compound was not accelerated by the addition of NaCl but was affected by pH. However, the details of the transporter mechanism of Mhp1 are unclear owing to a lack of structural information.…”

“…Recently, the membrane protein Mhp1, encoded by the hyuP gene from Microbacterium liquefaciens, has been cloned and expressed in Escherichia coli (Suzuki & Henderson, 2006). Mhp1 is composed of 489 amino acids with a molecular weight of 54.6 kDa and is predicted to have 12 transmembrane helices.…”

Finite Temperature String Method with Umbrella Sampling: Application on a Side Chain Flipping in Mhp1 Transporter

“…21 High-resolution crystal structures [22][23][24] are available for two major conformational states of Mhp1: the outward-facing (OF) and the inward-facing (IF) conformations. Several MD studies [23][24][25][26][27][28] were reported for the dynamics and conformational changes of Mhp1.…”