1887

Abstract

A single gene cluster encoding components of a putative ATP-binding cassette (ABC) transporter for basic amino acids was identified in the incomplete genome sequence of the thermophilic Gram-positive bacterium by searches. The cluster comprises three genes, and these were amplified from chromosomal DNA of , ligated into plasmid vectors and expressed in . The purified solute-binding protein (designated ArtJ) was demonstrated to bind -arginine with high affinity ( =0·39±0·06 μM). Competition experiments revealed only partial inhibition by excess -lysine (38 %) and -ornithine (46 %), while no inhibition was observed with -histidine or other amino acids tested. The membrane-associated transport complex, composed of a permease (designated ArtM) and an ATPase component (designated ArtP), was solubilized from membranes by decanoylsucrose and purified by metal-affinity chromatography. The ArtMP complex, when incorporated into liposomes formed from a crude extract of lipids, displayed ATPase activity in the presence of ArtJ only. Addition of -arginine further stimulated the activity twofold. ATP hydrolysis was optimal at 60 °C and sensitive to the specific inhibitor vanadate. Analysis of kinetic parameters revealed a maximal velocity of ATP hydrolysis of 0·71 μmol P min (mg protein) and a of 1·59 mM. Together, these results identify the ArtJMP complex as a high-affinity arginine ABC transporter.

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2005-03-01
2020-12-01
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