The activity of ribose-5-phosphate isomerases (RpiB) from Clostridium difficile for d-ribose isomerization was optimal at pH 7.5 and 40A degrees C, while that from Thermotoga maritima for l-talose isomerization was optimal at pH 8.0 and 70A degrees C. C. difficile RpiB exhibited activity only with aldose substrates possessing hydroxyl groups oriented in the right-handed configuration (Fischer projections) at the C2 and C3 positions, such as d-ribose, d-allose, l-talose, l-lyxose, d-gulose, and l-mannose. In contrast, T. maritima RpiB displayed activity only with aldose substrates possessing hydroxyl groups configured the same direction at the C2, C3, and C4 positions, such as the d- and l-forms of ribose, talose, and allose.