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Escherichia coli strain JM 109 harboring 6× His-tag l-rhamnose isomerase (l-RhI) from Pseudomonas stutzeri allowed a 20-fold increase in the volumetric yield of soluble enzyme compared to the value for the intrinsic yield. Detailed studies on the substrate specificity of the purified His-tagged protein revealed that it catalyzed previously unknown common and rare aldo/ketotetrose, aldo/ketopentose, and aldo/ketohexose substrates in both d- and l-forms, for instance, erythrose, threose, xylose, lyxose, ribose, glucose, mannose, galactose, altrose, tagatose, sorbose, psicose, and fructose. Using a high enzyme–substrate ratio in extended reactions, the enzyme-catalyzed interconversion reactions from which two different products from one substrate were formed: l-lyxose, l-glucose, l-tagatose and d-allose were isomerized to l-xylulose and l-xylose, l-fructose and l-mannose, l-galactose and l-talose, and d-psicose and d-altrose, in that order. Kinetic studies, however, showed that l-rhamnose with Km and Vmax values of 11 mM and 240 U/mg, respectively, was the most preferred substrate, followed by l-mannose, l-lyxose, d-ribose, and d-allose. Based on the observed catalytic mode of action, these new findings reflected a hitherto undetected interrelation between l-RhI and d-xylose isomerase (d-XI). [Copyright &y& Elsevier]