The Entner-Doudoroff (ED) pathway is a classic central pathway of D-glucose metabolism in all three phylogenetic domains. On the other hand, Archaea and/or bacteria possess several modified versions of the ED pathway, in which nonphosphorylated intermediates are involved. Several fungi, including Pichia stipitis and Debaryomyces hansenii, possess an alternative pathway of L-rhamnose metabolism, which is different from the known bacterial pathway. Gene cluster related to this hypothetical pathway was identified by bioinformatic analysis using the metabolic enzymes involved in analogous sugar pathways to the ED pathway. Furthermore, the homologous gene cluster was found not only in many other fungi but also several bacteria, including Azotobacter vinelandii. Four putative metabolic genes, LRA1-4, were cloned, overexpressed in Escherichia coli, and purified. Substrate specificity and kinetic analysis revealed that nonphosphorylated intermediates related to L-rhamnose are significant active substrates for the purified LRA1-4 proteins. Furthermore, L-2-keto-3-deoxyrhamnonate was structurally identified as both reaction products of dehydration by LRA3 and aldol condensation by LRA4. These results suggested that the LRA1-4 genes encode L-rhamnose 1-dehydrogenase, L-rhamnono-␥-lactonase, L-rhamnonate dehydratase, and L-KDR aldolase, respectively, by which L-rhamnose is converted into pyruvate and L-lactaldehyde through analogous reaction steps to the ED pathway. There was no evolutionary relationship between L-KDR aldolases from fungi and bacteria.The Embden-Meyerhof-Parnas pathway is a central metabolic pathway and is present, at least in part, in all organisms. Furthermore, Gram-negative bacteria possess the Entner-Dou- 2). Schematic sugar conversion is almost analogous to that of the ED pathway, whereas their equivalent metabolic enzymes possess no evolutionary relationship (3-5). Recently, several alternative and/or novel pathways related to D-arabinose (6), L-arabinose (7-10), D-xylose (11, 12), and L-fucose (13), differing from these well known pathways, were identified genetically and were partially analogous to ED and npED pathways. These nonphosphorylative sugar metabolic pathways are classified into two groups, in which sugar is commonly converted into 2-keto-3-deoxyacidsugar through the participation of dehydrogenase, lactonase, and dehydratase. In the "type I pathway" of D-galactose (14), D-fucose (15-18), and L-arabinose (19), 2-keto-3-deoxyacidsugar is cleaved through an aldolase reaction to aldehyde and pyruvate as well as the ED and archaeal npED pathways, although most metabolic genes have not yet been identified. In the bacterial D-gluconate pathway (20), D-gluconate enters the pentose-phosphate pathway (PPP in Fig. 1). In the recently identified bacterial L-fucose pathway (13), an intermediate of L-2-keto-3-deoxyfuconate is dehydrogenated and then cleaved to L-lactate and pyruvate. On the other hand, the "type II pathway" of the nonphosphorylative sugar metabolic pathway corresponds to an alternative...