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KMID : 1094720150200030462
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Biotechnology and Bioprocess Engineering
2015 Volume.20 No. 3 p.462 ~ p.472
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Identification and characterization of 3,6-anhydro-L-galactonate cycloisomerase belonging to theenolase superfamily
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Cho Sun-Ja
Kim Jeong-Ah
Lee Sun-Bok
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Abstract
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Recently, we have shown that Postechiella marina M091 degrades 3,6-anhydro-L-galactose (L-AnG) to pyruvate and D-glyceraldehyde-3-phosphate via six enzyme-catalyzed reactions and that the L-AnG dehydrogenase, an enzyme catalyzing the first step of L-AnG degradation, converts L-AnG to 3,6-anhydro-L-galactonate (L-AnGA). In this study, we report the identification and characterization of L-AnGA cycloisomerase (L-AnGACI), a novel enzyme that catalyzes the second step of L-AnG metabolism in agar-degrading microorganisms. To characterize this enzyme, the L-AnGACI gene (M091_0722) from P. marina (Pm_LAnGACI) was cloned and expressed in E. coli. The recombinant Pm_L-AnGACI catalyzed conversion of LAnGA to 2-keto-3-deoxy-L-galactonate (L-KDGal), which was confirmed by the L-KDGal aldolase reaction and the LC-MS analysis of the aldolase reaction products. The enzyme showed activity only towards L-AnGA (100%) and galactarate (1.8%) among the 12 sugar acids and carboxylates tested, and the enzyme activity was maximal at 30°C and pH 8.0. Enzyme activity was enhanced by addition of divalent ions such as Co2+ and Mg2+, as is observed from enzymes of the enolase superfamily. L-AnGACI conserves several residues commonly found in the enolase superfamily, including three metal-ion binding ligands (Asp198, Glu224, Glu250) and five active-site residues (Lys167, Lys169, Asp273, His300, Glu320). Phylogenetic analysis of amino acid sequences indicates that Pm_L-AnGACI belongs to a novel family within the mandelate racemase subgroup of the enolase superfamily. A reaction mechanism for cycloisomerization of L-AnGA to L-KDGal is proposed, which implies that the absolute configuration does not change during the reaction. To our knowledge, this is the first report on the characterization of L-AnGACI.
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KEYWORD
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3,6-anhydro-L-galactonate cycloisomerase, enolase superfamily, mandelate racemase, 3,6-anhydro-L-galactose, agarose metabolism
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