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7-Hydroxymethyl chlorophyll a reductase (HCAR) catalyzes the second half-reaction in chlorophyll b to chlorophyll a conversion. HCAR is required for the degradation of light-harvesting complexes and is necessary for efficient photosynthesis by balancing the chlorophyll a/b ratio. Reduction of the hydroxymethyl group uses redox cofactors [4Fe-4S] cluster and FAD to transfer electrons and is difficult because of the strong carbonoxygen bond. Here, we report the crystal structure of Arabidopsis HCAR at 2.7-Å resolution and reveal that two [4Fe-4S] clusters and one FAD within a very short distance form a consecutive electron pathway to the substrate pocket. In vitro kinetic analysis confirms the ferredoxin-dependent electron transport chain, thus supporting a proton-activated electron transfer mechanism. HCAR resembles a partial reconstruction of an archaeal F420-reducing [NiFe] hydrogenase, which suggests a common mode of efficient proton-coupled electron transfer through conserved cofactor arrangements. Furthermore, the trimeric form ofHCARprovides a biological clue of its interaction with light-harvesting complex II. [ABSTRACT FROM AUTHOR]

Members of glycosyltransferase family 75 (GT75) not only reversibly catalyze the autoglycosylation of a conserved arginine residue with specific NDP-sugars but also exhibit NDPpyranose mutase activity that reversibly converts specific NDP-pyranose to NDP-furanose. The latter activity provides valuable NDP-furanosyl donors for glycosyltransferases and requires a divalent cation as a cofactor instead of FAD used by UDP-D-galactopyranose mutase. However, details of the mechanism for NDP-pyranose mutase activity are not clear. Here we report the first crystal structures of GT75 family NDPpyranose mutases. The novel structures of GT75 member MtdL in complex with Mn2+ and GDP, GDP-D-glucopyranose, GDPL-fucopyranose, GDP-L-fucofuranose, respectively, combined with site-directed mutagenesis studies, reveal key residues involved in Mn2+ coordination, substrate binding, and catalytic reactions. We also provide a possible catalytic mechanism for this unique type of NDP-pyranose mutase. Taken together, our results highlight key elements of an enzyme family important for furanose biosynthesis. [ABSTRACT FROM AUTHOR]