Crystal structure and mutational analyses of ribokinase from Arabidopsis thaliana.

Nitrogen remobilization is a key issue in plants. Recent studies in Arabidopsis thaliana have revealed that nucleoside catabolism supplies xanthine, a nitrogen-rich compound, to the purine ring catabolic pathway, which liberates ammonia from xanthine for reassimilation into amino acids. Similarly, pyrimidine nuclosides are degraded and the pyrimidine bases are fully catabolized. During nucleoside hydrolysis, ribose is released, and ATP-dependent ribokinase (RBSK) phosphorylates ribose to ribose-5'-phosphate to allow its entry into central metabolism recycling the sugar carbons from nucleosides. In this study, we report the crystal structure of RBSK from Arapidopsis thaliana (AtRBSK) in three different ligation states: an unliganded state, a ternary complex with ribose and ATP, and a binary complex with ATP in the presence of Mg2+. In the monomeric conformation, AtRBSK is highly homologous to bacterial RBSKs, including the binding sites for a monovalent cation, ribose, and ATP. Its dimeric conformation, however, does not exhibit the noticeable ligand-induced changes that were observed in bacterial orthologs. Only in the presence of Mg2+, ATP in the binary complex adopts a catalytically competent conformation, providing a mode of action for Mg2+ in AtRBSK activity. The structural data combined with activity analyses of mutants allowed assignment of functional roles for the active site residues. Overall, this study provides the first structural characterization of plant RBSK, and experimentally validates a previous hypothetical model concerning the general reaction mechanism of RBSK.

Crystal structure of chloramphenicol-metabolizing enzyme EstDL136 from a metagenome.

Metagenomes often convey novel biological activities and therefore have gained considerable attention for use in biotechnological applications. Recently, metagenome-derived EstDL136 was found to possess chloramphenicol (Cm)-metabolizing features. Sequence analysis showed EstDL136 to be a member of the hormone-sensitive lipase (HSL) family with an Asp-His-Ser catalytic triad and a notable substrate specificity. In this study, we determined the crystal structures of EstDL136 and in a complex with Cm. Consistent with the high sequence similarity, the structure of EstDL136 is homologous to that of the HSL family. The active site of EstDL136 is a relatively shallow pocket that could accommodate Cm as a substrate as opposed to the long acyl chain substrates typical of the HSL family. Mutational analyses further suggested that several residues in the vicinity of the active site play roles in the Cm-binding of EstDL136. These results provide structural and functional insights into a metagenome-derived EstDL136.