Catalytic mechanism of ancestral L-lysine oxidase assigned by sequence data mining.

A large number of protein sequences are registered in public databases such as PubMed. Functionally uncharacterized enzymes are included in these databases, some of which likely have potential for industrial applications. However, assignment of the enzymes remained difficult tasks for now. In this study, we assigned a total of 28 original sequences to uncharacterized enzymes in the FAD-dependent oxidase family expressed in some species of bacteria including Chryseobacterium, Flavobacterium, and Pedobactor. Progenitor sequence of the assigned 28 sequences was generated by ancestral sequence reconstruction, and the generated sequence exhibited L-lysine oxidase activity; thus, we named the enzyme AncLLysO. Crystal structures of ligand-free and ligand-bound forms of AncLLysO were determined, indicating that the enzyme recognizes L-Lys by hydrogen bond formation with R76 and E383. The binding of L-Lys to AncLLysO induced dynamic structural change at a plug loop formed by residues 251 to 254. Biochemical assays of AncLLysO variants revealed the functional importance of these substrate recognition residues and the plug loop. R76A and E383D variants were also observed to lose their activity, and the kcat/Km value of G251P and Y253A mutations were approximately 800- to 1800-fold lower than that of AncLLysO, despite the indirect interaction of the substrates with the mutated residues. Taken together, our data demonstrate that combinational approaches to sequence classification from database and ancestral sequence reconstruction may be effective not only to find new enzymes using databases of unknown sequences but also to elucidate their functions.

Gateway binary vectors with organelle-targeted fluorescent proteins for highly sensitive reporter assay in gene expression analysis of plants.

Fluorescent proteins are valuable tools in the bioscience field especially in subcellular localization analysis of proteins and expression analysis of genes. Fusion with organelle-targeting signal accumulates fluorescent proteins in specific organelles, increases local brightness, and highlights the signal of fluorescent proteins even in tissues emitting a high background of autofluorescence. For these advantages, organelle-targeted fluorescent proteins are preferably used for promoter:reporter assay to define organ-, tissue-, or cell-specific expression pattern of genes in detail. In this study, we have developed a new series of Gateway cloning technology-compatible binary vectors, pGWBs (attR1-attR2 acceptor sites) and R4L1pGWB (attR4-attL1 acceptor sites), carrying organelle-targeted synthetic green fluorescent protein with S65T mutation (sGFP) (ER-, nucleus-, peroxisome-, and mitochondria-targeted sGFP) and organelle-targeted tag red fluorescent protein (TagRFP) (nucleus-, peroxisome-, and mitochondria-targeted TagRFP). These are available for preparation of promoter:reporter constructs by an LR reaction with a promoter entry clone attL1-promoter-attL2 (for pGWBs) or attL4-promoter-attR1 (for R4L1pGWBs), respectively. A transient expression experiment with particle bombardment using cauliflower mosaic virus 35S promoter-driven constructs has confirmed the correct localization of newly developed organelle-targeted TagRFPs by a co-localization analysis with the previously established organelle-targeted sGFPs. More intense and apparent fluorescence signals were detected by the nucleus- and peroxisome-targeted sGFPs than by the normal sGFPs in the promoter assay using transgenic Arabidopsis thaliana. The new pGWBs and R4L1pGWBs developed here are highly efficient and may serve as useful platforms for more accurate observation of GFP and RFP signals in gene expression analyses of plants.