Structural and functional studies of casein kinase I-like protein from rice.

Casein kinase I (CKI) is a protein serine/threonine kinase that is highly conserved from plants to animals. It performs various functions in both the cytoplasm and nucleus, such as DNA repair, cell cycle, cytokinesis, vesicular trafficking, morphogenesis and circadian rhythm. CKI proteins contain a highly conserved kinase domain responsible for catalytic activity at the N-terminus and a highly diverse regulatory domain responsible for determining substrate specificity at the C-terminus. CKI-like protein has been identified in plants, including in rice, but its function and structure have not been reported. Here, we report the 2.0 Å crystal structure of the kinase domain of CKI-like protein from rice. Although the structure adopts the typical bi-lobal kinase architecture, the length and orientation of the glycine-rich ATP-binding motif are dynamic within the CKI family. A loop between α5 and α6 (the α5-α6 loop), which was previously not detected in the CKI family because of flexibility, was clearly detected in our structure. In addition, we identified a lipase as a substrate of CKI-like protein from rice. Phosphorylation of the lipase dramatically reduced its catalytic activity, suggesting that CKI may play a role in the regulation of lipase activity.

Crystallization and preliminary X-ray crystallographic studies of casein kinase I-like protein from rice.

Casein kinase I (CKI) is a serine/threonine protein kinase that performs various functions in the cell, such as DNA repair, cell-cycle regulation, cytokinesis, vesicular trafficking, morphogenesis and circadian-rhythm regulation. CKI proteins contain a highly conserved catalytic domain at the N-terminus and a highly diverse regulatory domain that is responsible for substrate specificity at the C-terminus. In this study, CKI from rice (riceCKI) was overexpressed in Escherichia coli with an engineered C-terminal His tag. RiceCKI was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 2.0 Å from a crystal belonging to the monoclinic space group C2, with unit-cell parameters a = 108.83, b = 69.60, c = 55.85 Å, ß = 109.47°. The asymmetric unit was estimated to contain one monomer.

Crystallization and preliminary X-ray crystallographic studies of ß-transaminase from Mesorhizobium sp. strain LUK.

ß-Transaminase (ß-TA) catalyzes the transamination reaction between ß-aminocarboxylic acids and keto acids. This enzyme is a particularly suitable candidate for use as a biocatalyst for the asymmetric synthesis of enantiochemically pure ß-amino acids for pharmaceutical purposes. The ß-TA from Mesorhizobium sp. strain LUK (ß-TAMs) belongs to a novel class in that it shows ß-transaminase activity with a broad and unique substrate specificity. In this study, ß-TAMs was overexpressed in Escherichia coli with an engineered C-terminal His tag. ß-TAMs was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 2.5 Šfrom a crystal that belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 90.91, b = 192.17, c = 52.75 Å.