A leaf-specific 27 kDa protein of potato Kunitz-type proteinase inhibitor is induced in response to abscisic acid, ethylene, methyl jasmonate, and water deficit

The 22 kDa Kunitz-type potato proteinase inhibitor (22 kDa KPPI) was induced in tubers. However, the 27 kDa protein, which is immunologically related to the 22 kDa KPPI, was induced in leaves by wounding, hormones, and environmental stresses. The leaf-specific 27 kDa protein was induced in leaves that were treated with exogenous abscisic acid (ABA), ethephon, methyl jasmonate (MeJA), and water deficit. These results indicate that the 27 kDa protein in leaves could function as a defense protein against mechanical damages by herbivorous animals and abiotic environmental stresses that could induce plant hormones.     

Crystal structure of the MJ0490 gene product of the hyperthermophilic archaebacterium Methanococcus jannaschii, a novel member of the lactate/malate family of dehydrogenases

The MJ0490 gene, one of the only two genes of Methanococcus jannaschii showing sequence similarity to the lactate/malate family of dehydrogenases, was classified initially as coding for a putative l-lactate dehydrogenase (LDH). It has been re-classified as a malate dehydrogenase (MDH) gene, because it shows significant sequence similarity to MT0188, MDH II from Methanobacterium thermoautotrophicum strain DeltaH. The three-dimensional structure of its gene product has been determined in two crystal forms: a "dimeric" structure in the orthorhombic crystal at 1.9 A resolution and a "tetrameric" structure in the tetragonal crystal at 2.8 A. These structures share a similar subunit fold with other LDHs and MDHs. The tetrameric structure resembles typical tetrameric LDHs. The dimeric structure is equivalent to the P-dimer of tetrameric LDHs, unlike dimeric MDHs, which correspond to the Q-dimer. The structure reveals that the cofactor NADP(H) is bound at the active site, despite the fact that it was not intentionally added during protein purification and crystallization. The preference of NADP(H) over NAD(H) has been supported by activity assays. The cofactor preference is explained by the presence of a glycine residue in the cofactor binding pocket (Gly33), which replaces a conserved aspartate (or glutamate) residue in other NAD-dependent LDHs or MDHs. Preference for NADP(H) is contributed by hydrogen bonds between the oxygen atoms of the monophosphate group and the ribose sugar of adenosine in NADP(H) and the side-chains of Ser9, Arg34, His36, and Ser37. The MDH activity of MJ0490 is made possible by Arg86, which is conserved in MDHs but not in LDHs. The enzymatic assay showed that the MJ0490 protein possesses the fructose-1,6-bisphosphate-activated LDH activity (reduction). Thus the MJ0490 gene product appears to be a novel member of the lactate/malate dehydrogenase family, displaying an LDH scaffold and exhibiting a relaxed substrate and cofactor specificities in NADP(H) and NAD(H)-dependent malate and lactate dehydrogenase reactions.     

Subtype-specific role of phospholipase C-β in bradykinin and LPA signaling through differential binding of different PDZ scaffold proteins

Among phospholipase C (PLC) isozymes (β, γ, δ, ε, ζ and η), PLC-β plays a key role in G-protein coupled receptor (GPCR)-mediated signaling. PLC-β subtypes are often overlapped in their distribution, but have unique knock-out phenotypes in organism, suggesting that each subtype may have the different role even within the same type of cells. In this study, we examined the possibility of the differential coupling of each PLC-β subtype to GPCRs, and explored the molecular mechanism underlying the specificity. Firstly, we found that PLC-β1 and PLC-β3 are activated by bradykinin (BK) or lysophosphatidic acid (LPA), respectively. BK-triggered phosphoinositides hydrolysis and subsequent Ca²⁺ mobilization were abolished specifically by PLC-β1 silencing, whereas LPA-triggered events were by PLC-β3 silencing. Secondly, we showed the evidence that PDZ scaffold proteins is a key mediator for the selective coupling between PLC-β subtype and GPCR. We found PAR-3 mediates physical interaction between PLC-β1 and BK receptor, while NHERF2 does between PLC-β3 and LPA₂ receptor. Consistently, the silencing of PAR-3 or NHERF2 blunted PLC signaling induced by BK or LPA respectively. Taken together, these data suggest that each subtype of PLC-β is selectively coupled to GPCR via PDZ scaffold proteins in given cell types and plays differential role in the signaling of various GPCRs.     

Expression, purification, and biochemical characterization of the intron-encoded endonuclease, I-CreII

The ORF of the Cr.psbA4 intron of Chlamydomonas reinhardtii mediates efficient intron homing, and contains an H-N-H and possibly a GIY-YIG motif. The ORF was over-expressed in Escherichia coli without non-native amino acids, but was mostly insoluble. However, co-over-expression of E. coli chaperonins GroEL/GroES solubilized approximately 50% of the protein, which was purified by ion-exchange and heparin-affinity chromatography. Biochemical characterization showed that the protein is a double-strand-specific endonuclease that cleaves fused psbA exon 4-exon 5 DNA, and was named I-CreII. I-CreII has a relatively relaxed divalent metal ion requirement (Mg(2+), Mn(2+), Ca(2+), and Fe(2+) supported cleavage), is insensitive to salt <350 mM, and is stabilized by DNA. Cleavage of target DNA occurs close (4 nt on the top strand) to the intron-insertion site, and leaves 2-nt 3'-OH overhangs, similar to GIY-YIG endonucleases. The boundaries of the recognition sequence span approximately 30 bp, and encompass the cleavage and intron-insertion sites. Cleavage of heterologous psbA DNAs indicates the enzyme can tolerate multiple, but not all, substitutions in the recognition site. This work will facilitate further study of this novel endonuclease, which may also find use in site-specific manipulation of chloroplast DNA.     

Regioselective substitution of 6,7-dichloroquinoline-5,8-dione: synthesis and X-ray crystal structure of 4a,10,11-triazabenzo[3,2-a]fluorene-5,6-diones

6,7-Dichloroquinoline-5,8-dione (1) was reacted with a number of 2-aminopyridine derivatives. Of the several possible products of this reaction, 4a,10,11-triazabenzo[3,2-a]fluorene-5,6-dione (6), produced by condensation and rearrangement, was obtained as the major product, and its structure was subsequently unambigously determined by X-ray crystallographic study. Ortho-quinones were produced via nucleophilic substitution at position C7, which was unexpected, considering that para-quinones were produced via C6 substitution in the reaction between compound 1 and ethyl acetoacetate in our previous work. Such unexpected nucleophilic substitution at C7 provides an effective, yet simple route, to the preparation of biologically active ortho-quinone derivatives.