No abstract

Nine potential caspase counterparts, designated metacaspases, were identified in the Arabidopsis thaliana genome. Sequence analysis revealed two types of metacaspases, one with (type I) and one without (type II) a proline-or glutamine-rich N-terminal extension, possibly representing a prodomain. Production of recombinant Arabidopsis type II metacaspases in Escherichia coli resulted in cysteine-dependent autocatalytic processing of the proform into large and small subunits, in analogy to animal caspases. A detailed biochemical characterization with a broad range of synthetic oligopeptides and several protease inhibitors of purified recombinant proteins of both metacaspase 4 and 9 showed that both metacaspases are arginine/lysine-specific cysteine proteases and did not cleave caspase-specific synthetic substrates. These findings suggest that type II metacaspases are not directly responsible for earlier reported caspase-like activities in plants.

The first enzyme of the phenylpropanoid pathway, Phe ammonia-lyase (PAL), is encoded by four genes in Arabidopsis thaliana. Whereas PAL function is well established in various plants, an insight into the functional significance of individual gene family members is lacking. We show that in the absence of clear phenotypic alterations in the Arabidopsis pal1 and pal2 single mutants and with limited phenotypic alterations in the pal1 pal2 double mutant, significant modifications occur in the transcriptome and metabolome of the pal mutants. The disruption of PAL led to transcriptomic adaptation of components of the phenylpropanoid biosynthesis, carbohydrate metabolism, and amino acid metabolism, revealing complex interactions at the level of gene expression between these pathways. Corresponding biochemical changes included a decrease in the three major flavonol glycosides, glycosylated vanillic acid, scopolin, and two novel feruloyl malates coupled to coniferyl alcohol. Moreover, Phe overaccumulated in the double mutant, and the levels of many other amino acids were significantly imbalanced. The lignin content was significantly reduced, and the syringyl/guaiacyl ratio of lignin monomers had increased. Together, from the molecular phenotype, common and specific functions of PAL1 and PAL2 are delineated, and PAL1 is qualified as being more important for the generation of phenylpropanoids.

Dux, P.; Hard, K.; Devreese, B.; Nugteren-Roodzand, I.M.; Crielaard, W.; Boelens, R.; Beeumen, J.; Kaptein, R.; Hellingwerf, K.J. Published in: Biochemistry DOI:10.1021/bi00251a001Link to publication Citation for published version (APA):Hoff, W. D., Dux, P., Hard, K., Devreese, B., Nugteren-Roodzand, I. M., Crielaard, W., ... Hellingwerf, K. J. (1994). p-Coumaric acid, a new photoactive chromophore of a yellow photoreceptor protein with rhodopsin-like characteristics. Biochemistry, 33, 13959-13963. DOI: 10.1021/bi00251a001 General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. would make it the first eubacterial rhodopsin. Here we report the chemical structure of this chromophoric group to be p-coumaric acid, which is covalently bound to a unique cysteine in the apoprotein via a thiol ester bond, and thus not retinal. This makes PYP the first example of a protein containing p-coumaric acid, a metabolite previously found only in plants, as a prosthetic group and establishes the photoactive yellow proteins as a new type of photochemically active receptor molecule.The photoactive yellow proteins (PYP) constitute a responsible for the yellow color of the protein have been advanced (Meyer, 1985;McRee at al, 1989;Van Beeumen et al., 1993), but the true nature of this chromophore et al, 1993) and crystal structure ( M~R~~ et al., 1989) of pyp at 2.4-ij resolution have been and show that the protein is composed of two perpendicular plates of P-sheet, forming a p-clam structure very similar to the fold homologous group of proteins found in many Eubacteria (Meyer, 1985; M e w et al., 1990;Hoff et al., 1994a). The isolated from Ecfothiorhodospira halophila have been studied in some detail. Since PYP was isolated in 1985, a number of proposals concerning the chemical structure of the cofactor structural and photochemical characteristics Of the PYP remained unclear. The amino acid sequence (Van Beeumen

SummaryThe penicillin-binding protein (PBP) 1b of Escherichia coli catalyses the assembly of lipid-transported Nacetyl glucosaminyl-b-1,4 -N-acetylmuramoyl-L-alanyl-g-D-glutamyl-(L)-meso-diaminopimelyl-(L)-D-alanyl-D-alanine disaccharide pentapeptide units into polymeric peptidoglycan. These units are phosphodiester linked, at C1 of muramic acid, to a C55 undecaprenyl carrier. PBP1b has been puri®ed in the form of His tag (M46-N844) PBP1bg. This derivative provides the host cell in which it is produced with a functional wall peptidoglycan. His tag (M46-N844) PBP1bg possesses an amino-terminal hydrophobic segment, which serves as transmembrane spanner of the native PBP. This segment is linked, via an h 100-amino-acid insert, to a D198-G435 glycosyl transferase module that possesses the ®ve motifs characteristic of the PBPs of class A. In in vitro assays, the glycosyl transferase of the PBP catalyses the synthesis of linear glycan chains from the lipid carrier with an ef®ciency of h 39 000 M À1 s À1 . Glu-233, of motif 1, is central to the catalysed reaction. It is proposed that the Glu-233 g-COOH donates its proton to the oxygen atom of the scissile phosphoester bond of the lipid carrier, leading to the formation of an oxocarbonium cation, which then undergoes attack by the 4-OH group of a nucleophile N-acetylglucosamine. Asp-234 of motif 1 or Glu-290 of motif 3 could be involved in the stabilization of the oxocarbonium cation and the activation of the 4-OH group of the N-acetylglucosamine. In turn, Tyr-310 of motif 4 is an important component of the amino acid sequence-folding information. The glycosyl transferase module of PBP1b, the lysozymes and the lytic transglycosylase Slt70 have much the same catalytic machinery. They might be members of the same superfamily. The glycosyl transferase module is linked, via a short junction site, to the amino end of a Q447-N844 acyl transferase module, which possesses the catalytic centre-de®ning motifs of the penicilloyl serine transferases superfamily. In in vitro assays with the lipid precursor and in the presence of penicillin at concentrations suf®cient to derivatize the active-site serine 510 of the acyl transferase, the rate of glycan chain synthesis is unmodi®ed, showing that the functioning of the glycosyl transferase is acyl transferase independent. In the absence of penicillin, the products of the Ser-510-assisted double-proton shuttle are glycan strands substituted by cross-linked tetrapeptide±pentapeptide and tetrapeptide±tetrapeptide dimers and uncross-linked pentapeptide and tetrapeptide monomers. The acyl transferase of the PBP also catalyses aminolysis and hydrolysis of properly structured thiolesters, but it lacks activity on D-alanyl-D-alanine-terminated peptides. This substrate speci®city suggests that carbonyl donor activity requires the attachment of the pentapeptides to the glycan chains made by the glycosyl transferase, and it implies that one and the same PBP molecule catalyses transglycosylation and peptide cross-linking in a sequential manner. Att...