Evolution of DMSP (dimethylsulfoniopropionate) biosynthesis pathway: Origin and phylogenetic distribution in polyploid Spartina (Poaceae, Chloridoideae).

DMSP (dimethylsulfoniopropionate) is an ecologically important sulfur metabolite commonly produced by marine algae and by some higher plant lineages, including the polyploid salt marsh genus Spartina (Poaceae). The molecular mechanisms and genes involved in the DMSP biosynthesis pathways are still unknown. In this study, we performed comparative analyses of DMSP amounts and molecular phylogenetic analyses to decipher the origin of DMSP in Spartina that represents one of the major source of terrestrial DMSP in coastal marshes. DMSP content was explored in 14 Spartina species using 1H Nuclear Magnetic Resonance (NMR) spectroscopy and Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS). Putative genes encoding the four enzymatic steps of the DMSP biosynthesis pathway in Spartina were examined and their evolutionary dynamics were studied. We found that the hexaploid lineage containing S. alterniflora, S. foliosa and S. maritima and their derived hybrids and allopolyploids are all able to produce DMSP, in contrast to species in the tetraploid clade. Thus, examination of DMSP synthesis in a phylogenetic context implicated a single origin of this physiological innovation, which occurred in the ancestor of the hexaploid Spartina lineage, 3-6MYA. Candidate genes specific to the Spartina DMSP biosynthesis pathway were also retrieved from Spartina transcriptomes, and provide a framework for future investigations to decipher the molecular mechanisms involved in this plant phenotypic novelty that has major ecological impacts in saltmarsh ecosystems.

Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590.

l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli. The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.

Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase.

AAA+ ATPases are ubiquitous proteins that employ the energy obtained from ATP hydrolysis to remodel proteins, DNA, or RNA. The MoxR family of AAA+ proteins is widespread throughout bacteria and archaea but is largely uncharacterized. Limited work with specific members has suggested a potential role as molecular chaperones involved in the assembly of protein complexes. As part of an effort aimed at determining the function of novel AAA+ chaperones in Escherichia coli, we report the characterization of a representative member of the MoxR family, YieN, which we have renamed RavA (regulatory ATPase variant A). We show that the ravA gene exists on an operon with another gene encoding a protein, YieM, of unknown function containing a Von Willebrand Factor Type A domain. RavA expression is under the control of the sigmaS transcription factor, and its levels increase toward late log/early stationary phase, consistent with its possible role as a general stress-response protein. RavA functions as an ATPase and forms hexameric oligomers. Importantly, we demonstrate that RavA interacts strongly with inducible lysine decarboxylase (LdcI or CadA) forming a large cage-like structure consisting of two LdcI decamers linked by a maximum of five RavA oligomers. Surprisingly, the activity of LdcI does not appear to be affected by binding to RavA in a number of in vitro and in vivo assays, however, complex formation results in the stimulation of RavA ATPase activity. Data obtained suggest that the RavA-LdcI interaction may be important for the regulation of RavA activity against its targets.

Specific antiserum and monoclonal antibodies against the taurine biosynthesis enzyme cysteine sulfinate decarboxylase: identity of brain and liver enzyme.

Cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme for taurine, was purified 1,800-fold with a 1% yield from rat liver, where it was found to be 20-fold enriched compared with brain. The final fraction was homogeneous, as ascertained through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase HPLC. An antiserum was raised in the rabbit that (a) quantitatively immunoprecipitated CSD activity and (b) immunolabeled only one band (MW = 51,000) on an immunoblot from liver homogenate. Monoclonal antibodies were also raised that recognized the CSD protein and immunolabeled the same 51-kilodalton protein on an immunoblot from liver homogenate. In a brain extract, two CSD activities had been previously found and named CSDI and CSDII, according to their chromatographic elution patterns. We have compared the properties of CSDI from brain--the most likely enzyme involved in the biosynthesis of taurine in the brain, according to previous investigations-and CSD from liver: Both activities (a) were similarly eluted on ion-exchange and hydroxyapatite chromatographies, (b) showed the same elution pattern on gel filtration with an apparent native molecular weight of approximately 63,000, and (c) were immunoprecipitated in a strictly identical manner by the antiserum against liver CSD. Moreover, this antiserum as well as the monoclonal antibodies immunolabeled a single band (51 kilodaltons) on an immunoblot from brain CSD-enriched fraction or liver fraction. All these data show that CSDI from brain and liver CSD are the same monomeric enzyme. They also indicate that a specific antiserum against rat liver CSD has been raised that can be used for immunocytochemical visualization of CSD-containing cells in the brain.