Subject(s)
Calcium Signaling/drug effects , Calcium/metabolism , Cell Nucleus/drug effects , Cyclopentanes/chemistry , Cytosol/drug effects , Nicotiana/chemistry , Plant Growth Regulators/chemistry , Cell Compartmentation , Cell Nucleus/metabolism , Cells, Cultured , Cyclopentanes/pharmacology , Cytosol/metabolism , Fatty Acids, Unsaturated/chemistry , Fatty Acids, Unsaturated/pharmacology , Inflammation Mediators/chemistry , Inflammation Mediators/pharmacology , Oxylipins , Plant Growth Regulators/pharmacologyABSTRACT
Sequence analysis of the metabolically rich 8.7-Mbp genome of the model actinomycete Streptomyces coelicolor A3(2) revealed three genes encoding predicted type III polyketide synthases (PKSs). We report the inactivation, expression, and characterization of the type III PKS homologous SCO1206 gene product as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). Incubation of recombinant THNS with malonyl-CoA showed THN production, as demonstrated by UV and HPLC analyses. The K(m) value for malonyl-CoA and the k(cat) value for THN synthesis were determined spectrophotometrically to be 3.58+/-0.85 micro M and 0.48+/-0.03 min(-1), respectively. The C-terminal region of S. coelicolor THNS, which is longer than most other bacterial and plant type III PKSs, was shortened by 25 amino acid residues and the resulting mutant was shown to be slightly more active (K(m)=1.97+/-0.19 micro M, k(cat)=0.75+/-0.04 min(-1)) than the wild-type enzyme.
Subject(s)
Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Naphthols/metabolism , Streptomyces/genetics , Streptomyces/metabolism , Acyltransferases/genetics , Acyltransferases/metabolism , Amino Acid Sequence , Industrial Microbiology , Kinetics , Molecular Sequence Data , Mutagenesis , Phylogeny , Pigmentation/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Although phenylpropanoids and flavonoids are common plant natural products, these major classes of biologically active secondary metabolites are largely absent from bacteria. The ubiquitous plant enzymes phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) are key biosynthetic catalysts in phenylpropanoid and flavonoid assembly, respectively. Until recently, few bacterial counterparts were known, thus reflecting the dearth of these plant natural products in bacteria. This review highlights our progress on the biochemical and genetic characterization of recently identified streptomycete biosynthetic pathways to benzoic acid and type III polyketide synthase (PKS)-derived products. The sediment-derived bacterium "Streptomyces maritimus" produces benzoyl-CoA in a plant-like manner from phenylalanine involving a PAL-mediated reaction through cinnamic acid during the biosynthesis of the polyketide antibiotic enterocin. All but one of the genes encoding benzoyl-CoA biosynthesis in "S. maritimus" have been cloned, sequenced, and inactivated, providing a model for benzoate biosynthesis not only in this bacterium, but in plants where benzoic acid is an important constituent of many products. The recent discovery that bacteria harbor homodimeric PKSs belonging to the plant CHS superfamily of condensing enzymes has further linked the biosynthetic capabilities of plants and bacteria. A bioinformatics approach led to the prediction that the model actinomycete Streptomyces coelicolor A3(2) contains up to three type III PKSs. Biochemical analysis of one of the recombinant type III PKSs from S. coelicolor demonstrated activity as a 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). A homology model of THNS based upon the known three-dimensional structure of CHS was constructed to explore the structural and mechanistic details of this new subclass of bacterial PKSs.
Subject(s)
Acyltransferases/metabolism , Benzoic Acid/chemistry , Chalcone/chemistry , Multienzyme Complexes/metabolism , Phenylalanine Ammonia-Lyase/metabolism , Polyketide Synthases/metabolism , Streptomyces/enzymology , Streptomyces/genetics , Acyl Coenzyme A/biosynthesis , Acyl Coenzyme A/genetics , Acyl Coenzyme A/metabolism , Bridged-Ring Compounds/chemistry , Bridged-Ring Compounds/metabolism , Catalysis , Flavonoids/metabolism , Models, Molecular , Molecular Structure , Multienzyme Complexes/classification , Phenylpropionates/metabolism , Plants/chemistry , Plants/enzymology , Polyketide Synthases/classification , Protein ConformationABSTRACT
Sponges (class Porifera) are evolutionarily ancient metazoans that populate the tropical oceans in great abundances but also occur in temperate regions and even in freshwater. Sponges contain large numbers of bacteria that are embedded within the animal matrix. The phylogeny of these bacteria and the evolutionary age of the interaction are virtually unknown. In order to provide insights into the species richness of the microbial community of sponges, we performed a comprehensive diversity survey based on 190 sponge-derived 16S ribosomal DNA (rDNA) sequences. The sponges Aplysina aerophoba and Theonella swinhoei were chosen for construction of the bacterial 16S rDNA library because they are taxonomically distantly related and they populate nonoverlapping geographic regions. In both sponges, a uniform microbial community was discovered whose phylogenetic signature is distinctly different from that of marine plankton or marine sediments. Altogether 14 monophyletic, sponge-specific sequence clusters were identified that belong to at least seven different bacterial divisions. By definition, the sequences of each cluster are more closely related to each other than to a sequence from nonsponge sources. These monophyletic clusters comprise 70% of all publicly available sponge-derived 16S rDNA sequences, reflecting the generality of the observed phenomenon. This shared microbial fraction represents the smallest common denominator of the sponges investigated in this study. Bacteria that are exclusively found in certain host species or that occur only transiently would have been missed. A picture emerges where sponges can be viewed as highly concentrated reservoirs of so far uncultured and elusive marine microorganisms.
