ABSTRACT
Taxonomy of mud crabs genus Scylla has been misidentified for several years due to their high morphological plasticity. Several reports concerning mud crab have been published with misleading identification in Bangladesh. In this study, partial fragments of nuclear and mitochondrial DNA of Scylla species obtained from four locations along the Bangladesh coast were used to resolve taxonomical ambiguity of mud crab species. A single PCR product from the nuclear first internal transcribed spacer (ITS-1) marker and phylogenetic trees constructed based on 16S rDNA sequences indicated that all Scylla species obtained in this study were S. olivacea. Both molecular data and morphological characters revealed that S. olivacea is the only major species in Bangladesh coastal waters. Further, the 16S rDNA haplotypes significantly differed with known S. serrata by 33%. From this study it is clear that 'S. serrata' commonly reported from Bangladesh should be S. olivacea.
Subject(s)
Brachyura/genetics , Phylogeny , Animals , Bangladesh , Brachyura/classification , Cell Nucleus/genetics , Genetic Markers , Mitochondria/genetics , Polymerase Chain ReactionABSTRACT
Aquatic crustaceans and some bivalve mollusks contain a large amount of free D-alanine (up to 100 mumol/g wet wt.) in their tissues. Under high salinity stress, crustaceans and bivalve mollusks largely accumulate D- and L-alanine irrespective of species examined, together with L-glutamine, L-proline, and glycine of which increases are species dependent. These data indicate that D-alanine is one of the major compatible osmolytes responsible for the intracellular isosmotic regulation in the tissues of crustaceans and bivalves. Alanine racemase has been proven to catalyze the interconversion of D- and L-alanine in these invertebrates. The enzyme has been isolated to homogeneity from the muscle of black tiger prawn Penaeus monodon and its cDNA has been cloned from the muscle and hepatopancreas of kuruma prawn Penaeus japonicus for the first time in eukaryotes other than yeast. Several fish species fed on crustaceans and mollusks contain D-amino acid and D-aspartate oxidases that catalyze the decomposition of D-amino acids. A cDNA of D-amino acid oxidase has been cloned from the hepatopancreas of omnivorous common carp Cyprinus carpio. During oral administration of free D-alanine to carp, the activity and mRNA of D-amino acid oxidase increased rapidly in hepatopancreas and the increases were highest in intestine followed by hepatopancreas and kidney. These data suggest that D-amino acid oxidase is inducible in carp and an important enzyme responsible for the efficient utilization of carbon skeleton of D-alanine in their feeds.
Subject(s)
Alanine/metabolism , Alanine/physiology , Fishes/metabolism , Invertebrates/metabolism , Alanine/biosynthesis , Animals , Hypoxia/metabolism , Osmolar ConcentrationABSTRACT
D-amino acid oxidase of carp (Cyprinus carpio) hepatopancreas was overexpressed in Escherichia coli cells and purified to homogeneity for the first time in animal tissues other than pig kidney. The purified preparation had a specific activity of 293 units mg(-1) protein toward D-alanine as a substrate. It showed the highest activity toward D-alanine with a low Km of 0.23 mM and a high kcat of 190 s(-1) compared to 10 s(-1) of the pig kidney enzyme. Nonpolar and polar uncharged D-amino acids were preferable substrates to negatively or positively charged amino acids. The enzyme exhibited better thermal and pH stabilities than several yeast counterparts or the pig kidney enzyme. Secondary structure topology consisted of 11 alpha-helices and 17 beta-strands that differed slightly from pig kidney and Rhodotorula gracilis enzymes. A three-dimensional model of the carp enzyme constructed from a deduced amino acid sequence resembled that of pig kidney D-amino acid oxidase but with a shorter active site loop and a longer C-terminal loop. Judging from these characteristics, carp D-amino acid oxidase is close to the pig kidney enzyme structurally, but analogous to the R. gracilis enzyme enzymatically in turnover rate and pH and temperature stabilities.
Subject(s)
Carps/metabolism , D-Amino-Acid Oxidase/chemistry , Hepatopancreas/enzymology , Alanine/chemistry , Alanine/metabolism , Animals , Cloning, Molecular , D-Amino-Acid Oxidase/antagonists & inhibitors , D-Amino-Acid Oxidase/genetics , Enzyme Inhibitors/pharmacology , Enzyme Stability , Escherichia coli/enzymology , Escherichia coli/genetics , Hydrogen-Ion Concentration , Protein Conformation , Protein Structure, Secondary , Substrate Specificity , TemperatureABSTRACT
A full-length cDNA encoding D-amino acid oxidase (DAO, EC 1.4.3.3) was cloned and sequenced from the hepatopancreas of carp fed a diet supplemented with D-alanine. This clone contained an open reading frame encoding 347 amino acid residues. The deduced amino acid sequence exhibited about 60 and 19-29% identity to mammalian and microbial DAOs, respectively. The expression of full-length carp DAO cDNA in Escherichia coli resulted in a significant level of protein with DAO activity. In carp fed the diet with D-alanine for 14 days, DAO mRNA was strongly expressed in intestine followed by hepatopancreas and kidney, but not in muscle. During D-alanine administration, DAO gene was expressed quickly in hepatopancreas with the increase of DAO activity. The inducible nature of carp DAO indicates that it plays an important physiological role in metabolizing exogenous D-alanine that is abundant in their prey invertebrates, crustaceans, and mollusks.
Subject(s)
Alanine/administration & dosage , Carps/genetics , Carps/metabolism , D-Amino-Acid Oxidase/chemistry , D-Amino-Acid Oxidase/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Sequence Alignment , Administration, Oral , Amino Acid Oxidoreductases/chemistry , Amino Acid Oxidoreductases/classification , Amino Acid Oxidoreductases/genetics , Amino Acid Oxidoreductases/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , Conserved Sequence , D-Amino-Acid Oxidase/classification , D-Amino-Acid Oxidase/genetics , D-Aspartate Oxidase , Dose-Response Relationship, Drug , Enzyme Activation , Hepatopancreas/chemistry , Hepatopancreas/drug effects , Hepatopancreas/enzymology , Humans , Mice , Molecular Sequence Data , Organ Specificity , Recombinant Proteins/chemistry , Recombinant Proteins/classification , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Species Specificity , SwineABSTRACT
The distributions of D-amino acid oxidase (D-AAO, EC 1.4.3.3) and D-aspartate oxidase (D-AspO, EC 1.4.3.1) activities were examined on several tissues of various fish species. Both enzyme activities were commonly high in kidney and liver and low in intestine with some exceptions. After oral administration of D-alanine at 5 micromol /g body weight(-1)day(-1) to carp for 30 days, D-AAO activity increased by about 8-, 3-, and 1.5-fold in intestine, hepatopancreas, and kidney, respectively, whereas no increase was found in brain. In contrast, oral administration of D-glutamate or D-aspartate did not show any increase of D-AspO activity in any tissues. D-AAO and D-AspO of common carp kidney and hepatopancreas were subcellularly localized in peroxisomes, as clarified in mammals. D-proline was the best substrate for D-AAO in rainbow trout kidney, common carp kidney, and hepatopancreas, followed by D-alanine and D-phenylalanine. N-methyl-D-aspartate was the best substrate for D-AspO in rainbow trout kidney and common carp hepatopancreas. The optimal pH for D-AAO in rainbow trout kidney was broad, from 7.4 to 8.2, and that for D-AspO was around 10. D-AAO was inhibited by benzoate known as D-AAO inhibitor and D-AspO was strongly inhibited by meso-tartarate as D-AspO inhibitor. From these results, at least D-AAO in fish is considered to work as a metabolizing agent of exogenous and endogenous free D-alanine that is abundant in aquatic invertebrates such as crustaceans and bivalve mollusks, which are potential food sources of these fishes.
