Crystal Structure and Substrate Specificity of Drosophila 3,4-Dihydroxyphenylalanine Decarboxylase

3,4-Dihydroxyphenylalanine decarboxylase (DDC), also known as aromatic L-amino acid decarboxylase, catalyzes the decarboxylation of a number of aromatic L-amino acids. Physiologically, DDC is responsible for the production of dopamine and serotonin through the decarboxylation of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively. In insects, both dopamine and serotonin serve as classical neurotransmitters, neuromodulators, or neurohormones, and dopamine is also involved in insect cuticle formation, eggshell hardening, and immune responses.In this study, we expressed a typical DDC enzyme from Drosophila melanogaster, critically analyzed its substrate specificity and biochemical properties, determined its crystal structure at 1.75 Angstrom resolution, and evaluated the roles residues T82 and H192 play in substrate binding and enzyme catalysis through site-directed mutagenesis of the enzyme. Our results establish that this DDC functions exclusively on the production of dopamine and serotonin, with no activity to tyrosine or tryptophan and catalyzes the formation of serotonin more efficiently than dopamine.The crystal structure of Drosophila DDC and the site-directed mutagenesis study of the enzyme demonstrate that T82 is involved in substrate binding and that H192 is used not only for substrate interaction, but for cofactor binding of drDDC as well. Through comparative analysis, the results also provide insight into the structure-function relationship of other insect DDC-like proteins

Current Advances on Structure-Function Relationships of Pyridoxal 5′-Phosphate-Dependent Enzymes

Pyridoxal 5′-phosphate (PLP) functions as a coenzyme in many enzymatic processes, including decarboxylation, deamination, transamination, racemization, and others. Enzymes, requiring PLP, are commonly termed PLP-dependent enzymes, and they are widely involved in crucial cellular metabolic pathways in most of (if not all) living organisms. The chemical mechanisms for PLP-mediated reactions have been well elaborated and accepted with an emphasis on the pure chemical steps, but how the chemical steps are processed by enzymes, especially by functions of active site residues, are not fully elucidated. Furthermore, the specific mechanism of an enzyme in relation to the one for a similar class of enzymes seems scarcely described or discussed. This discussion aims to link the specific mechanism described for the individual enzyme to the same types of enzymes from different species with aminotransferases, decarboxylases, racemase, aldolase, cystathionine β-synthase, aromatic phenylacetaldehyde synthase, et al. as models. The structural factors that contribute to the reaction mechanisms, particularly active site residues critical for dictating the reaction specificity, are summarized in this review

Kynurenine aminotransferase 3/glutamine transaminase L/cysteine conjugate beta-lyase 2 is a major glutamine transaminase in the mouse kidney

AbstractBackgroundKynurenine aminotransferase 3 (KAT3) catalyzes the transamination of Kynurenine to kynurenic acid, and is identical to cysteine conjugate beta-lyase 2 (CCBL2) and glutamine transaminase L (GTL). GTL was previously purified from the rat liver and considered as a liver type glutamine transaminase. However, because of the substrate overlap and high sequence similarity of KAT3 and KAT1, it was difficult to assay the specific activity of each KAT and to study the enzyme localization in animals.MethodsKAT3 transcript and protein levels as well as enzyme activity in the liver and kidney were analyzed by regular reverse transcription-polymerase chain reaction (RT-PCR), real time RT-PCR, biochemical activity assays combined with a specific inhibition assay, and western blotting using a purified and a highly specific antibody, respectively.ResultsThis study concerns the comparative biochemical characterization and localization of KAT 3 in the mouse. The results showed that KAT3 was present in both liver and kidney of the mouse, but was much more abundant in the kidney than in the liver. The mouse KAT3 is more efficient in transamination of glutamine with indo-3-pyruvate or oxaloacetate as amino group acceptor than the mouse KAT1.ConclusionsMouse KAT3 is a major glutamine transaminase in the kidney although it was named a liver type transaminase.General significanceOur data highlights KAT3 as a key enzyme for studying the nephrotoxic mechanism of some xenobiotics and the formation of chemopreventive compounds in the mouse kidney. This suggests tissue localizations of KAT3/GTL/CCBL2 in other animals may be carefully checked

Inhibition of Aflatoxin Synthesis in Aspergillus flavus by Three Structurally Modified Lentinans

The chemical properties of β-glucans leading to their inhibition on aflatoxin (AF) production by Aspergillus flavus remain unclear. In this study, structurally modified lentinan derivatives were prepared by carboxymethylation, sulfation, and phosphorylation to explore their inhibition activity to AF synthesis. The results demonstrated that inhibitory activity of lentinan decreased at higher or lower concentrations than 200 μg/mL. Compared with lentinan, the sulphated derivatives only performed a reduced optimal inhibition rate at a higher concentration. The phosphorylated derivatives achieved complete inhibition of AF production at 50 μg/mL, but the inhibitory activity was attenuated with an increase of concentration. The minimum concentration of carboxymethylated derivatives to completely inhibit AF synthesis was the same as that of the original lentinan, whereas their inhibition activity was not reduced at the increasing concentration. RT-PCR analyses were conducted to understand the effects of lentinan and its carboxymethylated derivatives on the transcription of certain genes associated with AF biosynthesis. The results showed that lentinan delayed the transcription of aflQ, whereas its carboxymethylated derivatives promoted the transcriptions of all the tested genes. Our results revealed that some chemical group features apart from the β-bond could play the vital role in the prevention of AF formation by polysaccharide, and highlighted the structural modifications which could promote its practicability in the control of aflatoxin contamination

Identification of Drosophila melanogaster yellow-f and yellow-f2 proteins as dopachrome-conversion enzymes.

This study describes the identification of Drosophila yellow-f and yellow-f2 as dopachrome-conversion enzymes responsible for catalysing the conversion of dopachrome into 5,6-dihydroxyindole in the melanization pathway. Drosophila yellow -y gene and yellow -b, -c, -f and -f2 genes were expressed in an insect cell/baculovirus expression system and their corresponding recombinant proteins were screened for dopachrome-conversion enzyme activity. Among the yellow and yellow -related genes, the yellow -f and yellow -f2 genes were identified as the genes coding for Drosophila dopachrome-conversion enzyme based on the high activity of their recombinant proteins in catalysing the production of 5,6-dihydroxyindole from dopachrome. Both yellow-f and yellow-f2 are capable of mediating a decarboxylative structural rearrangement of dopachrome, as well as an isomerization/tautomerization of dopamine chrome and dopa methyl ester chrome. Northern hybridization revealed the transcription of yellow -f in larvae and pupae, but a high abundance of mRNA was observed in later larval and early pupal stages. In contrast, yellow-f2 transcripts were present at all stages, but high abundance of its mRNA was observed in later-stage pupae and adults. These data indicate that yellow-f and yellow-f2 complement each other during Drosophila development and that the yellow-f is involved in larval and pupal melanization, and yellow-f2 plays a major role in melanization reactions in Drosophila during later pupal and adult development. Results from this study provide the groundwork towards a better understanding of the physiological roles of the Drosophila yellow gene family

The mitochondrial genomes of the Geometroidea (Lepidoptera) and their phylogenetic implications

Abstract The Geometroidea is a large superfamily of Lepidoptera in species composition and contains numerous economically important pest species that cause great loss in crop and forest production. However, understanding of mitogenomes remains limited due to relatively fewer mitogenomes previously reported for this megadiverse group. Here, we sequenced and annotated nine mitogenomes for Geometridae and further analyzed the mitogenomic evolution and phylogeny of the whole superfamily. All nine mitogenomes contained 37 mitochondrial genes typical in insects, and gene organization was conserved except for Somatina indicataria. In S. indicataria, the positions of two tRNAs were rearranged. The trnR was located before trnA instead of after trnA typical in Lepidoptera, whereas the trnE was detected rarely on the minority strand (N‐strand). This trnR‐trnA‐trnN‐trnS1‐trnE‐trnF newly recognized in S. indicataria represents the first gene rearrangement reported for Geometroidea and is also unique in Lepidoptera. Besides, nucleotide composition analyses showed little heterogeneity among the four geometrid subfamilies involved herein, and overall, nad6 and atp8 have higher nucleotide diversity and Ka/Ks rate in Geometridae. In addition, the taxonomic assignments of the nine species, historically defined by morphological studies, were confirmed by various phylogenetic analyses based on the hitherto most extensive mitogenomic sampling in Geometroidea

The drDDC active site.

<p>A stereo view of the active site in the drDDC structure. The LLP and the protein residues within a 4 Å distance of the cofactor are shown. Only the 2<i>F</i>_o - <i>F</i>_c electron density map covering the LLP is shown contoured at the 1.8 sigma. Hydrogen bonds are shown in dashed lines.</p

Kinetic parameters of drDDC WT and mutant proteins towards different substrates.

<p>The activities were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008826#s4" target="_blank">Materials and Methods</a>. The K_M and <i>k</i>_cat for different substrates were derived by using varying concentrations (0.1 to 12 mM)) of individual substrates. The parameters were calculated by fitting the Michaelis–Menten equation to the experimental data using the enzyme kinetics module. Results are means ± SE.</p