An Appended Domain Results in an Unusual Architecture for Malaria Parasite Tryptophanyl-tRNA Synthetase

Specific activation of amino acids by aminoacyl-tRNA synthetases (aaRSs) is essential for maintaining fidelity during protein translation. Here, we present crystal structure of malaria parasite Plasmodium falciparum tryptophanyl-tRNA synthetase (Pf-WRS) catalytic domain (AAD) at 2.6 Å resolution in complex with L-tryptophan. Confocal microscopy-based localization data suggest cytoplasmic residency of this protein. Pf-WRS has an unusual N-terminal extension of AlaX-like domain (AXD) along with linker regions which together seem vital for enzymatic activity and tRNA binding. Pf-WRS is not proteolytically processed in the parasites and therefore AXD likely provides tRNA binding capability rather than editing activity. The N-terminal domain containing AXD and linker region is monomeric and would result in an unusual overall architecture for Pf-WRS where the dimeric catalytic domains have monomeric AXDs on either side. Our PDB-wide comparative analyses of 47 WRS crystal structures also provide new mechanistic insights into this enzyme family in context conserved KMSKS loop conformations.     

Quinone and quinone methide as transient intermediates involved in the side chain hydroxylation of N-acyldopamine derivatives by soluble enzymes from Manduca sexta cuticle.

Proteins solubilized from the pharate cuticle of Manduca sexta were fractionated by ammonium sulfate precipitation and activated by the endogenous enzymes. The activated fraction readily converted exogenously supplied N-acetyldopamine (NADA) to N-acetylnorepinephrine (NANE). Either heat treatment (70 degrees C for 10 min) or addition of phenylthiourea (2.5 microM) caused total inhibition of the side chain hydroxylation. If chemically prepared NADA quinone was supplied instead of NADA to the enzyme solution containing phenylthiourea, it was converted to NANE. Presence of a quinone trap such as N-acetylcysteine in the NADA-cuticular enzyme reaction not only prevented the accumulation of NADA quinone, but also abolished NANE production. In such reaction mixtures, the formation of a new compound characterized as NADA-quinone-N-acetylcysteine adduct could be readily witnessed. These studies indicate that NADA quinone is an intermediate during the side chain hydroxylation of NADA by Manduca cuticular enzyme(s). Since such a conversion calls for the isomerization of NADA quinone to NADA quinone methide and subsequent hydration of NADA quinone methide, attempts were also made to trap the latter compound by performing the enzymatic reaction in methanol. These attempts resulted in the isolation of beta-methoxy NADA (NADA quinone methide methanol adduct) as an additional product. Similarly, when the N-beta-alanyldopamine (NBAD)-Manduca enzyme reaction was carried out in the presence of L-kynurenine, two diastereoisomers of NBAD quinone methide-kynurenine adduct (= papiliochrome IIa and IIb) could be isolated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a new phenoloxidase inhibitor from the cuticle of Manduca sexta

Melanin, the phenolic biopolymer that serves as a skin- and hair pigment-protecting agent against harmful solar radiation and a free radical trap, is biosynthesized in animals mainly by the action of tyrosinase also known as phenoloxidase. Regulation of tyrosinase and hence melanogenesis is vital for all animals. In this report, we present the isolation and characterization of a new, heat-labile glycoprotein inhibitor of phenoloxidase from the larvae of Manduca sexta. The inhibitor was isolated from the live larval cuticle by buffer extraction and purified to homogeneity employing ammonium sulfate precipitation, dialysis, and concanavalin A-Sepharose chromatography. It migrated with a molecular weight of 380,000 on SDS-PAGE gels and inhibited the activity of insect and plant as well as fungal phenoloxidases. Inhibitor formed a tight complex with phenoloxidases, which resisted dissociation even by 1% Triton X-100 or SDS. Selective inhibition of phenoloxidase, while acting on certain but not all different substrates, was observed. The physiological importance of this newly discovered high-molecular-weight phenoloxidase inhibitor is discussed.     

Serum amyloid A, an acute-phase protein, modulates proteoglycan synthesis in cultured murine peritoneal macrophages.

Cultured peritoneal macrophages obtained from azocasein-injected mice were found to produce several fold more cell-associated and medium proteoglycans than peritoneal macrophages from untreated mice. Since serum amyloid A (an acute-phase protein) is also upregulated following injections of azocasein, we questioned whether its production was the immediate agent stimulating proteoglycan formation. Cultured peritoneal macrophages from untreated mice were then incubated with varying concentrations of SAA, resulting in a similar dose-dependent several fold increase in proteoglycan production. Of particular note was a disproportionate increase in cell-associated heparan sulfate proteoglycans in both experimental groups and of dermatan sulfate and chondroitin sulfate proteoglycans when cells were incubated in the presence of SAA in the culture medium. These results indicate a potentially important function of SAA in directing specific modifications in inflammatory conditions where increase in macrophage proteoglycans may play direct roles.     

Structural and functional analysis of the anti-malarial drug target prolyl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRSs) drive protein translation in cells and hence these are essential enzymes across life. Inhibition of these enzymes can halt growth of an organism by stalling protein translation. Therefore, small molecule targeting of aaRS active sites is an attractive avenue from the perspective of developing anti-infectives. Febrifugine and its derivatives like halofuginone (HF) are known to inhibit prolyl-tRNA synthetase of malaria parasite Plasmodium falciparum. Here, we present functional and crystallographic data on P. falciparum prolyl-tRNA synthetase (PfPRS). Using immunofluorescence data, we show that PfPRS is exclusively resident in the parasite cytoplasm within asexual blood stage parasites. The inhibitor HF interacts strongly with PfPRS in a non-competitive binding mode in presence or absence of ATP analog. Intriguingly, the two monomers that constitute dimeric PfPRS display significantly different conformations in their active site regions. The structural analyses presented here provide a framework for development of febrifugine derivatives that can seed development of new anti-malarials.     

Molecular mechanisms for mammalian melanogenesis. Comparison with insect cuticular sclerotization.

Melanogenesis is an important biochemical process for the production of skin pigments which protect many animals from the damage of solar radiation. The abnormalities in melanogenesis are associated with albinism, vitiligo, as well as malignant melanoma in humans. In the lower forms of animals viz., insects, the exoskeleton is hardened to protect their soft bodies by a process called sclerotization, which is often accompanied by melanization. Recent advances in the biochemistry of sclerotization and melanization reveal remarkable similarity between these two processes. The seven stages of sclerotization are: (a) enzymatic oxidation of N-acyldopamine, (b) Michael-1,4-addition reactions of N-acyldopamine quinone, (c) tautomerization of quinone to quinone methide, (d) Michael-1,6-addition of quinone methides, (e) tautomerization of N-acyldopamine quinone methide to 1,2-dehydro-N-acyldopamine, (f) enzymatic oxidation of 1,2-dehydro-N-acyldopamine, and (g) the reactions of resultant quinonoid compounds. Amazingly, striking similarities in the reaction sequences are found in the melanization process starting from dopa. These comparisons predict a central role for quinone methides as reactive intermediates during melanization. Accordingly, recent studies provide increasing evidence in favor of this proposition.