Lowering pH optimum of activity of SshEstI, a slightly alkaliphilic archaeal esterase of the hormone-sensitive lipase family.

SshEstI, a carboxylesterase from the thermoacidophilic archaeon Saccharolobus shibatae, is a member of the hormone-sensitive lipase family that displays slightly alkaliphilic activity with an optimum activity at pH 8.0. In this study, three distinct strategies were explored to confer acidophilic properties to SshEstI. The first strategy involved engineering the oxyanion hole by replacing Gly81 with serine or aspartic acid. The G81S mutant showed optimum activity at pH 7.0, whereas the aspartic acid mutant (G81D) rendered the enzyme slightly acidophilic with optimum activity observed at pH 6.0; however, kcat and kcat/Km values were reduced by these substitutions. The second strategy involved examining the effects of surfactant additives on the pH-activity profiles of SshEstI. The results showed that cetyltrimethylammonium bromide (CTAB) enhanced wild-type enzyme (WT) activity at acidic pH values. In the presence of 0.1 mM CTAB, G81S and G81D were acidophilic enzymes with optimum activity at pH 6.0 and 4.0, respectively, although their enzyme activities were low. The third strategy involved engineering the active site to resemble that of kumamolisin-As (kuma-As), an acidophilic peptidase of the sedolisin family. The catalytic triad of kuma-As was exchanged into SshEstI using site-directed mutagenesis. X-ray crystallographic analysis of the mutants (H274D and H274E) revealed that the potential hydrogen donor-acceptor distances around the active site of WT were fully maintained in these mutants. However, these mutants were inactive at pH 4-8.

Structural insights into catalytic promiscuity of chalcone synthase from Glycine max (L.) Merr.: Coenzyme A-induced alteration of product specificity.

Catalytic promiscuity of enzymes plays a pivotal role in driving the evolution of plant specialized metabolism. Chalcone synthase (CHS) catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC), a common precursor of plant flavonoids, from p-coumaroyl-coenzyme A (-CoA) and three malonyl-CoA molecules. CHS has promiscuous product specificity, producing a significant amount of p-coumaroyltriacetic lactone (CTAL) in vitro. However, mechanistic aspects of this CHS promiscuity remain to be clarified. Here, we show that the product specificity of soybean CHS (GmCHS1) is altered by CoA, a reaction product, which selectively inhibits THC production (IC50, 67 µM) and enhances CTAL production. We determined the structure of a ternary GmCHS1/CoA/naringenin complex, in which CoA is bound to the CoA-binding tunnel via interactions with Lys55, Arg58, and Lys268. Replacement of these residues by alanine resulted in an enhanced THC/CTAL production ratio, suggesting the role of these residues in the CoA-mediated alteration of product specificity. In the ternary complex, a mobile loop ("the K-loop"), which contains Lys268, was in a "closed conformation" placing over the CoA-binding tunnel, whereas in the apo and binary complex structures, the K-loop was in an "open conformation" and remote from the tunnel. We propose that the production of THC involves a transition of the K-loop conformation between the open and closed states, whereas synthesis of CTAL is independent of it. In the presence of CoA, an enzyme conformer with the closed K-loop conformation becomes increasingly dominant, hampering the transition of K-loop conformations to result in decreased THC production and increased CTAL production.

Cell-free translation system with artificial lipid-monolayer particles as a unique tool for characterizing lipid-monolayer binding proteins.

Methods for functional analysis of proteins specifically localizing to lipid monolayers such as rubber particles and lipid droplets are limited. We have succeeded in establishing a system in which artificially prepared lipid monolayer particles are added to a cell-free translation system to confirm the properties of proteins that specifically bind to lipid monolayers in a translation-coupled manner.

Structural-Functional Correlations between Unique N-terminal Region and C-terminal Conserved Motif in Short-chain cis-Prenyltransferase from Tomato.

Neryl diphosphate (C10) synthase (NDPS1), a homodimeric soluble cis-prenyltransferase from tomato, contains four disulfide bonds, including two inter-subunit S-S bonds in the N-terminal region. Mutagenesis studies demonstrated that the S-S bond formation affects not only the stability of the dimer but also the catalytic efficiency of NDPS1. Structural polymorphs in the crystal structures of NDPS1 complexed with its substrate and substrate analog were identified by employing massive data collections and hierarchical clustering analysis. Heterogeneity of the C-terminal region, including the conserved RXG motifs, was observed in addition to the polymorphs of the binding mode of the ligands. One of the RXG motifs covers the active site with an elongated random coil when the ligands are well-ordered. Conversely, the other RXG motif was located away from the active site with a helical structure. The heterogeneous C-terminal regions suggest alternating structural transitions of the RXG motifs that result in closed and open states of the active sites. Site-directed mutagenesis studies demonstrated that the conserved glycine residue cannot be replaced. We propose that the putative structural transitions of the order/disorder of N-terminal regions and the closed/open states of C-terminal regions may cooperate and be important for the catalytic mechanism of NDPS1.

Some nutritional and metabolic aspects of calves maintained under two different dietary protein and experimentally infected with Haemonchus placei / Alguns aspectos nutricionais e metabólicos de bezerros mantidos com dois níveis diferentes de proteína na dieta e infectados experimentalmente com Haemonchus placei

The experiment was carried out to study the effects of the dietary protein and the infection with H. placei on nutritional parameters in six-months-old calves, based on their response to an infection of 100,000 infective larvae. Treatments consisted of two protein levels, 97.8 (G1) and 175.3 (G2) g of crude protein per kg of dry matter. After three monthsreceiving the diet, the animals were infected. Five days prior and 30 d after infection, animals were kept in metabolic cages to measure diet digestibility, water and N balance. Thirty-five days after infection, animals were slaughtered, abomasum removed and the worm burdens counted. There were no statistical differences in live weight changes, butanimals from G1 showed a tendency to decrease weight after infection. The protein level in diet showed an effect (p 0.01) on plasma, urea and protein. The infection affected (p 0.05) haematocrit, haemoglobin, albumin and total plasma protein. Crude protein digestibility was lower (p 0.05) in G1. Dry matter and crude protein digestibilities werenot affected by infection. Nitrogen balance was lower for G1 and urine nitrogen excretion was increased (p 0.01) by infection. There was no effect on water balance. It is suggested that some of the nutritive parameters studied were affected by protein level in diet, as well as by the infection with H. placei.

O presente experimento teve por objetivo o estudo dos parâmetros nutricionais em bezerros mantidos sob dietas com dois níveis protéicos e infectados com 100.000 larvas infectantes de H. placei. Os animais do grupo 1 (G1) receberam uma dieta com 97,8 g de proteína bruta (PB) por kg de matéria seca (MS) e os do grupo 2 (G2), 175,3 g PB kg 1 MS.A infecção foi realizada três meses após o início da dieta. Cinco dias antes e 30 dias após a infecção, os animais foram colocados em gaiolas metabólicas para estudos de digestibilidade e balanços hídrico e de nitrogênio. Trinta e cinco dias pós-infecção os bezerros foram abatidos e os vermes recuperados. A variação de peso nos dois grupos não foi estatisticamente diferente (p > 0,05) e houve uma tendência, nos animais do G1, a reduzir o ganho de peso após a infecção. O nível protéico da dieta teve efeito significativo (p 0,01) nos teores de uréia e proteína plasmática; por outro lado, a infecção afetou os teores de hematócrito, hemoglobina, albumina e proteína total (p 0,05). Adigestibilidade aparente da PB foi inferior (p 0,05) no G1 e a digestibilidade da MS e PB não foram afetadas pela infecção (p > 0,05). O balanço de nitrogênio foi inferior no G1 (p 0,01) e a infecção alterou a excreção de nitrogênio via urina (p 0,01). O balanço hídrico não mostrou significância (p > 0,05) para as fontes de variação