8 research outputs found
A tRNA-Dependent Two-Enzyme Pathway for the Generation of Singly and Doubly Methylated Ditryptophan 2,5-Diketopiperazines
A large
number of bioactive natural products containing a 2,5-diketopiperazine
(DKP) moiety have been isolated from various microbial sources. Especially
tryptophan-containing cyclic dipeptides (CDPs) show great structural
and functional diversity, while little is known about their biosynthetic
pathways. Here, we describe the bioinformatic analysis of a cyclodipeptide
synthase (CDPS)-containing gene cluster from <i>Actinosynnema
mirum</i> spanning 2.9 kb that contains two putative DKP-modifying
enzymes. We establish the biosynthetic pathway leading to two methylated
ditryptophan CDPs through <i>in vivo</i> and <i>in
vitro</i> analyses. Our studies identify the first CDPS (Amir_4627)
that shows high substrate specificity synthesizing only one main product,
cycloÂ(Trp-Trp) (cWW). It is the first member of the CDPS family that
can form ditryptophan DKPs and the first prokaryotic CDPS whose main
product constituents differ from the four amino acids (Phe, Leu, Tyr,
and Met) usually found in CDPS-dependent CDPs. We show that after
cWW formation a <i>S</i>-adenosyl-l-methionine-dependent <i>N</i>-methyltransferase (Amir_4628) conducts two successive
methylations at the DKP-ring nitrogens and additionally show that
it is able to methylate four other phenylalanine-containing CDPs.
This makes Amir_4628 the first identified DKP-ring-modifying methyltransferase.
The large number of known modifying enzymes of bacterial and fungal
origin known to act upon Trp-containing DKPs makes the identification
of a potent catalyst for cWW formation, encoded by a small gene, valuable
for combinatorial <i>in vivo</i> as well as chemoenzymatic
approaches, with the aim of generating derivatives of known CDP natural
products or entirely new chemical entities with potentially improved
or new biological activities
Effect of Tetraphenylborate on Physicochemical Properties of Bovine Serum Albumin
The binding interactions of bovine serum albumin (BSA) with tetraphenylborate ions ([B(Ph)4]â) have been investigated by a set of experimental methods (isothermal titration calorimetry, steady-state fluorescence spectroscopy, differential scanning calorimetry and circular dichroism spectroscopy) and molecular dynamics-based computational approaches. Two sets of structurally distinctive binding sites in BSA were found under the experimental conditions (10 mM cacodylate buffer, pH 7, 298.15 K). The obtained results, supported by the competitive interactions experiments of SDS with [B(Ph)4]â for BSA, enabled us to find the potential binding sites in BSA. The first site is located in the subdomain I A of the protein and binds two [B(Ph)4]â ions (logK(ITC)1 = 7.09 ± 0.10; ÎG(ITC)1 = â9.67 ± 0.14 kcal molâ1; ÎH(ITC)1 = â3.14 ± 0.12 kcal molâ1; TÎS(ITC)1 = â6.53 kcal molâ1), whereas the second site is localized in the subdomain III A and binds five ions (logK(ITC)2 = 5.39 ± 0.06; ÎG(ITC)2 = â7.35 ± 0.09 kcal molâ1; ÎH(ITC)2 = 4.00 ± 0.14 kcal molâ1; TÎS(ITC)2 = 11.3 kcal molâ1). The formation of the {[B(Ph)4]â}âBSA complex results in an increase in the thermal stability of the alfa-helical content, correlating with the saturation of the particular BSA binding sites, thus hindering its thermal unfolding
Applications of isothermal titration calorimetry in pure and applied research from 2016 to 2020
The last 5 years have seen a series of advances in the application of isothermal titration microcalorimetry (ITC) and interpretation of ITC data. ITC has played an invaluable role in understanding multiprotein complex formation including proteolysis-targeting chimeras (PROTACS), and mitochondrial autophagy receptor Nix interaction with LC3 and GABARAP. It has also helped elucidate complex allosteric communication in protein complexes like trp RNA-binding attenuation protein (TRAP) complex. Advances in kinetics analysis have enabled the calculation of kinetic rate constants from pre-existing ITC data sets. Diverse strategies have also been developed to study enzyme kinetics and enzyme-inhibitor interactions. ITC has also been applied to study small molecule solvent and solute interactions involved in extraction, separation, and purification applications including liquid-liquid separation and extractive distillation. Diverse applications of ITC have been developed from the analysis of protein instability at different temperatures, determination of enzyme kinetics in suspensions of living cells to the adsorption of uremic toxins from aqueous streams