Search Results (18)

Ornithine aminotransferase (OAT) is overexpressed in hepatocellular carcinoma (HCC), and we previously showed that inactivation of OAT inhibits the growth of HCC. Recently, we found that (3S,4S)-3-amino-4-fluorocyclopentenecarboxylic acid (5) was a potent inactivator of γ-aminobutyric acid aminotransferase (GABA-AT), proceeding by an enamine mechanism. Here we describe our investigations into the activity and mechanism of 5 as an inactivator of human OAT. We have found that 5 exhibits 10-fold less inactivation efficiency (k_inact/K_I) against hOAT than GABA-AT. A comprehensive mechanistic study was carried out to understand its inactivation mechanism with hOAT. pK_a and electrostatic potential calculations were performed to further support the notion that the α,β-unsaturated alkene of 5 is critical for enhancing acidity and nucleophilicity of the corresponding intermediates and ultimately responsible for the improved inactivation efficiency of 5 over the corresponding saturated analogue (4). Intact protein mass spectrometry and the crystal structure complex with hOAT provide evidence to conclude that 5 mainly inactivates hOAT through noncovalent interactions, and that, unlike with GABA-AT, covalent binding with hOAT is a minor component of the total inhibition which is unique relative to other monofluoro-substituted derivatives. Furthermore, based on the results of transient-state measurements and free energy calculations, it is suggested that the α,β-unsaturated carboxylate group of PLP-bound 5 may be directly involved in the inactivation cascade by forming an enolate intermediate. Overall, compound 5 exhibits unusual structural conversions which are catalyzed by specific residues within hOAT, ultimately leading to an enamine mechanism-based inactivation of hOAT through noncovalent interactions and covalent modification. Full article

A novel peptide, Cm39, was identified in the venom of the scorpion Centruroides margaritatus. Its primary structure was determined. It consists of 37 amino acid residues with a MW of 3980.2 Da. The full chemical synthesis and proper folding of Cm39 was obtained. Based on amino acid sequence alignment with different K⁺ channel inhibitor scorpion toxin (KTx) families and phylogenetic analysis, Cm39 belongs to the α-KTx 4 family and was registered with the systematic number of α-KTx 4.8. Synthetic Cm39 inhibits the voltage-gated K⁺ channel hK_V1.2 with high affinity (K_d = 65 nM). The conductance–voltage relationship of K_V1.2 was not altered in the presence of Cm39, and the analysis of the toxin binding kinetics was consistent with a bimolecular interaction between the peptide and the channel; therefore, the pore blocking mechanism is proposed for the toxin–channel interaction. Cm39 also inhibits the Ca²⁺-activated K_Ca2.2 and K_Ca3.1 channels, with K_d = 502 nM, and K_d = 58 nM, respectively. However, the peptide does not inhibit hK_V1.1, hK_V1.3, hK_V1.4, hK_V1.5, hK_V1.6, hK_V11.1, mK_Ca1.1 K⁺ channels or the hNa_V1.5 and hNa_V1.4 Na⁺ channels at 1 μM concentrations. Understanding the unusual selectivity profile of Cm39 motivates further experiments to reveal novel interactions with the vestibule of toxin-sensitive channels. Full article

Sialidase preparations are applied in structural and functional studies on sialoglycans, in the production of sialylated therapeutic proteins and synthetic substrates for use in biochemical research, etc. They are obtained mainly from pathogenic microorganisms; therefore, the demand for apathogenic producers of sialidase is of exceptional importance for the safe production of this enzyme. Here, we report for the first time the presence of a sialidase gene and enzyme in the saprophytic actinomycete Oerskovia paurometabola strain O129. An electrophoretically pure, glycosylated enzyme with a molecular weight of 70 kDa was obtained after a two-step chromatographic procedure using DEAE cellulose and Q-sepharose. The biochemical characterization showed that the enzyme is extracellular, inductive, and able to cleave α(2→3,6,8) linked sialic acids with preference for α(2→3) bonds. The enzyme production was strongly induced by glycomacropeptide (GMP) from milk whey, as well as by sialic acid. Investigation of the deduced amino acid sequence revealed that the protein molecule has the typical six-bladed β-propeller structure and contains all features of bacterial sialidases, i.e., an YRIP motif, five Asp-boxes, and the conserved amino acids in the active site. The presence of an unusual signal peptide of 40 amino acids was predicted. The sialidase-producing O. paurometabola O129 showed high and constant enzyme production. Together with its saprophytic nature, this makes it a reliable producer with high potential for industrial application. Full article

The endogenous hemorphins are bioactive peptides with activity on opioid receptors. They are extensively studied and summarized in numerous reviews. During the last decade, several research teams have synthesized, characterized, and pharmacologically evaluated synthetic hemorphin analogs containing unusual amino acids, D-amino acids, α-aminophosphonic acids, and their derivatives. The present review summarizes the current studies on short-chain synthetic hemorphin peptide derivates containing non-natural amino acids. This review focuses on the structure–activity relationship analysis, details on specific methods for their characterization, and the advantage of synthetic hemorphin analogs compared to endogenous peptides as potent biologically active compounds with a complex mechanism of action. Full article

Pentameric ligand-gated ion channels (pLGIC) play important roles in fast neuronal signal transmission. Functional receptors are pentamers, with each subunit having an extracellular domain (ECD), a transmembrane domain (TMD) and an intracellular domain. The binding of the agonist to the ECD induces a structural change that is transduced to the TMD to open the channel. Molecular details of this process are emerging, but a comprehensive understanding is still lacking. Proline (Pro) is one amino acid that has attracted much interest; its unusual features generate bends in loops and kinks and bulges in helices, which can be essential for function in some pLGICs. Here, we explore the roles of four conserved Pros in the glycine receptor (GlyR), creating substitutions with canonical and noncanonical amino acids, characterizing them using two electrode voltage clamp electrophysiology in Xenopus oocytes, and interpreting changes in receptor parameters using structural data from the open and closed states of the receptor. The data reveal that for efficient function, the Pro in the α1β1 loop is needed to create a turn and to be the correct size and shape to interact with nearby residues; the peptide bond of the Pro in the Cys-loop requires the cis conformation; and the Pros in loop A and M1 allow efficient function because of their reduced hydrogen bonding capacity. These data are broadly consistent with data from other pLGICs, and therefore likely represent the important features of these Pros in all members of the family. Full article

A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium Exiguobacterium sibiricum from Siberian permafrost soil was cloned and expressed in Escherichia coli. The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features characteristic for the cold-active enzymes while possessing an unusually high number of proline residues in the loops—a typical feature of thermophilic enzymes. The activity of the purified recombinant protein was tested with p-nitrophenyl α-D-glucopyranoside as a substrate. The enzyme displayed a plateau-shaped temperature-activity profile with the optimum at 25 °C and a pronounced activity at low temperatures (50% of maximum activity at 5 °C). To improve the thermal stability at temperatures above 40 °C, we have introduced proline residues into four positions of EsOgl by site-directed mutagenesis according to “the proline rule”. Two of the mutants, S130P and A109P demonstrated a three- and two-fold increased half-life at 45 °C. Moreover, S130P mutation led to a 60% increase in the catalytic rate constant. Combining the mutations resulted in a further increase in stability transforming the temperature-activity profile to a typical mesophilic pattern. In the most thermostable variant A109P/S130P/E176P, the half-life at 45 °C was increased from 11 min (wild-type) to 129 min. Full article

Genetic decoding is flexible, due to programmed deviation of the ribosomes from standard translational rules, globally termed “recoding”. In Archaea, recoding has been unequivocally determined only for termination codon readthrough events that regulate the incorporation of the unusual amino acids selenocysteine and pyrrolysine, and for −1 programmed frameshifting that allow the expression of a fully functional α-l-fucosidase in the crenarchaeon Saccharolobus solfataricus, in which several functional interrupted genes have been identified. Increasing evidence suggests that the flexibility of the genetic code decoding could provide an evolutionary advantage in extreme conditions, therefore, the identification and study of interrupted genes in extremophilic Archaea could be important from an astrobiological point of view, providing new information on the origin and evolution of the genetic code and on the limits of life on Earth. In order to shed some light on the mechanism of programmed −1 frameshifting in Archaea, here we report, for the first time, on the analysis of the transcription of this recoded archaeal α-l-fucosidase and of its full-length mutant in different growth conditions in vivo. We found that only the wild type mRNA significantly increased in S. solfataricus after cold shock and in cells grown in minimal medium containing hydrolyzed xyloglucan as carbon source. Our results indicated that the increased level of fucA mRNA cannot be explained by transcript up-regulation alone. A different mechanism related to translation efficiency is discussed. Full article

Unusual α-amino acids (UAAs) are important fundamental building blocks and play a key role in medicinal chemistry. Here, we constructed a hydrogen-borrowing dual-enzyme cascade for efficient synthesis of UAAs from α-hydroxy acids (α-HAs). D-mandelate dehydrogenase from Lactobacillus brevis (LbMDH) was screened for the catalysis of α-HAs to α-keto acids but with low activity towards aliphatic α-HAs. Therefore, we rational engineered LbMDH to improve its activity towards aliphatic α-HAs. The substitution of residue Leu243 located in the substrate entrance channel with nonpolar amino acids like Met, Trp, and Ile significantly influenced the enzyme activity towards different α-HAs. Compared with wild type (WT), variant L243W showed 103 U/mg activity towards D-α-hydroxybutyric acid, 1.7 times of the WT’s 60.2 U/mg, while its activity towards D-mandelic acid decreased. Variant L243M showed 2.3 times activity towards D-mandelic acid compared to WT, and its half-life at 40 °C increased to 150.2 h comparing with 98.5 h of WT. By combining LbMDH with L-leucine dehydrogenase from Bacillus cereus, the synthesis of structurally diverse range of UAAs from α-HAs was constructed. We achieved 90.7% conversion for L-phenylglycine production and 66.7% conversion for L-α-aminobutyric acid production. This redox self-sufficient cascade provided high catalytic efficiency and generated pure products. Full article

The discovery of liquid water at several locations in the solar system raises the possibility that microbial life may have evolved outside Earth and as such could be accidently introduced into the Earth’s ecosystem. Unusual sugars or amino acids, like non-proteinogenic isovaline and α-aminoisobutyric acid that are vanishingly rare or absent from life forms on Earth, have been found in high abundance on non-terrestrial carbonaceous meteorites. It is therefore conceivable that exo-microorganisms might contain proteins that include these rare amino acids. We therefore asked whether the mammalian immune system would be able to recognize and induce appropriate immune responses to putative proteinaceous antigens that include these rare amino acids. To address this, we synthesised peptide antigens based on a backbone of ovalbumin and introduced isovaline and α-aminoisobutyric acid residues and demonstrated that these peptides can promote naïve OT-I cell activation and proliferation, but did so less efficiently than the canonical peptides. This is relevant to the biosecurity of missions that may retrieve samples from exoplanets and moons that have conditions that may be permissive for life, suggesting that accidental contamination and exposure to exo-microorganisms with such distinct proteomes might pose an immunological challenge. Full article

Synthetic oligomers and polymers inspired by the multifunctional tethering system (byssus) of the common mussel (genus Mytilus) have emerged since the 1980s as a very active research domain within the wider bioinspired and biomimetic materials arena. The unique combination of strong underwater adhesion, robust mechanical properties and self-healing capacity has been linked to a large extent to the presence of the unusual α-amino acid derivative l-DOPA (l-3,4-dihydroxyphenylalanine) as a building block of the mussel byssus proteins. This paper provides a short overview of marine biofouling, discussing the different marine biofouling species and natural defenses against these, as well as biomimicry as a concept investigated in the marine antifouling context. A detailed discussion of the literature on the Mytilus mussel family follows, covering elements of their biology, biochemistry and the specific measures adopted by these mussels to utilise their l-DOPA-rich protein sequences (and specifically the ortho-bisphenol (catechol) moiety) in their benefit. A comprehensive account is then given of the key catechol chemistries (covalent and non-covalent/intermolecular) relevant to adhesion, cohesion and self-healing, as well as of some of the most characteristic mussel protein synthetic mimics reported over the past 30 years and the related polymer functionalisation strategies with l-DOPA/catechol. Lastly, we review some of the most recent advances in such mussel-inspired synthetic oligomers and polymers, claimed as specifically aimed or intended for use in marine antifouling coatings and/or tested against marine biofouling species. Full article

Polysialic acid (polySia) is an unusual glycan that posttranslational modifies neural cell adhesion molecule (NCAM) proteins in mammalian cells. The up-regulated expression of polySia-NCAM is associated with tumor progression in many metastatic human cancers and in neurocognitive processes. Two members of the ST8Sia family of α2,8-polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST) both catalyze synthesis of polySia when activated cytidine monophosphate(CMP)-Sialic acid (CMP-Sia) is translocate into the lumen of the Golgi apparatus. Two key polybasic domains in the polySTs, the polybasic region (PBR) and the polysialyltransferase domain (PSTD) areessential forpolysialylation of the NCAM proteins. However, the precise molecular details to describe the interactions required for polysialylation remain unknown. In this study, we hypothesize that PSTD interacts with both CMP-Sia and polySia to catalyze polysialylation of the NCAM proteins. To test this hypothesis, we synthesized a 35-amino acid-PSTD peptide derived from the ST8Sia IV gene sequence and used it to study its interaction with CMP-Sia, and polySia. Our results showed for the PSTD-CMP-Sia interaction, the largest chemical-shift perturbations (CSP) were in amino acid residues V251 to A254 in the short H1 helix, located near the N-terminus of PSTD. However, larger CSP values for the PSTD-polySia interaction were observed in amino acid residues R259 to T270 in the long H2 helix. These differences suggest that CMP-Sia preferentially binds to the domain between the short H1 helix and the longer H2 helix. In contrast, polySia was principally bound to the long H2 helix of PSTD. For the PSTD-polySia interaction, a significant decrease in peak intensity was observed in the 20 amino acid residues located between the N-and C-termini of the long H2 helix in PSTD, suggesting a slower motion in these residues when polySia bound to PSTD. Specific features of the interactions between PSTD-CMP-Sia, and PSTD-polySia were further confirmed by comparing their 800 MHz-derived HSQC spectra with that of PSTD-Sia, PSTD-TriSia (DP 3) and PSTD-polySia. Based on the interactions between PSTD-CMP-Sia, PSTD-polySia, PBR-NCAM and PSTD-PBR, these findingsprovide a greater understanding of the molecular mechanisms underlying polySia-NCAM polysialylation, and thus provides a new perspective for translational pharmacological applications and development by targeting the two polysialyltransferases. Full article

Microorganisms, especially those that survive in extremely cold places such as Antarctica, have gained research attention since they produce a unique feature of the protein, such as being able to withstand at extreme temperature, salinity, and pressure, that make them desired for biotechnological application. Here, we report the first hormone-sensitive lipase (HSL)-like esterase from a Glaciozyma species, a psychrophilic yeast designated as GlaEst12-like esterase. In this study, the putative lipolytic enzyme was cloned, expressed in E. coli, purified, and characterised for its biochemical properties. Protein sequences analysis showed that GlaEst12 shared about 30% sequence identity with chain A of the bacterial hormone-sensitive lipase of E40. It belongs to the H group since it has the conserved motifs of Histidine-Glycine-Glycine-Glycine (HGGG)and Glycine-Aspartate-Serine-Alanine-Glycine (GDSAG) at the amino acid sequences. The recombinant GlaEst12 was successfully purified via one-step Ni-Sepharose affinity chromatography. Interestingly, GlaEst12 showed unusual properties with other enzymes from psychrophilic origin since it showed an optimal temperature ranged between 50–60 °C and was stable at alkaline pH conditions. Unlike other HSL-like esterase, this esterase showed higher activity towards medium-chain ester substrates rather than shorter chain ester. The 3D structure of GlaEst12, predicted by homology modelling using Robetta software, showed a secondary structure composed of mainly α/β hydrolase fold, with the catalytic residues being found at Ser²³², Glu³⁴¹, and His³⁷¹. Full article

Serine proteases constitute the major protein content of mast cell (MC) secretory granules. These proteases can generally be subdivided into chymases and tryptases based on their primary cleavage specificity. Here, we presented the extended cleavage specificities of a rabbit β-chymase and a guinea pig α-chymase. Analyses by phage display screening and a panel of recombinant substrates showed a marked similarity in catalytic activity between the enzymes, both being strict Leu-ases (cleaving on the carboxyl side of Leu). Amino acid sequence alignment of a panel of mammalian chymotryptic MC proteases and 3D structural modeling identified an unusual residue in the rabbit enzyme at position 216 (Thr instead of more common Gly), which is most likely critical for the Leu-ase specificity. Almost all mammals studied, except rabbit and guinea pig, express classical chymotryptic enzymes with similarly extended specificities, indicating an important role of chymase in MC biology. The rabbit and guinea pig are the only two mammalian species currently known to lack a classical MC chymase. Key questions are now how this major difference affects their MC function, and if genes of other loci can rescue the loss of a chymotryptic activity in MCs of these two species. Full article

Trichoderma koningiopsis and T. gamsii belong to clade Viride of Trichoderma, the largest and most diverse group of this genus. They produce a wide range of bioactive secondary metabolites, including peptaibols with antibacterial, antifungal, and antiviral properties. The unusual amino acid residues of peptaibols, i.e., α-aminoisobutyric acid (Aib), isovaline (Iva), and the C-terminal 1,2-amino alcohol make them unique among peptides. In this study, the peptaibiomes of T. koningiopsis and T. gamsii were investigated by HPLC-ESI-MS. The examined strains appeared to produce 19-residue peptaibols, most of which are unknown from literature, but their amino acid sequences are similar to those of trikoningins, tricholongins, trichostrigocins, trichorzianins, and trichorzins. A new group of peptaibols detected in T. koningiopsis are described here under the name “Koningiopsin”. Trikoningin KA V, the closest peptaibol compound to the peptaibols produced by these two strains, was selected for structural investigation by short MD simulation, which revealed that many residues show high preference for left handed helix formation. The bioactivity of the peptaibol mixtures produced by T. koningiopsis and T. gamsii was tested on agar plates against bacteria, yeasts, and filamentous fungi. The results revealed characteristic differences in bioactivities towards the different groups of target microorganisms, which can be explained with the differences in their cell wall structures. Full article

Reactive intermediate deaminase (Rid) protein family is a recently discovered group of enzymes that is conserved in all domains of life and is proposed to play a role in the detoxification of reactive enamines/imines. UK114, the mammalian member of RidA subfamily, was identified in the early 90s as a component of perchloric acid-soluble extracts from goat liver and exhibited immunomodulatory properties. Multiple activities were attributed to this protein, but its function is still unclear. This work addressed the question of whether UK114 is a Rid enzyme. Biochemical analyses demonstrated that UK114 hydrolyzes α-imino acids generated by l- or d-amino acid oxidases with a preference for those deriving from Ala > Leu = l-Met > l-Gln, whereas it was poorly active on l-Phe and l-His. Circular Dichroism (CD) analyses of UK114 conformational stability highlighted its remarkable resistance to thermal unfolding, even at high urea concentrations. The half-life of heat inactivation at 95 °C, measured from CD and activity data, was about 3.5 h. The unusual conformational stability of UK114 could be relevant in the frame of a future evaluation of its immunogenic properties. In conclusion, mammalian UK114 proteins are RidA enzymes that may play an important role in metabolism homeostasis also in these organisms. Full article