scholarly journals Structural and biochemical characterization of human orphan DHRS10 reveals a novel cytosolic enzyme with steroid dehydrogenase activity

2007 ◽  
Vol 402 (3) ◽  
pp. 419-427 ◽  
Author(s):  
Petra Lukacik ◽  
Brigitte Keller ◽  
Gabor Bunkoczi ◽  
Kathryn Kavanagh ◽  
Wen Hwa Lee ◽  
...  
Keyword(s):  
Dehydrogenase Activity ◽  
Active Site ◽  
Biochemical Characterization ◽  
Functional Characterization ◽  
Significant Proportion ◽  
Tissue Expression ◽  
Intact Cells ◽  
Variable Loops ◽  
The Central Nervous System

To this day, a significant proportion of the human genome remains devoid of functional characterization. In this study, we present evidence that the previously functionally uncharacterized product of the human DHRS10 gene is endowed with 17β-HSD (17β-hydroxysteroid dehydrogenase) activity. 17β-HSD enzymes are primarily involved in the metabolism of steroids at the C-17 position and also of other substrates such as fatty acids, prostaglandins and xenobiotics. In vitro, DHRS10 converts NAD+ into NADH in the presence of oestradiol, testosterone and 5-androstene-3β,17β-diol. Furthermore, the product of oestradiol oxidation, oestrone, was identified in intact cells transfected with a construct plasmid encoding the DHRS10 protein. In situ fluorescence hybridization studies have revealed the cytoplasmic localization of DHRS10. Along with tissue expression data, this suggests a role for DHRS10 in the local inactivation of steroids in the central nervous system and placenta. The crystal structure of the DHRS10 apoenzyme exhibits secondary structure of the SDR (short-chain dehydrogenase/reductase) family: a Rossmann-fold with variable loops surrounding the active site. It also reveals a broad and deep active site cleft into which NAD+ and oestradiol can be docked in a catalytically competent orientation.

scholarly journals Molecular and functional characterization of somatostatin-type signalling in a deuterostome invertebrate

Open Biology ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 200172
Author(s):  
Ya Zhang ◽  
Luis Alfonso Yañez Guerra ◽  
Michaela Egertová ◽  
Cleidiane G. Zampronio ◽  
Alexandra M. Jones ◽  
...  
Keyword(s):  
Functional Characterization ◽  
The Body ◽  
Pharmacological Characterization ◽  
Cardiac Stomach ◽  
Physiological Processes ◽  
Pharmacological Tests ◽  
The Central Nervous System

Somatostatin (SS) and allatostatin-C (ASTC) are structurally and evolutionarily related neuropeptides that act as inhibitory regulators of physiological processes in mammals and insects, respectively. Here, we report the first molecular and functional characterization of SS/ASTC-type signalling in a deuterostome invertebrate—the starfish Asterias rubens (phylum Echinodermata). Two SS/ASTC-type precursors were identified in A. rubens (ArSSP1 and ArSSP2) and the structures of neuropeptides derived from these proteins (ArSS1 and ArSS2) were analysed using mass spectrometry. Pharmacological characterization of three cloned A. rubens SS/ASTC-type receptors (ArSSR1–3) revealed that ArSS2, but not ArSS1, acts as a ligand for all three receptors. Analysis of ArSS2 expression in A. rubens using mRNA in situ hybridization and immunohistochemistry revealed stained cells/fibres in the central nervous system, the digestive system (e.g. cardiac stomach) and the body wall and its appendages (e.g. tube feet). Furthermore, in vitro pharmacological tests revealed that ArSS2 causes dose-dependent relaxation of tube foot and cardiac stomach preparations, while injection of ArSS2 in vivo causes partial eversion of the cardiac stomach. Our findings provide new insights into the molecular evolution of SS/ASTC-type signalling in the animal kingdom and reveal an ancient role of SS-type neuropeptides as inhibitory regulators of muscle contractility.

scholarly journals Synthesis and In Vitro Screening of Novel Heterocyclic β-d-Gluco- and β-d-Galactoconjugates as Butyrylcholinesterase Inhibitors

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2833
Author(s):  
Krešimir Baumann ◽  
Lorena Kordić ◽  
Marko Močibob ◽  
Goran Šinko ◽  
Srđanka Tomić
Keyword(s):  
Active Site ◽  
Kinetic Studies ◽  
Inhibitory Effect ◽  
In Vitro Screening ◽  
Sugar Moiety ◽  
Brain Barrier Permeability ◽  
The Central Nervous System ◽  
Key Residues ◽  
Micromolar Range

The development of selective butyrylcholinesterase (BChE) inhibitors may improve the treatment of Alzheimer’s disease by increasing lower synaptic levels of the neurotransmitter acetylcholine, which is hydrolysed by acetylcholinesterase, as well as by overexpressed BChE. An increase in the synaptic levels of acetylcholine leads to normal cholinergic neurotransmission and improved cognitive functions. A series of 14 novel heterocyclic β-d-gluco- and β-d-galactoconjugates were designed and screened for inhibitory activity against BChE. In the kinetic studies, 4 out of 14 compounds showed an inhibitory effect towards BChE, with benzimidazolium and 1-benzylbenzimidazolium substituted β-d-gluco- and β-d-galacto-derivatives in a 10–50 micromolar range. The analysis performed by molecular modelling indicated key residues of the BChE active site, which contributed to a higher affinity toward the selected compounds. Sugar moiety in the inhibitor should enable better blood–brain barrier permeability, and thus increase bioavailability in the central nervous system of these compounds.

scholarly journals Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis

2015 ◽  
Vol 112 (20) ◽  
pp. 6347-6352 ◽  
Author(s):  
Bradley M. Hover ◽  
Nam K. Tonthat ◽  
Maria A. Schumacher ◽  
Kenichi Yokoyama
Keyword(s):  
Active Site ◽  
Biochemical Characterization ◽  
De Novo ◽  
Specific Binding ◽  
Molybdenum Cofactor ◽  
Enzyme Active Site ◽  
Complex Rearrangement ◽  
Cofactor Biosynthesis ◽  
Molybdenum Cofactor Biosynthesis

The molybdenum cofactor (Moco) is essential for all kingdoms of life, plays central roles in various biological processes, and must be biosynthesized de novo. During Moco biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangement of guanosine 5′-triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC (molybdenum cofactor biosynthesis protein A and C, respectively). Conventionally, MoaA was considered to catalyze the majority of this transformation, with MoaC playing little or no role in the pyranopterin formation. Recently, this view was challenged by the isolation of 3′,8-cyclo-7,8-dihydro-guanosine 5′-triphosphate (3′,8-cH2GTP) as the product of in vitro MoaA reactions. To elucidate the mechanism of formation of Moco pyranopterin backbone, we performed biochemical characterization of 3′,8-cH2GTP and functional and X-ray crystallographic characterizations of MoaC. These studies revealed that 3′,8-cH2GTP is the only product of MoaA that can be converted to cPMP by MoaC. Our structural studies captured the specific binding of 3′,8-cH2GTP in the active site of MoaC. These observations provided strong evidence that the physiological function of MoaA is the conversion of GTP to 3′,8-cH2GTP (GTP 3′,8-cyclase), and that of MoaC is to catalyze the rearrangement of 3′,8-cH2GTP into cPMP (cPMP synthase). Furthermore, our structure-guided studies suggest that MoaC catalysis involves the dynamic motions of enzyme active-site loops as a way to control the timing of interaction between the reaction intermediates and catalytically essential amino acid residues. Thus, these results reveal the previously unidentified mechanism behind Moco biosynthesis and provide mechanistic and structural insights into how enzymes catalyze complex rearrangement reactions.

scholarly journals Functional Characterization of CTX-M-14 and CTX-M-15 β-Lactamases by In Vitro DNA Shuffling

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
Kathy Hiu Laam Po ◽  
Edward Wai Chi Chan ◽  
Sheng Chen
Keyword(s):  
Amino Acid ◽  
Structural Analysis ◽  
Active Site ◽  
Target Genes ◽  
Functional Characterization ◽  
Single Amino Acid ◽  
Dna Shuffling ◽  
Β Sheet ◽  
M 14

ABSTRACT This work investigated the molecular events driving the evolution of the CTX-M-type β-lactamases by the use of DNA shuffling of fragments of the bla CTX-M-14 and bla CTX-M-15 genes. Analysis of a total of 51 hybrid enzymes showed that enzymatic activity could be maintained in most cases, yet hybrids that were active possessed fewer amino acid substitutions than those that were inactive, suggesting that point mutations in the constructs rather than reshuffling of the fragments of the two target genes would more likely cause disruption of CTX-M activity. For example, the P67L and L261P changes in a CTX-M-14 fragment could completely abolish the activity of the enzyme on all antibiotics tested. Structural analysis showed that L216 was located in the active-site β sheet and might interact with the adjacent hydrophobic residues to stabilize the active-site β sheet and maintain the integrity of the enzyme active site. Likewise, a single amino acid substitution, E64K, was found to exhibit a significant suppressive effect on CTX-M-15 activity. Structural analysis showed that E64 might form a salt bridge with R44, disruption of which might affect CTX-M-15 activity. Further analysis of the structure-function relationship of a range of mutant enzymes confirmed that, as can be expected, unstable enzymes lose their activity and avoid selective events. These findings suggest that the distal pockets could also contribute to the activity of the enzymes and may be regarded as alternative targets for inhibitor development.

scholarly journals Phenotypic and functional characterization of competitive long-term repopulating hematopoietic stem cells enriched from 5-fluorouracil- treated murine marrow

Blood ◽  
1993 ◽  
Vol 81 (9) ◽  
pp. 2310-2320 ◽  
Author(s):  
SJ Szilvassy ◽  
S Cory
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Functional Characterization ◽  
Significant Proportion ◽  
Cell Types ◽  
Hematopoietic Stem ◽  
Clonogenic Cell

Lymphomyeloid stem cells from the bone marrow of C57BL/6 mice treated with 5-fluorouracil (5-FU) were characterized with respect to 12 parameters using fluorescence-activated cell sorting and a competitive long-term repopulation assay. Stem cells were larger than lymphocytes and exhibited side light-scatter characteristic of blast cells. Most expressed low levels of Thy-1.2, high levels of Sca-1 (Ly6-A/E), H-2Kb, and AA4.1 antigens and stained brightly with rhodamine-123. Significantly, most long-term repopulating cells also expressed CD4, some at high density. In addition, a significant proportion displayed low to medium levels of the “lineage-specific” markers CD45R (B220), Gr- 1, and TER-119. A simple and rapid multiparameter sorting procedure enriched the stem cells 100-fold and substantially removed most other clonogenic cell types, including day 12 spleen colony-forming cells. Cells able to generate cobblestone colonies on stromal cells in vitro were co-enriched. Lethally irradiated mice transplanted with limiting numbers of the sorted stem cells did not survive unless cotransplanted with “compromised” marrow cells prepared by prior serial transplantation and shown to be depleted of long-term repopulating activity. A significant number of recipients transplanted with 25 to 100 sorted cells contained donor-derived B and T lymphocytes and granulocytes in their peripheral blood for at least 6 months. Limiting dilution analysis in vivo indicated that the frequency of competitive long-term repopulating units (CRU) in the sorted population was at least 1 in 60 cells. The calculated frequency of CRU was largely independent of the time of recipient analysis between 10 and 52 weeks, indicating that highly enriched stem cells can be recruited relatively early in certain transplant settings. This simple enrichment and assay strategy for repopulating hematopoietic stem cells should facilitate further analysis of their regulation in vivo.

scholarly journals Phenotypic and functional characterization of competitive long-term repopulating hematopoietic stem cells enriched from 5-fluorouracil- treated murine marrow

Blood ◽  
1993 ◽  
Vol 81 (9) ◽  
pp. 2310-2320 ◽  
Author(s):  
SJ Szilvassy ◽  
S Cory
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Functional Characterization ◽  
Significant Proportion ◽  
Cell Types ◽  
Hematopoietic Stem ◽  
Clonogenic Cell

Abstract Lymphomyeloid stem cells from the bone marrow of C57BL/6 mice treated with 5-fluorouracil (5-FU) were characterized with respect to 12 parameters using fluorescence-activated cell sorting and a competitive long-term repopulation assay. Stem cells were larger than lymphocytes and exhibited side light-scatter characteristic of blast cells. Most expressed low levels of Thy-1.2, high levels of Sca-1 (Ly6-A/E), H-2Kb, and AA4.1 antigens and stained brightly with rhodamine-123. Significantly, most long-term repopulating cells also expressed CD4, some at high density. In addition, a significant proportion displayed low to medium levels of the “lineage-specific” markers CD45R (B220), Gr- 1, and TER-119. A simple and rapid multiparameter sorting procedure enriched the stem cells 100-fold and substantially removed most other clonogenic cell types, including day 12 spleen colony-forming cells. Cells able to generate cobblestone colonies on stromal cells in vitro were co-enriched. Lethally irradiated mice transplanted with limiting numbers of the sorted stem cells did not survive unless cotransplanted with “compromised” marrow cells prepared by prior serial transplantation and shown to be depleted of long-term repopulating activity. A significant number of recipients transplanted with 25 to 100 sorted cells contained donor-derived B and T lymphocytes and granulocytes in their peripheral blood for at least 6 months. Limiting dilution analysis in vivo indicated that the frequency of competitive long-term repopulating units (CRU) in the sorted population was at least 1 in 60 cells. The calculated frequency of CRU was largely independent of the time of recipient analysis between 10 and 52 weeks, indicating that highly enriched stem cells can be recruited relatively early in certain transplant settings. This simple enrichment and assay strategy for repopulating hematopoietic stem cells should facilitate further analysis of their regulation in vivo.

scholarly journals Indole Biodegradation in Acinetobacter sp. Strain O153: Genetic and Biochemical Characterization

2017 ◽  
Vol 83 (19) ◽  
Author(s):  
Mikas Sadauskas ◽  
Justas Vaitekūnas ◽  
Renata Gasparavičiūtė ◽  
Rolandas Meškys
Keyword(s):  
Biochemical Characterization ◽  
Functional Characterization ◽  
Future Research ◽  
Signal Molecule ◽  
Major Drawback ◽  
Content Type ◽  
Enzymatic Function ◽  
Wide Range ◽  
Microbial Biodegradation

ABSTRACT Indole is a molecule of considerable biochemical significance, acting as both an interspecies signal molecule and a building block of biological elements. Bacterial indole degradation has been demonstrated for a number of cases; however, very little is known about genes and proteins involved in this process. This study reports the cloning and initial functional characterization of genes (iif and ant cluster) responsible for indole biodegradation in Acinetobacter sp. strain O153. The catabolic cascade was reconstituted in vitro with recombinant proteins, and each protein was assigned an enzymatic function. Degradation starts with oxidation, mediated by the IifC and IifD flavin-dependent two-component oxygenase system. Formation of indigo is prevented by IifB, and the final product, anthranilic acid, is formed by IifA, an enzyme which is both structurally and functionally comparable to cofactor-independent oxygenases. Moreover, the iif cluster was identified in the genomes of a wide range of bacteria, suggesting the potential of widespread Iif-mediated indole degradation. This work provides novel insights into the genetic background of microbial indole biodegradation. IMPORTANCE The key finding of this research is identification of the genes responsible for microbial biodegradation of indole, a toxic N-heterocyclic compound. A large amount of indole is present in urban wastewater and sewage sludge, creating a demand for an efficient and eco-friendly means to eliminate this pollutant. A common strategy of oxidizing indole to indigo has the major drawback of producing insoluble material. Genes and proteins of Acinetobacter sp. strain O153 (DSM 103907) reported here pave the way for effective and indigo-free indole removal. In addition, this work suggests possible novel means of indole-mediated bacterial interactions and provides the basis for future research on indole metabolism.

scholarly journals Biochemical Characterization of the 20S Proteasome from the Methanoarchaeon Methanosarcina thermophila

1998 ◽  
Vol 180 (6) ◽  
pp. 1480-1487 ◽  
Author(s):  
Julie A. Maupin-Furlow ◽  
Henry C. Aldrich ◽  
James G. Ferry
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Biochemical Characterization ◽  
Sodium Dodecyl ◽  
20S Proteasome ◽  
Β Subunit ◽  
Basic Residue ◽  
Methanosarcina Thermophila ◽  
Monovalent Ions

ABSTRACT The 20S proteasome from the methanoarchaeon Methanosarcina thermophila was produced in Escherichia coli and characterized. The biochemical properties revealed novel features of the archaeal 20S proteasome. A fully active 20S proteasome could be assembled in vitro with purified native α ring structures and β prosubunits independently produced in Escherichia coli, which demonstrated that accessory proteins are not essential for processing of the β prosubunits or assembly of the 20S proteasome. A protein complex with a molecular mass intermediate to those of the α7 ring and the 20S proteasome was detected, suggesting that the 20S proteasome is assembled from precursor complexes. The heterologously produced M. thermophila 20S proteasome predominately catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residues Phe and Tyr (chymotrypsinlike activity) in short chromogenic and fluorogenic peptides. Low-level hydrolyzing activities were also detected carboxyl to the acidic residue Asp and the basic residue Arg (trypsinlike activity). Sodium dodecyl sulfate and divalent or monovalent ions stimulated chymotrypsinlike activity and inhibited postglutamyl activity, whereas ATP stimulated postglutamyl activity but had little effect on the chymotrypsinlike activity. The results suggest that the 20S proteasome is a flexible protein which adjusts to binding of substrates. The 20S proteasome also hydrolyzed large proteins. Replacement of the nucleophilic Thr1 residue with an Ala in the β subunit abolished all activities, which suggests that only one active site is responsible for the multisubstrate activity. Replacement of β subunit active-site Lys33 with Arg reduced all activities, which further supports the existence of one catalytic site; however, this result also suggests a role for Lys33 in polarization of the Thr1 N, which serves to strip a proton from the active-site Thr1 Oγ nucleophile. Replacement of Asp51 with Asn had no significant effect on trypsinlike activity, enhanced postglutamyl and trypsinlike activities, and only partially reduced lysozyme-hydrolyzing activity, which suggested that this residue is not essential for multisubstrate activity.

Biochemical and functional characterization of the human tissue kallikrein 9

2017 ◽  
Vol 474 (14) ◽  
pp. 2417-2433 ◽  
Author(s):  
Panagiota S. Filippou ◽  
Sofia Farkona ◽  
Davor Brinc ◽  
Yijing Yu ◽  
Ioannis Prassas ◽  
...  
Keyword(s):  
Human Tissue ◽  
Biochemical Characterization ◽  
Functional Characterization ◽  
Squamous Carcinoma ◽  
Biochemical Properties ◽  
Tissue Kallikrein ◽  
Related Family ◽  
Squamous Carcinoma Cells

Human tissue kallikrein 9 (KLK9) is a member of the kallikrein-related family of proteases. Despite its known expression profile, much less is known about the functional roles of this protease and its implications in normal physiology and disease. We present here the first data on the biochemical characterization of KLK9, investigate parameters that affect its enzymatic activity (such as inhibitors) and provide preliminary insights into its putative substrates. We show that mature KLK9 is a glycosylated chymotrypsin-like enzyme with strong preference for tyrosine over phenylalanine at the P1 cleavage position. The enzyme activity is enhanced by Mg2+ and Ca2+, but is reversibly attenuated by Zn2+. KLK9 is inhibited in vitro by many naturally occurring or synthetic protease inhibitors. Using a combination of degradomic and substrate specificity assays, we identified candidate KLK9 substrates in two different epithelial cell lines [the non-tumorigenic human keratinocyte cells (HaCaT) and the tumorigenic tongue squamous carcinoma cells (SCC9)]. Two potential KLK9 substrates [KLK10 and midkine (MDK)] were subjected to further validation. Taken together, our data delineate some functional and biochemical properties of KLK9 for future elucidation of the role of this enzyme in health and disease.

Roles of glutamine in neurotransmission

2010 ◽  
Vol 6 (4) ◽  
pp. 263-276 ◽  
Author(s):  
Jan Albrecht ◽  
Marta Sidoryk-Węgrzynowicz ◽  
Magdalena Zielińska ◽  
Michael Aschner
Keyword(s):  
Amino Acids ◽  
Significant Proportion ◽  
Brain Slices ◽  
Amino Butyric Acid ◽  
Pathological Conditions ◽  
The Central Nervous System ◽  
Activity Dependent ◽  
The Brain

Glutamine (Gln) is found abundantly in the central nervous system (CNS) where it participates in a variety of metabolic pathways. Its major role in the brain is that of a precursor of the neurotransmitter amino acids: the excitatory amino acids, glutamate (Glu) and aspartate (Asp), and the inhibitory amino acid, γ-amino butyric acid (GABA). The precursor–product relationship between Gln and Glu/GABA in the brain relates to the intercellular compartmentalization of the Gln/Glu(GABA) cycle (GGC). Gln is synthesized from Glu and ammonia in astrocytes, in a reaction catalyzed by Gln synthetase (GS), which, in the CNS, is almost exclusively located in astrocytes (Martinez-Hernandez et al., 1977). Newly synthesized Gln is transferred to neurons and hydrolyzed by phosphate-activated glutaminase (PAG) to give rise to Glu, a portion of which may be decarboxylated to GABA or transaminated to Asp. There is a rich body of evidence which indicates that a significant proportion of the Glu, Asp and GABA derived from Gln feed the synaptic, neurotransmitter pools of the amino acids. Depolarization-induced-, calcium- and PAG activity-dependent releases of Gln-derived Glu, GABA and Asp have been observed in CNS preparations in vitro and in the brain in situ. Immunocytochemical studies in brain slices have documented Gln transfer from astrocytes to neurons as well as the location of Gln-derived Glu, GABA and Asp in the synaptic terminals. Patch-clamp studies in brain slices and astrocyte/neuron co-cultures have provided functional evidence that uninterrupted Gln synthesis in astrocytes and its transport to neurons, as mediated by specific carriers, promotes glutamatergic and GABA-ergic transmission. Gln entry into the neuronal compartment is facilitated by its abundance in the extracellular spaces relative to other amino acids. Gln also appears to affect neurotransmission directly by interacting with the NMDA class of Glu receptors. Transmission may also be modulated by alterations in cell membrane polarity related to the electrogenic nature of Gln transport or to uncoupled ion conductances in the neuronal or glial cell membranes elicited by Gln transporters. In addition, Gln appears to modulate the synthesis of the gaseous messenger, nitric oxide (NO), by controlling the supply to the cells of its precursor, arginine. Disturbances of Gln metabolism and/or transport contribute to changes in Glu-ergic or GABA-ergic transmission associated with different pathological conditions of the brain, which are best recognized in epilepsy, hepatic encephalopathy and manganese encephalopathy.

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