scholarly journals Lanthanide-dependent alcohol dehydrogenases require an essential aspartate residue for metal coordination and enzymatic function

2020 ◽  
Vol 295 (24) ◽  
pp. 8272-8284 ◽  
Author(s):  
Nathan M. Good ◽  
Matthias Fellner ◽  
Kemal Demirer ◽  
Jian Hu ◽  
Robert P. Hausinger ◽  
...  
Keyword(s):  
Ethanol Oxidation ◽  
Methylotrophic Bacterium ◽  
Functional Importance ◽  
Catalytic Function ◽  
Lanthanide Elements ◽  
Enzymatic Function ◽  
Aspartate Residue ◽  
Active Site Region

The lanthanide elements (Ln3+), those with atomic numbers 57–63 (excluding promethium, Pm3+), form a cofactor complex with pyrroloquinoline quinone (PQQ) in bacterial XoxF methanol dehydrogenases (MDHs) and ExaF ethanol dehydrogenases (EDHs), expanding the range of biological elements and opening novel areas of metabolism and ecology. Other MDHs, known as MxaFIs, are related in sequence and structure to these proteins, yet they instead possess a Ca2+-PQQ cofactor. An important missing piece of the Ln3+ puzzle is defining what features distinguish enzymes that use Ln3+-PQQ cofactors from those that do not. Here, using XoxF1 MDH from the model methylotrophic bacterium Methylorubrum extorquens AM1, we investigated the functional importance of a proposed lanthanide-coordinating aspartate residue. We report two crystal structures of XoxF1, one with and another without PQQ, both with La3+ bound in the active-site region and coordinated by Asp320. Using constructs to produce either recombinant XoxF1 or its D320A variant, we show that Asp320 is needed for in vivo catalytic function, in vitro activity, and La3+ coordination. XoxF1 and XoxF1 D320A, when produced in the absence of La3+, coordinated Ca2+ but exhibited little or no catalytic activity. We also generated the parallel substitution in ExaF to produce ExaF D319S and found that this variant loses the capacity for efficient ethanol oxidation with La3+. These results provide evidence that a Ln3+-coordinating aspartate is essential for the enzymatic functions of XoxF MDHs and ExaF EDHs, supporting the notion that sequences of these enzymes, and the genes that encode them, are markers for Ln3+ metabolism.

scholarly journals Lanthanide-dependent alcohol dehydrogenases require an essential aspartate residue for metal coordination and function

2020 ◽  
Author(s):  
Nathan M. Good ◽  
Matthias Fellner ◽  
Kemal Demirer ◽  
Jian Hu ◽  
Robert P. Hausinger ◽  
...  
Keyword(s):  
Ethanol Oxidation ◽  
Methylotrophic Bacterium ◽  
Functional Importance ◽  
Catalytic Function ◽  
Lanthanide Elements ◽  
And Function ◽  
Aspartate Residue ◽  
Active Site Region

ABSTRACTThe presence of lanthanide elements (Ln3+) and pyrroloquinoline quinone (PQQ) containing cofactors in XoxF methanol dehydrogenases (MDHs) and ExaF ethanol dehydrogenases (EDHs) has expanded the list of biological elements and opened novel areas of metabolism and ecology. Other MDHs known as MxaFIs are related in sequence and structure to these proteins, yet they instead possess a Ca2+-PQQ cofactor. An important missing piece of the Ln3+ puzzle is defining what protein features distinguish enzymes using Ln3+-PQQ cofactors from those that do not. In this study, we investigated the functional importance of a proposed lanthanide-coordinating aspartate using XoxF1 MDH from the model methylotrophic bacterium Methylorubrum extorquens AM1. We report two crystal structures of XoxF1, one containing PQQ and the other free of this organic molecule, both with La3+ bound in the active site region and coordinated by Asp320. Using constructs to produce either recombinant XoxF1 or its D320A variant, we show Asp320 is needed for in vivo catalytic function, in vitro activity of purified enzyme, and coordination of La3+. XoxF1 and XoxF1 D320A, when produced in the absence of La3+, coordinate Ca2+, but exhibit little or no catalytic activity. In addition, we generated the parallel substitution to produce ExaF D319S, and showed the enzyme loses the capacity for efficient ethanol oxidation with La3+. These results provide empirical evidence of an essential Ln3+-coordinating aspartate for the function of XoxF MDHs and ExaF EDHs; thus, supporting the suggestion that sequences of these enzymes, and the genes that encode them, are markers for Ln3+ metabolism.

Human Pluripotent Stem Cell-Derived Organoids as a Model of Intestinal Xenobiotic Metabolism

StemJournal ◽  
2020 ◽  
pp. 1-10
Author(s):  
Kengo Sasaki ◽  
Makoto Inoue ◽  
Masakazu Machida ◽  
Tomoyuki Kawasaki ◽  
Satoru Tsuruta ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Drug Transporters ◽  
Oral Intake ◽  
Human Pluripotent Stem Cell ◽  
Catalytic Function ◽  
Enzymatic Function ◽  
Intestinal Organoids

Background: The human intestine is the site of absorption and first-pass metabolism for oral intake. Assessment of absorption, distribution, metabolism, excretion, and toxicity (ADMET) of xenobiotics has transformed the understanding of in vivo pharmacology. However, these processes are difficult torecapitulate in vitro. Objective: We have developed a simple protocol for the generation of mature functional intestinal organoids from human pluripotent stem cells (hPSCs)under xenogeneic-free conditions. We sought to characterize transcription level in drug transporters and metabolism and evaluate CYP3A4 catalytic function of the organoids. Methods: Human pluripotent stem cell-derived intestinal organoids were generated and evaluated the expression of drug transporters and metabolizing enzymes. We examined the induction of CYP3A4 and ABCB1 gene expression in the organoids. Furthermore, we analyzed the CYP3A4 enzyme activity of the organoids by the p450-Glo CYP3A4 assay kit with luciferin isopropyl acetal. Results: Stem cell-derived intestinal organoids had an outward polarized intestinal epithelial layer and showed similar expression levels of drug transporters and metabolism genes as the adult healthy intestine. They also exhibited CYP3A4 enzymatic function in vitro. Conclusion: This model provides a novel platform for pharmacological testing and can enhance human ADMET studies in drug development.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals The STAGA Subunit ADA2b Is an Important Regulator of Human GCN5 Catalysis

2008 ◽  
Vol 29 (1) ◽  
pp. 266-280 ◽  
Author(s):  
Armin M. Gamper ◽  
Jaehoon Kim ◽  
Robert G. Roeder
Keyword(s):  
Saga Complex ◽  
Histone Tails ◽  
Functional Roles ◽  
Enzymatic Function ◽  
Core Histones ◽  
Human Proteins ◽  
Important Regulator ◽  
And Function

ABSTRACT Human STAGA is a multisubunit transcriptional coactivator containing the histone acetyltransferase GCN5L. Previous studies of the related yeast SAGA complex have shown that the yeast Gcn5, Ada2, and Ada3 components form a heterotrimer that is important for the enzymatic function of SAGA. Here, we report that ADA2a and ADA2b, two human homologues of yeast Ada2, each have the ability to form a heterotrimer with ADA3 and GCN5L but that only the ADA2b homologue is found in STAGA. By comparing the patterns of acetylation of several substrates, we found context-dependent requirements for ADA2b and ADA3 for the efficient acetylation of histone tails by GCN5. With human proteins, unlike yeast proteins, the acetylation of free core histones by GCN5 is unaffected by ADA2b or ADA3. In contrast, the acetylation of mononucleosomal substrates by GCN5 is enhanced by ADA2b, with no significant additional effect of ADA3, and the efficient acetylation of nucleosomal arrays (chromatin) by GCN5 requires both ADA2b and ADA3. Thus, ADA2b and ADA3 appear to act at two different levels of histone organization within chromatin to facilitate GCN5 function. Interestingly, although ADA2a forms a complex(es) with GCN5 and ADA3 both in vitro and in vivo, ADA2a-containing complexes are unable to acetylate nucleosomal H3. We have also shown the preferential recruitment of ADA2b, relative to ADA2a, to p53-dependent genes. This finding indicates that the previously demonstrated presence and function of GCN5 on these promoters reflect the action of STAGA and that the ADA2a and ADA2b paralogues have nonredundant functional roles.

scholarly journals BTB Protein Keap1 Targets Antioxidant Transcription Factor Nrf2 for Ubiquitination by the Cullin 3-Roc1 Ligase

2005 ◽  
Vol 25 (1) ◽  
pp. 162-171 ◽  
Author(s):  
Manabu Furukawa ◽  
Yue Xiong
Keyword(s):  
Transcription Factor ◽  
Ring Finger ◽  
Negative Regulator ◽  
Protein Accumulation ◽  
Protein Motif ◽  
Terminal Domain ◽  
Catalytic Function ◽  
Cullin 3

ABSTRACT The concentrations and functions of many eukaryotic proteins are regulated by the ubiquitin pathway, which consists of ubiquitin activation (E1), conjugation (E2), and ligation (E3). Cullins are a family of evolutionarily conserved proteins that assemble by far the largest family of E3 ligase complexes. Cullins, via a conserved C-terminal domain, bind with the RING finger protein Roc1 to recruit the catalytic function of E2. Via a distinct N-terminal domain, individual cullins bind to a protein motif present in multiple proteins to recruit specific substrates. Cullin 3 (Cul3), but not other cullins, binds directly with BTB domains to constitute a potentially large number of BTB-CUL3-ROC1 E3 ubiquitin ligases. Here we report that the human BTB-Kelch protein Keap1, a negative regulator of the antioxidative transcription factor Nrf2, binds to CUL3 and Nrf2 via its BTB and Kelch domains, respectively. The KEAP1-CUL3-ROC1 complex promoted NRF2 ubiquitination in vitro and knocking down Keap1 or CUL3 by short interfering RNA resulted in NRF2 protein accumulation in vivo. We suggest that Keap1 negatively regulates Nrf2 function in part by targeting Nrf2 for ubiquitination by the CUL3-ROC1 ligase and subsequent degradation by the proteasome. Blocking NRF2 degradation in cells expressing both KEAP1 and NRF2 by either inhibiting the proteasome activity or knocking down Cul3, resulted in NRF2 accumulation in the cytoplasm. These results may reconcile previously observed cytoplasmic sequestration of NRF2 by KEAP1 and suggest a possible regulatory step between KEAP1-NRF2 binding and NRF2 degradation.

A Mathematical Treatment of the Blood Dissociation Curve for Oxygen

1963 ◽  
Vol 9 (6) ◽  
pp. 745-762 ◽  
Author(s):  
Rodolfo Margaria
Keyword(s):  
Dissociation Constants ◽  
Quantitative Description ◽  
Mathematical Treatment ◽  
Physiological Effects ◽  
Functional Importance ◽  
Acidic Group ◽  
Oxygen Dissociation ◽  
Dissociation Curves

Abstract A quantitative description of Bohr's effect can be made from the oxygen dissociation curves of hemoglobin at different pHs, and the dissociation constants of the 02-linked acidic group of Hb (KR) and Hb02 (Ko) have been calculated as, respectively, 1.29 1O-8 and 3.42 10-7 at37°. On the assumption that the oxygenation of hemoglobin takes place in four successive steps, the constant for each equilibrium can easily be calculated, and the values given. It appears that the oxygenation takes place with the same affinity for the first three steps, while the affinity for the fourth oxygenation is 125 times greater. On the basis of these results a simplification of Adair's formula is given, containing only two constants, one (K) being representative of the affinity of the oxygen for the first three hemes, and the other (in)being the increase of affinity for the fourth oxygenation. This formula seems to fit most data in the literature of hemoglobin solutions and of blood in vivo and in vitro. The physiological effects and the functional importance of the increased affinity for the fourth oxygenation are described, and the possibility that disturbances of the Hb oxygenation process may be due to the lack of this process is considered.

Carbohydrate metabolism in human skeletal muscle during exercise is not regulated by G-1,6-P2

1988 ◽  
Vol 65 (1) ◽  
pp. 487-489 ◽  
Author(s):  
A. Katz ◽  
K. Sahlin ◽  
J. Henriksson
Keyword(s):  
Skeletal Muscle ◽  
Human Skeletal Muscle ◽  
Work Load ◽  
Short Term ◽  
Catalytic Function ◽  
Muscle Ph ◽  
Induced Changes ◽  
Femoris Muscle

Glucose 1,6-bisphosphate (G-1,6-P2) is a potent activator of phosphofructokinase (PFK) and an inhibitor of hexokinase in vitro. It has been suggested that increases in G-1,6-P2 are a main means by which PFK can achieve significant catalytic function in vivo despite falling pH and that increases in G-1,6-P2 will inhibit hexokinase in vivo. The purpose of the present study was to determine whether contraction-induced changes in flux through PFK and hexokinase are associated with changes in G-1,6-P2 in skeletal muscle. Ten men performed bicycle exercise for 10 min at 40 and 75% of maximal O2 uptake (VO2max) and to fatigue [4.8 +/- 0.6 (SE) min] at 100% VO2max. Biopsies were obtained from the quadriceps femoris muscle at rest and after each work load and analyzed for G-1,6-P2. G-1,6-P2 averaged 111 +/- 13 mumol/kg dry wt at rest and 121 +/- 16, 123 +/- 15, and 123 +/- 11 mumol/kg dry wt after the low-, moderate-, and high-intensity exercise bouts, respectively (P less than 0.05 for all means vs. rest). Flux through PFK was estimated to increase exponentially as the exercise intensity increased and muscle pH decreased at the higher work loads, whereas flux through hexokinase was estimated to increase during exercise at 40 and 75% VO2max but decrease sharply at 100% VO2max. These data demonstrate that flux through neither PFK nor hexokinase is mediated by changes in G-1,6-P2 in human skeletal muscle during short-term dynamic exercise.

An active immunization approach to generate protective catalytic antibodies

2001 ◽  
Vol 360 (1) ◽  
pp. 151-157 ◽  
Author(s):  
Jun WANG ◽  
Yunqing HAN ◽  
Miles F. WILKINSON
Keyword(s):  
Active Immunization ◽  
Catalytic Antibodies ◽  
Toxic Substances ◽  
Mouse Blood ◽  
Physiological Conditions ◽  
Catalytic Function ◽  
Carbamate Insecticide ◽  
Hydrolysis Of

We report that mice immunized with a phosphate immunogen produced polyclonal catalytic antibodies (PCAbs) that catalysed the hydrolysis of carbaryl, a widely used broad-spectrum carbamate insecticide that exerts toxic effects in animals and humans. The reaction catalysed by the PCAbs (IgGs) obeyed Michaelis–Menten kinetics in vitro with the following values at pH8.0 and 25°C: Km≈ 8.0μM, kcat = 4.8×10−3–5.8×10−1, kcat/knon-cat = 5.6×101–6.8×103 (where knon-cat is the rate constant of the reaction in the absence of added catalyst). The PCAbs were also active in whole sera under physiological conditions in vitro. The PCAbs induced in vivo were also active in vivo, as immunization with the phosphate immunogen decreased the mouse blood concentration of carbaryl. To our knowledge, this is the first report demonstrating that active immunization generates antibodies possessing therapeutic catalytic function in vivo. We propose that active immunization schemes that induce enzymically active antibodies may provide a highly specific therapeutic approach for degrading toxic substances.

scholarly journals Negative regulation of Raf-1 by phosphorylation of serine 621.

1996 ◽  
Vol 16 (10) ◽  
pp. 5409-5418 ◽  
Author(s):  
H Mischak ◽  
T Seitz ◽  
P Janosch ◽  
M Eulitz ◽  
H Steen ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Negative Regulation ◽  
Catalytic Function ◽  
Dependent Protein ◽  
The One

The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.

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