scholarly journals Alkaline ceramidase catalyzes the hydrolysis of ceramides via a catalytic mechanism shared by Zn2+-dependent amidases

2018 ◽  
Author(s):  
Jae Kyo Yi ◽  
Ruijuan Xu ◽  
Lina M. Obeid ◽  
Yusuf A. Hannun ◽  
Michael V. Airola ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Trichostatin A ◽  
Membrane Lipid ◽  
Yeast Cells ◽  
Adiponectin Receptors ◽  
Wild Type Enzyme ◽  
Zinc Coordination ◽  
Hydrolysis Of ◽  
Insight Into

ABSTRACTHuman alkaline ceramidase 3 (ACER3) is one of three alkaline ceramidases (ACERs) that catalyze the conversion of ceramide to sphingosine. ACERs are the members of the CREST superfamily of integral-membrane lipid hydrolases, including the adiponectin receptors which play roles in energy metabolism. All CREST members conserve a set of three Histidine, one Aspartate, and one Serine residue. However, the structural and catalytic roles for these residues are unclear. Here, we use ACER3 as a prototype enzyme to gain insight into this unique class of enzymes. Recombinant ACER3 was expressed in yeast cells that lack endogenous ceramidase activity, and microsomes were used for biochemical characterization. Six point mutantions of the conserved CREST motif were developed that are predicted to form a Zn-dependent active site based on homology with the human adiponectin receptors, whose crystal structures were recently determined. Five mutations completely lost their activity, except for S77A, which showed a 600-fold decrease compared with the wild-type enzyme. The activity of S77C mutation was pH sensitive, with neutral pH partially recovering ACER3 activity. This suggested a role for S77 in stabilizing the oxyanion of the transition state and differs from the proposed role in Zinc coordination for the adiponectin receptors (Vasiliauskaité-Brooks et. al., Nature, 2017). Together, these data suggest ACER3 is a Zn2+-dependent amidase that uses a catalytic mechanism for ceramide hydrolysis that is similar to other soluble Zn-based amidases. Consistent with this mechanism, ACER3 was specifically inhibited by trichostatin A, an HDAC inhibitor, which is a strong chelator of Zinc.

scholarly journals A Thermolabile Phospholipase B from Talaromyces marneffei GD-0079: Biochemical Characterization and Structure Dynamics Study

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 231 ◽  
Author(s):  
Rabia Durrani ◽  
Faez Iqbal Khan ◽  
Shahid Ali ◽  
Yonghua Wang ◽  
Bo Yang
Keyword(s):  
Fatty Acids ◽  
Conformational Changes ◽  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Structural Information ◽  
Expression System ◽  
Talaromyces Marneffei ◽  
Structure Dynamics ◽  
Phospholipase B ◽  
Hydrolysis Of

Phospholipase B (EC 3.1.1.5) are a distinctive group of enzymes that catalyzes the hydrolysis of fatty acids esterified at the sn-1 and sn-2 positions forming free fatty acids and lysophospholipids. The structural information and catalytic mechanism of phospholipase B are still not clear. Herein, we reported a putative phospholipase B (TmPLB1) from Talaromyces marneffei GD-0079 synthesized by genome mining library. The gene (TmPlb1) was expressed and the TmPLB1 was purified using E. coli shuffle T7 expression system. The putative TmPLB1 was purified by affinity chromatography with a yield of 13.5%. The TmPLB1 showed optimum activity at 35 °C and pH 7.0. The TmPLB1 showed enzymatic activity using Lecithin (soybean > 98% pure), and the hydrolysis of TmPLB1 by 31P NMR showed phosphatidylcholine (PC) as a major phospholipid along with lyso-phospholipids (1-LPC and 2-LPC) and some minor phospholipids. The molecular modeling studies indicate that its active site pocket contains Ser125, Asp183 and His215 as the catalytic triad. The structure dynamics and simulations results explained the conformational changes associated with different environmental conditions. This is the first report on biochemical characterization and structure dynamics of TmPLB1 enzyme. The present study could be helpful to utilize TmPLB1 in food industry for the determination of food components containing phosphorus. Additionally, such enzyme could also be useful in Industry for the modifications of phospholipids.

Copper–oxygen Dynamics in Tyrosinase

2019 ◽  
Author(s):  
Nobutaka Fujieda ◽  
Sachiko Yanagisawa ◽  
Minoru Kubo ◽  
Genji Kurisu ◽  
Shinobu Itoh
Keyword(s):  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Active Form ◽  
Copper Ion ◽  
Atomic Resolution ◽  
Oxygen Species ◽  
X Ray ◽  
Oxygen Dynamics ◽  
Insight Into ◽  
Copper Centre

To unveil the activation of dioxygen on the copper centre (Cu<sub>2</sub>O<sub>2</sub>core) of tyrosinase, we performed X-ray crystallograpy with active-form tyrosinase at near atomic resolution. This study provided a novel insight into the catalytic mechanism of the tyrosinase, including the rearrangement of copper-oxygen species as well as the intramolecular migration of copper ion induced by substrate-binding.<br>

scholarly journals Cold-Active β-Galactosidases: Insight into Cold Adaption Mechanisms and Biotechnological Exploitation

Marine Drugs ◽  
2021 ◽  
Vol 19 (1) ◽  
pp. 43
Author(s):  
Marco Mangiagalli ◽  
Marina Lotti
Keyword(s):  
Low Temperature ◽  
Molecular Mechanisms ◽  
Building Blocks ◽  
Cold Active ◽  
Cold Adaption ◽  
Glycosyl Donor ◽  
Pharmaceutical Industries ◽  
Biotechnological Processes ◽  
Hydrolysis Of ◽  
Insight Into

β-galactosidases (EC 3.2.1.23) catalyze the hydrolysis of β-galactosidic bonds in oligosaccharides and, under certain conditions, transfer a sugar moiety from a glycosyl donor to an acceptor. Cold-active β-galactosidases are identified in microorganisms endemic to permanently low-temperature environments. While mesophilic β-galactosidases are broadly studied and employed for biotechnological purposes, the cold-active enzymes are still scarcely explored, although they may prove very useful in biotechnological processes at low temperature. This review covers several issues related to cold-active β-galactosidases, including their classification, structure and molecular mechanisms of cold adaptation. Moreover, their applications are discussed, focusing on the production of lactose-free dairy products as well as on the valorization of cheese whey and the synthesis of glycosyl building blocks for the food, cosmetic and pharmaceutical industries.

Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants

2001 ◽  
Vol 356 (1) ◽  
pp. 217-222 ◽  
Author(s):  
Ricardo FRANCO ◽  
Alice S. PEREIRA ◽  
Pedro TAVARES ◽  
Arianna MANGRAVITA ◽  
Michael J. BARBER ◽  
...  
Keyword(s):  
Absorption Spectra ◽  
Catalytic Mechanism ◽  
Protoporphyrin Ix ◽  
Three Dimensional ◽  
Iron Chelation ◽  
Enzymic Activity ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Flow Experiments

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.

Catalytically active holo Homo sapiens adenosine deaminase I adopts a closed conformation

2022 ◽  
Vol 78 (1) ◽  
Author(s):  
Minh Thu Ma ◽  
Maria Rain Jennings ◽  
John Blazeck ◽  
Raquel L. Lieberman
Keyword(s):  
Adenosine Deaminase ◽  
T Cell Activation ◽  
Cell Activation ◽  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Bos Taurus ◽  
Homo Sapiens ◽  
Severe Combined Immunodeficiency ◽  
Closed Conformation ◽  
Catalytically Active

Homo sapiens adenosine deaminase 1 (HsADA1; UniProt P00813) is an immunologically relevant enzyme with roles in T-cell activation and modulation of adenosine metabolism and signaling. Patients with genetic deficiency in HsADA1 suffer from severe combined immunodeficiency, and HsADA1 is a therapeutic target in hairy cell leukemias. Historically, insights into the catalytic mechanism and the structural attributes of HsADA1 have been derived from studies of its homologs from Bos taurus (BtADA) and Mus musculus (MmADA). Here, the structure of holo HsADA1 is presented, as well as biochemical characterization that confirms its high activity and shows that it is active across a broad pH range. Structurally, holo HsADA1 adopts a closed conformation distinct from the open conformation of holo BtADA. Comparison of holo HsADA1 and MmADA reveals that MmADA also adopts a closed conformation. These findings challenge previous assumptions gleaned from BtADA regarding the conformation of HsADA1 that may be relevant to its immunological interactions, particularly its ability to bind adenosine receptors. From a broader perspective, the structural analysis of HsADA1 presents a cautionary tale for reliance on homologs to make structural inferences relevant to applications such as protein engineering or drug development.

Yeast cells lacking 5'-->3' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5' cap structure

1993 ◽  
Vol 13 (8) ◽  
pp. 4826-4835
Author(s):  
C L Hsu ◽  
A Stevens
Keyword(s):  
Full Length ◽  
Yeast Cells ◽  
Mrna Turnover ◽  
Turnover Rates ◽  
Wild Type ◽  
Mrna Species ◽  
Turnover Process ◽  
Hydrolysis Of ◽  
Extension Analysis ◽  
Wild Type Yeast

Analysis of the slowed turnover rates of several specific mRNA species and the higher cellular levels of some of these mRNAs in Saccharomyces cerevisiae lacking 5'-->3' exoribonuclease 1 (xrn1 cells) has led to the finding that these yeast contain higher amounts of essentially full-length mRNAs that do not bind to oligo(dT)-cellulose. On the other hand, the length of mRNA poly(A) chains found after pulse-labeling of cells lacking the exoribonuclease, the cellular rate of synthesis of oligo(dT)-bound mRNA, and the initial rate of its deadenylation appeared quite similar to the same measurements in wild-type yeast cells. Examination of the 5' cap structure status of the poly(A)-deficient mRNAs by comparative analysis of the m7G content of poly(A)- and poly(A)+ RNA fractions of wild-type and xrn1 cells suggested that the xrn1 poly(A)- mRNA fraction is low in cap structure content. Further analysis of the 5' termini by measurements of the rate of 5'-->3' exoribonuclease 1 hydrolysis of specific full-length mRNA species showed that approximately 50% of the xrn1 poly(A)-deficient mRNA species lack the cap structure. Primer extension analysis of the 5' terminus of ribosomal protein 51A (RP51A) mRNA showed that about 30% of the poly(A)-deficient molecules of the xrn1 cells are slightly shorter at the 5' end. The finding of some accumulation of poly(A)-deficient mRNA species partially lacking the cap structure together with the reduction of the rate of mRNA turnover in cells lacking the enzyme suggest a possible role for 5'-->3' exoribonuclease 1 in the mRNA turnover process.

scholarly journals Crystallographic insight into the catalytic mechanism of subunit A of the A-ATP synthase and the P-loop switch in evolution

2010 ◽  
Vol 1797 ◽  
pp. 32-33
Author(s):  
Anil Kumar ◽  
Malathy Sony Subramanian Manimekalai ◽  
Asha Manikkoth Balakrishna ◽  
Gerhard Grüber
Keyword(s):  
Atp Synthase ◽  
Catalytic Mechanism ◽  
Subunit A ◽  
Insight Into

The crystal structure of human mitochondrial 3-ketoacyl-CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities

2014 ◽  
Vol 70 (12) ◽  
pp. 3212-3225 ◽  
Author(s):  
Tiila-Riikka Kiema ◽  
Rajesh K. Harijan ◽  
Malgorzata Strozyk ◽  
Toshiyuki Fukao ◽  
Stefan E. H. Alexson ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Reaction Mechanism ◽  
Active Site ◽  
Quaternary Structure ◽  
Fatty Acyl ◽  
Physiological Importance ◽  
Loop Domain ◽  
Solution Studies ◽  
Hydrolysis Of ◽  
Insight Into

Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of theZoogloea ramigerabiosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.

scholarly journals Structure of a Talaromyces pinophilus GH62 arabinofuranosidase in complex with AraDNJ at 1.25 Å resolution

2018 ◽  
Vol 74 (8) ◽  
pp. 490-495 ◽  
Author(s):  
Olga V. Moroz ◽  
Lukasz F. Sobala ◽  
Elena Blagova ◽  
Travis Coyle ◽  
Wei Peng ◽  
...  
Keyword(s):  
Plant Biomass ◽  
Cellulosic Biomass ◽  
Structural Basis ◽  
Side Chain ◽  
Polymeric Substrates ◽  
Hydrolysis Of ◽  
Insight Into ◽  
Positively Charged ◽  
Talaromyces Pinophilus ◽  
Resolution Structure

The enzymatic hydrolysis of complex plant biomass is a major societal goal of the 21st century in order to deliver renewable energy from nonpetroleum and nonfood sources. One of the major problems in many industrial processes, including the production of second-generation biofuels from lignocellulose, is the presence of `hemicelluloses' such as xylans which block access to the cellulosic biomass. Xylans, with a polymeric β-1,4-xylose backbone, are frequently decorated with acetyl, glucuronyl and arabinofuranosyl `side-chain' substituents, all of which need to be removed for complete degradation of the xylan. As such, there is interest in side-chain-cleaving enzymes and their action on polymeric substrates. Here, the 1.25 Å resolution structure of the Talaromyces pinophilus arabinofuranosidase in complex with the inhibitor AraDNJ, which binds with a K d of 24 ± 0.4 µM, is reported. Positively charged iminosugars are generally considered to be potent inhibitors of retaining glycosidases by virtue of their ability to interact with both acid/base and nucleophilic carboxylates. Here, AraDNJ shows good inhibition of an inverting enzyme, allowing further insight into the structural basis for arabinoxylan recognition and degradation.

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