Structural studies on the catalytic mechanism of Diaminopropionate ammonia lyase

Diaminopropionate ammonia lyase (DAPAL) is a non-stereo specific fold-type II pyridoxal 5' phosphate (PLP) dependent enzyme that catalyzes the conversion of both D/L isoforms of the nonstandard amino acid Diaminopropionate (DAP) to pyruvate and ammonia. DAP is important for the synthesis of nonribosomal peptide antibiotics such as viomycin and capreomycin. Earlier structural studies on EcDAPAL bound to a reaction intermediate (aminoacrylate) suggested that the enzyme follows a two base mechanism, where Asp120 and Lys77 function as general bases to abstract proton from D-DAP and L-DAP respectively. A novel disulfide was observed near the active site, although its functional significance was not clear. In the present study, structural and biochemical characterization of active site mutants Asp120 (Asp120Asn/Ser/Thr/Cys) and Lys77 (Lys77His/ Thr/Ala/Val) of EcDAPAL has been carried out. Reduction of catalytic efficiency (Kcat/Km) of D120N EcDAPAL for D-DAP by 140 fold and presence of the uncatalyzed ligand at the active site in the crystal structure suggested that Asp120 indeed abstracts proton from D-DAP. Lys77, which was speculated to be important for proton abstraction from L DAP, however seemed to be crucial for PLP binding only. Presence of non-covalently bound PLP in the L77W mutant and occurence of both the ketoenamine, enolimine forms of internal aldimine in L77R mutant provided an in depth insight into the unique chemistry of internal aldimine formation in PLP dependent enzymes. To investigate the role of the novel disulfide bond near the active site, C265 and C291 were mutated to Serine. Studies on these mutants show that this disulfide bond gives additional stability to the protein and might regulate the entry of substrates to the active site. Thus, these studies provide deeper insights into the reaction mechanism of EcDAPAL, representing the overall reaction mechanism followed by several other fold-type II PLP pendent enzymes.

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Structure based protein engineering to confer selectivity of guanine deaminase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331409562x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C437-C437

Author(s):

Aruna Bitra ◽

Ruchi Anand

Keyword(s):

Crystal Structures ◽

Active Site ◽

Catalytic Mechanism ◽

Catalytic Efficiency ◽

Structural Basis ◽

Active Site Residues ◽

X Ray ◽

Secondary Activity ◽

Guanine Deaminase ◽

Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

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The Role of Glutathione in the Isomerization of Δ5-Androstene- 3,17-dione Catalyzed by Human Glutathione Transferase A1-1

Journal of Biological Chemistry ◽

10.1074/jbc.m009146200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 11698-11704 ◽

Cited By ~ 37

Author(s):

Pär L. Pettersson ◽

Bengt Mannervik

Keyword(s):

Active Site ◽

Active Sites ◽

Catalytic Mechanism ◽

Glutathione Transferase ◽

Ph Dependence ◽

Enzymatic Catalysis ◽

Catalytic Efficiency ◽

Protonated Form ◽

Hydroxyl Group ◽

Isomerization Reaction

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Δ5-androstene-3,17-dione (AD) into Δ4-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect.S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr9into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pKavalue of the enzyme-bound glutathione thiol. Thus, Tyr9promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr9residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3β-hydroxysteroid dehydrogenase.

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Structural studies reveal flexible roof of active site responsible for ω-transaminase CrmG overcoming by-product inhibition

Communications Biology ◽

10.1038/s42003-020-01184-w ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Jinxin Xu ◽

Xiaowen Tang ◽

Yiguang Zhu ◽

Zhijun Yu ◽

Kai Su ◽

...

Keyword(s):

Pharmaceutical Industry ◽

Active Site ◽

Catalytic Mechanism ◽

Product Inhibition ◽

Strong Interactions ◽

Structural Studies ◽

Biochemical Studies ◽

Fundamental Challenge ◽

Amine Compounds

AbstractAmine compounds biosynthesis using ω-transaminases has received considerable attention in the pharmaceutical industry. However, the application of ω-transaminases was hampered by the fundamental challenge of severe by-product inhibition. Here, we report that ω-transaminase CrmG from Actinoalloteichus cyanogriseus WH1-2216-6 is insensitive to inhibition from by-product α-ketoglutarate or pyruvate. Combined with structural and QM/MM studies, we establish the detailed catalytic mechanism for CrmG. Our structural and biochemical studies reveal that the roof of the active site in PMP-bound CrmG is flexible, which will facilitate the PMP or by-product to dissociate from PMP-bound CrmG. Our results also show that amino acceptor caerulomycin M (CRM M), but not α-ketoglutarate or pyruvate, can form strong interactions with the roof of the active site in PMP-bound CrmG. Based on our results, we propose that the flexible roof of the active site in PMP-bound CrmG may facilitate CrmG to overcome inhibition from the by-product.

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The reaction mechanism of retaining glycosyltransferases

Biochemical Society Transactions ◽

10.1042/bst20150177 ◽

2016 ◽

Vol 44 (1) ◽

pp. 51-60 ◽

Cited By ~ 24

Author(s):

Albert Ardèvol ◽

Javier Iglesias-Fernández ◽

Víctor Rojas-Cervellera ◽

Carme Rovira

Keyword(s):

Quantum Mechanics ◽

Reaction Mechanism ◽

Molecular Mechanics ◽

Active Site ◽

Molecular Mechanisms ◽

Catalytic Mechanism ◽

Strong Support ◽

Front Face ◽

Displacement Reaction ◽

Displacement Mechanism

The catalytic mechanism of retaining glycosyltransferases (ret-GTs) remains a controversial issue in glycobiology. By analogy to the well-established mechanism of retaining glycosidases, it was first suggested that ret-GTs follow a double-displacement mechanism. However, only family 6 GTs exhibit a putative nucleophile protein residue properly located in the active site to participate in catalysis, prompting some authors to suggest an unusual single-displacement mechanism [named as front-face or SNi (substitution nucleophilic internal)-like]. This mechanism has now received strong support, from both experiment and theory, for several GT families except family 6, for which a double-displacement reaction is predicted. In the last few years, we have uncovered the molecular mechanisms of several retaining GTs by means of quantum mechanics/molecular mechanics (QM/MM) metadynamics simulations, which we overview in the present work.

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Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis alkylhydroperoxidase C

Biochemical Journal ◽

10.1042/bj20020545 ◽

2002 ◽

Vol 367 (1) ◽

pp. 255-261 ◽

Cited By ~ 32

Author(s):

Radha CHAUHAN ◽

Shekhar C. MANDE

Keyword(s):

Mycobacterium Tuberculosis ◽

Reaction Mechanism ◽

Kinetic Parameters ◽

Active Site ◽

Double Mutant ◽

Catalytic Mechanism ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Directed Mutagenesis ◽

Resistant Strains

Mycobacterium tuberculosis alkylhydroperoxidase C (AhpC) belongs to the peroxiredoxin family, but unusually contains three cysteine residues in its active site. It is overexpressed in isoniazid-resistant strains of M. tuberculosis. We demonstrate that AhpC is capable of acting as a general antioxidant by protecting a range of substrates including supercoiled DNA. Active-site Cys to Ala mutants show that all three cysteine residues are important for activity. Cys-61 plays a central role in activity and Cys-174 also appears to be crucial. Interestingly, the C174A mutant is inactive, but double mutant C174/176A shows significant revertant activity. Kinetic parameters indicate that the C176A mutant is active, although much less efficient. We suggest that M. tuberculosis AhpC therefore belongs to a novel peroxiredoxin family and might follow a unique disulphide-relay reaction mechanism.

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Purification, Characterization, and Structural Studies of a Sulfatase from Pedobacter yulinensis

Molecules ◽

10.3390/molecules27010087 ◽

2021 ◽

Vol 27 (1) ◽

pp. 87

Author(s):

Caleb R. Schlachter ◽

Andrea O’Malley ◽

Linda L. Grimes ◽

John J. Tomashek ◽

Maksymilian Chruszcz ◽

...

Keyword(s):

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Active Site ◽

Drugs Of Abuse ◽

Biochemical Characterization ◽

Genomic Data ◽

Organic Substrates ◽

Structural Studies ◽

Biotechnological Applications ◽

Sulfatase Activity

Sulfatases are ubiquitous enzymes that hydrolyze sulfate from sulfated organic substrates such as carbohydrates, steroids, and flavones. These enzymes can be exploited in the field of biotechnology to analyze sulfated metabolites in humans, such as steroids and drugs of abuse. Because genomic data far outstrip biochemical characterization, the analysis of sulfatases from published sequences can lead to the discovery of new and unique activities advantageous for biotechnological applications. We expressed and characterized a putative sulfatase (PyuS) from the bacterium Pedobacter yulinensis. PyuS contains the (C/S)XPXR sulfatase motif, where the Cys or Ser is post-translationally converted into a formylglycine residue (FGly). His-tagged PyuS was co-expressed in Escherichia coli with a formylglycine-generating enzyme (FGE) from Mycobacterium tuberculosis and purified. We obtained several crystal structures of PyuS, and the FGly modification was detected at the active site. The enzyme has sulfatase activity on aromatic sulfated substrates as well as phosphatase activity on some aromatic phosphates; however, PyuS did not have detectable activity on 17α-estradiol sulfate, cortisol 21-sulfate, or boldenone sulfate.

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Biochemical Characterization and Crystal Structure of a Novel NAD+-Dependent Isocitrate Dehydrogenase from Phaeodactylum tricornutum

International Journal of Molecular Sciences ◽

10.3390/ijms21165915 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5915

Author(s):

Shi-Ping Huang ◽

Lu-Chun Zhou ◽

Bin Wen ◽

Peng Wang ◽

Guo-Ping Zhu

Keyword(s):

Isocitrate Dehydrogenase ◽

Phaeodactylum Tricornutum ◽

Biochemical Characterization ◽

Model Organism ◽

Catalytic Efficiency ◽

Marine Diatom ◽

Type Ii ◽

Dimeric Structure ◽

New Information ◽

Ef Hand

The marine diatom Phaeodactylum tricornutum originated from a series of secondary symbiotic events and has been used as a model organism for studying diatom biology. A novel type II homodimeric isocitrate dehydrogenase from P. tricornutum (PtIDH1) was expressed, purified, and identified in detail through enzymatic characterization. Kinetic analysis showed that PtIDH1 is NAD+-dependent and has no detectable activity with NADP+. The catalytic efficiency of PtIDH1 for NAD+ is 0.16 μM−1·s−1 and 0.09 μM−1·s−1 in the presence of Mn2+ and Mg2+, respectively. Unlike other bacterial homodimeric NAD-IDHs, PtIDH1 activity was allosterically regulated by the isocitrate. Furthermore, the dimeric structure of PtIDH1 was determined at 2.8 Å resolution, and each subunit was resolved into four domains, similar to the eukaryotic homodimeric NADP-IDH in the type II subfamily. Interestingly, a unique and novel C-terminal EF-hand domain was first defined in PtIDH1. Deletion of this domain disrupted the intact dimeric structure and activity. Mutation of the four Ca2+-binding sites in the EF-hand significantly reduced the calcium tolerance of PtIDH1. Thus, we suggest that the EF-hand domain could be involved in the dimerization and Ca2+-coordination of PtIDH1. The current report, on the first structure of type II eukaryotic NAD-IDH, provides new information for further investigation of the evolution of the IDH family.

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Hydroperoxide and peroxynitrite reductase activity of poplar thioredoxin-dependent glutathione peroxidase 5: kinetics, catalytic mechanism and oxidative inactivation

Biochemical Journal ◽

10.1042/bj20111378 ◽

2012 ◽

Vol 442 (2) ◽

pp. 369-380 ◽

Cited By ~ 32

Author(s):

Benjamin Selles ◽

Martin Hugo ◽

Madia Trujillo ◽

Vaibhav Srivastava ◽

Gunnar Wingsle ◽

...

Keyword(s):

Disulfide Bond ◽

Conformational Changes ◽

Catalytic Mechanism ◽

Reductase Activity ◽

Populus Trichocarpa ◽

Catalytic Efficiency ◽

Cysteine Residue ◽

Midpoint Potential ◽

Oxidative Inactivation

Gpxs (glutathione peroxidases) constitute a family of peroxidases, including selenocysteine- or cysteine-containing isoforms (SeCys-Gpx or Cys-Gpx), which are regenerated by glutathione or Trxs (thioredoxins) respectively. In the present paper we show new data concerning the substrates of poplar Gpx5 and the residues involved in its catalytic mechanism. The present study establishes the capacity of this Cys-Gpx to reduce peroxynitrite with a catalytic efficiency of 106 M−1·s−1. In PtGpx5 (poplar Gpx5; Pt is Populus trichocarpa), Glu79, which replaces the glutamine residue usually found in the Gpx catalytic tetrad, is likely to be involved in substrate selectivity. Although the redox midpoint potential of the Cys44–Cys92 disulfide bond and the pKa of Cys44 are not modified in the E79Q variant, it exhibited significantly improved kinetic parameters (Kperoxide and kcat) with tert-butyl hydroperoxide. The characterization of the monomeric Y151R variant demonstrated that PtGpx5 is not an obligate homodimer. Also, we show that the conserved Phe90 is important for Trx recognition and that Trx-mediated recycling of PtGpx5 occurs via the formation of a transient disulfide bond between the Trx catalytic cysteine residue and the Gpx5 resolving cysteine residue. Finally, we demonstrate that the conformational changes observed during the transition from the reduced to the oxidized form of PtGpx5 are primarily determined by the oxidation of the peroxidatic cysteine into sulfenic acid. Also, MS analysis of in-vitro-oxidized PtGpx5 demonstrated that the peroxidatic cysteine residue can be over-oxidized into sulfinic or sulfonic acids. This suggests that some isoforms could have dual functions potentially acting as hydrogen-peroxide- and peroxynitrite-scavenging systems and/or as mediators of peroxide signalling as proposed for 2-Cys peroxiredoxins.

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Biochemical characterization of the chondroitinase ABC I active site

Biochemical Journal ◽

10.1042/bj20050532 ◽

2005 ◽

Vol 390 (2) ◽

pp. 395-405 ◽

Cited By ~ 38

Author(s):

Vikas Prabhakar ◽

Rahul Raman ◽

Ishan Capila ◽

Carlos J. Bosques ◽

Kevin Pojasek ◽

...

Keyword(s):

Active Site ◽

Structural Model ◽

Catalytic Mechanism ◽

Biochemical Characterization ◽

Chondroitin Sulphate ◽

Critical Role ◽

Site Directed Mutagenesis ◽

Chondroitinase Abc ◽

Enzyme Substrate

cABC I (chondroitinase ABC I) from Proteus vulgaris is a GalAG (galactosaminoglycan) depolymerizing lyase that cleaves its substrates at the glycosidic bond via β-elimination. cABC I cleaves a particularly broad range of GalAG substrates, including CS (chondroitin sulphate), DS (dermatan sulphate) and hyaluronic acid. We recently cloned and recombinantly expressed cABC I in Escherichia coli, and completed a preliminary biochemical characterization of the enzyme. In the present study, we have coupled site-directed mutagenesis of the recombinant cABC I with a structural model of the enzyme–substrate complex in order to investigate in detail the roles of active site amino acids in the catalytic action of the enzyme. The putative catalytic residues His-501, Tyr-508, Arg-560 and Glu-653 were probed systematically via mutagenesis. Assessment of these mutants in kinetic and end-point assays provided direct evidence on the catalytic roles of these active-site residues. The crystal structure of the native enzyme provided a framework for molecular docking of representative CS and DS substrates. This enabled us to construct recombinant enzyme–substrate structural complexes. These studies together provided structural insights into the effects of the mutations on the catalytic mechanism of cABC I and the differences in its processing of CS and DS substrates. All His-501 mutants were essentially inactive and thereby implicating this amino acid to play the critical role of proton abstraction during catalysis. The kinetic data for Glu-653 mutants indicated that it is involved in a hydrogen bonding network in the active site. The proximity of Tyr-508 to the glycosidic oxygen of the substrate at the site of cleavage suggested its potential role in protonating the leaving group. Arg-560 was proximal to the uronic acid C-5 proton, suggesting its possible role in the stabilization of the carbanion intermediate formed during catalysis.

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Probing the Plasticity in the Active Site of Protein N-terminal Methyltransferase 1 Using Bisubstrate Analogs

10.1101/2020.04.13.039990 ◽

2020 ◽

Author(s):

Dongxing Chen ◽

Cheng Dong ◽

Guangping Dong ◽

Karthik Srinivasan ◽

Jinrong Min ◽

...

Keyword(s):

Active Site ◽

Binding Sites ◽

Biochemical Characterization ◽

Potent Inhibitor ◽

Structural Studies ◽

Cofactor Binding ◽

Bisubstrate Analogs ◽

Bisubstrate Inhibitors ◽

General Guidance ◽

Methyltransferase 1

AbstractThe interactions of a series of bisubstrate analogs with protein N-terminal methyltransferase 1 (NTMT1) were examined to probe the molecular properties of the NTMT1 active site through biochemical characterization and structural studies. Our results indicate that a 2-C to 4-C atom linker enables its respective bisubstrate analog to occupy both substrate and cofactor binding sites of NTMT1, but the bisubstrate analog with a 5-C atom linker only interacts with the substrate binding site and functions as a substrate. Furthermore, the 4-C atom linker is the optimal and produces the most potent inhibitor (Ki, app = 130 ± 40 pM) for NTMT1 to date, displaying over 100,000-fold selectivity over other methyltransferases and 3,000-fold even to its homolog NTMT2. This study reveals the molecular basis for the plasticity of the NTMT1 active site. Additionally, our study outlines a general guidance on the development of bisubstrate inhibitors for any methyltransferases.

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