Structural insights into dihydroxylation of terephthalate, a product of polyethylene terephthalate degradation

2022 ◽  
Author(s):  
Jai Krishna Mahto ◽  
Neetu Neetu ◽  
Monica Sharma ◽  
Monika Dubey ◽  
Bhanu Prakash Vellanki ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Polyethylene Terephthalate ◽  
Active Site ◽  
Catalytic Domain ◽  
Structural Features ◽  
Comamonas Testosteroni ◽  
Plastic Pollution ◽  
Microbial Utilization ◽  
Steady State Kinetics

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

scholarly journals A trapped human PPM1A–phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity

2018 ◽  
Vol 293 (21) ◽  
pp. 7993-8008 ◽  
Author(s):  
Subrata Debnath ◽  
Dalibor Kosek ◽  
Harichandra D. Tagad ◽  
Stewart R. Durell ◽  
Daniel H. Appella ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Metal Ions ◽  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Catalytic Domain ◽  
Protein Phosphatases ◽  
Random Order ◽  
Structural Features

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro. The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E–c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.

Crystal structure of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis

2021 ◽  
Vol 77 (11) ◽  
Author(s):  
Kohei Sasamoto ◽  
Tomoki Himiyama ◽  
Kunihiko Moriyoshi ◽  
Takashi Ohmoto ◽  
Koichi Uegaki ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cellulose Acetate ◽  
Electron Density ◽  
Active Site ◽  
Catalytic Domain ◽  
Crystal Packing ◽  
Signal Sequence ◽  
Industrial Applications ◽  
Side Chain ◽  
Active Site Conformation

The acetylxylan esterases (AXEs) classified into carbohydrate esterase family 4 (CE4) are metalloenzymes that catalyze the deacetylation of acetylated carbohydrates. AXE from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866), which belongs to CE4, is composed of three parts: a signal sequence (residues 1–22), an N-terminal region (NTR; residues 23–135) and a catalytic domain (residues 136–324). TTE0866 catalyzes the deacetylation of highly substituted cellulose acetate and is expected to be useful for industrial applications in the reuse of resources. In this study, the crystal structure of TTE0866 (residues 23–324) was successfully determined. The crystal diffracted to 1.9 Å resolution and belonged to space group I212121. The catalytic domain (residues 136–321) exhibited a (β/α)7-barrel topology. However, electron density was not observed for the NTR (residues 23–135). The crystal packing revealed the presence of an intermolecular space without observable electron density, indicating that the NTR occupies this space without a defined conformation or was truncated during the crystallization process. Although the active-site conformation of TTE0866 was found to be highly similar to those of other CE4 enzymes, the orientation of its Trp264 side chain near the active site was clearly distinct. The unique orientation of the Trp264 side chain formed a different-shaped cavity within TTE0866, which may contribute to its reactivity towards highly substituted cellulose acetate.

scholarly journals Crystal structure of full‐length human collagenase 3 (MMP‐13) with peptides in the active site defines exosites in the catalytic domain

2013 ◽  
Vol 27 (11) ◽  
pp. 4395-4405 ◽  
Author(s):  
Enrico A. Stura ◽  
Robert Visse ◽  
Philippe Cuniasse ◽  
Vincent Dive ◽  
Hideaki Nagase
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Catalytic Domain ◽  
Full Length

scholarly journals Crystal Structure of Inorganic Pyrophosphatase From Schistosoma japonicum Reveals the Mechanism of Chemicals and Substrate Inhibition

2021 ◽  
Vol 9 ◽  
Author(s):  
Qun-Feng Wu ◽  
Wei-Si Wang ◽  
Shen-Bo Chen ◽  
Bin Xu ◽  
Yong-Dong Li ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Schistosoma Japonicum ◽  
Active Site ◽  
Substrate Inhibition ◽  
Structural Features ◽  
Inorganic Pyrophosphatase ◽  
Metabolic Pathway Analysis ◽  
Inorganic Pyrophosphate ◽  
X Ray Crystallography ◽  
Pi Complex

Soluble inorganic pyrophosphatases (PPases) are essential for facilitating the growth and development of organisms, making them attractive functional proteins. To provide insight into the molecular basis of PPases in Schistosoma japonicum (SjPPase), we expressed the recombinant SjPPase, analyzed the hydrolysis mechanism of inorganic pyrophosphate (PPi), and measured its activity. Moreover, we solved the crystal structure of SjPPase in complex with orthophosphate (Pi) and performed PPi and methylene diphosphonic acid (MDP) docking into the active site. Our results suggest that the SjPPase possesses PPi hydrolysis activity, and the activity declines with increased MDP or NaF concentration. However, the enzyme shows unexpected substrate inhibition properties. Through PPi metabolic pathway analysis, the physiological action of substrate inhibition might be energy saving, adaptably cytoprotective, and biosynthetic rate regulating. Furthermore, the structure of apo-SjPPase and SjPPase with Pi has been solved at 2.6 and 2.3 Å, respectively. The docking of PPi into the active site of the SjPPase-Pi complex revealed that substrate inhibition might result from blocking Pi exit due to excess PPi in the SjPPase-Pi complex of the catalytic cycle. Our results revealed the structural features of apo-SjPPase and the SjPPase-Pi complex by X-ray crystallography, providing novel insights into the physiological functions of PPase in S. japonicum without the PPi transporter and the mechanism of its substrate inhibition.

scholarly journals The Crystal Structure of a Streptomyces thermoviolaceus Thermophilic Chitinase Known for Its Refolding Efficiency

2020 ◽  
Vol 21 (8) ◽  
pp. 2892
Author(s):  
Piotr H. Malecki ◽  
Magdalena Bejger ◽  
Wojciech Rypniewski ◽  
Constantinos E. Vorgias
Keyword(s):  
Crystal Structure ◽  
Catalytic Domain ◽  
Structural Features ◽  
Salt Bridges ◽  
Aromatic Rings ◽  
Tim Barrel ◽  
Trimmed Surface ◽  
Binding Cleft ◽  
Surface Loops ◽  
Structure Of Proteins

Analyzing the structure of proteins from extremophiles is a promising way to study the rules governing the protein structure, because such proteins are results of structural and functional optimization under well-defined conditions. Studying the structure of chitinases addresses an interesting aspect of enzymology, because chitin, while being the world’s second most abundant biopolymer, is also a recalcitrant substrate. The crystal structure of a thermostable chitinase from Streptomyces thermoviolaceus (StChi40) has been solved revealing a β/α-barrel (TIM-barrel) fold with an α+β insertion domain. This is the first chitinase structure of the multi-chitinase system of S. thermoviolaceus. The protein is also known to refold efficiently after thermal or chemical denaturation. StChi40 is structurally close to the catalytic domain of psychrophilic chitinase B from Arthrobacter TAD20. Differences are noted in comparison to the previously examined chitinases, particularly in the substrate-binding cleft. A comparison of the thermophilic enzyme with its psychrophilic homologue revealed structural features that could be attributed to StChi40’s thermal stability: compactness of the structure with trimmed surface loops and unique disulfide bridges, one of which is additionally stabilized by S–π interactions with aromatic rings. Uncharacteristically for thermophilic proteins, StChi40 has fewer salt bridges than its mesophilic and psychrophilic homologues.

scholarly journals The genome of a Bacteroidetes inhabitant of the human gut encodes a structurally distinct enoyl-acyl carrier protein reductase (FabI)

2020 ◽  
Vol 295 (22) ◽  
pp. 7635-7652
Author(s):  
Christopher D. Radka ◽  
Matthew W. Frank ◽  
Jiangwei Yao ◽  
Jayaraman Seetharaman ◽  
Darcie J. Miller ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Change ◽  
Conformational Changes ◽  
Active Site ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Binding Pocket ◽  
Structural Features ◽  
Antibacterial Drug ◽  
Carrier Protein

Enoyl-acyl carrier protein reductase (FabI) catalyzes a rate-controlling step in bacterial fatty-acid synthesis and is a target for antibacterial drug development. A phylogenetic analysis shows that FabIs fall into four divergent clades. Members of clades 1–3 have been structurally and biochemically characterized, but the fourth clade, found in members of phylum Bacteroidetes, is uncharacterized. Here, we identified the unique structure and conformational changes that distinguish clade 4 FabIs. Alistipes finegoldii is a prototypical Bacteroidetes inhabitant of the gut microbiome. We found that A. finegoldii FabI (AfFabI) displays cooperative kinetics and uses NADH as a cofactor, and its crystal structure at 1.72 Å resolution showed that it adopts a Rossmann fold as do other characterized FabIs. It also disclosed a carboxyl-terminal extension that forms a helix–helix interaction that links the protomers as a unique feature of AfFabI. An AfFabI·NADH crystal structure at 1.86 Å resolution revealed that this feature undergoes a large conformational change to participate in covering the NADH-binding pocket and establishing the water channels that connect the active site to the central water well. Progressive deletion of these interactions led to catalytically compromised proteins that fail to bind NADH. This unique conformational change imparted a distinct shape to the AfFabI active site that renders it refractory to a FabI drug that targets clade 1 and 3 pathogens. We conclude that the clade 4 FabI, found in the Bacteroidetes inhabitants of the gut, have several structural features and conformational transitions that distinguish them from other bacterial FabIs.

scholarly journals The Crystal Structure of the Catalytic Domain of the NF-κB Inducing Kinase Reveals a Narrow but Flexible Active Site

Structure ◽  
2012 ◽  
Vol 20 (10) ◽  
pp. 1704-1714 ◽  
Author(s):  
Gladys de Leon-Boenig ◽  
Krista K. Bowman ◽  
Jianwen A. Feng ◽  
Terry Crawford ◽  
Christine Everett ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Catalytic Domain

Conformational Changes Facilitate FXI Autoactivation to FXIa

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 19-19 ◽  
Author(s):  
Wenman Wu ◽  
Heinrich Roder ◽  
Peter N. Walsh
Keyword(s):  
Crystal Structure ◽  
Energy Transfer ◽  
Conformational Changes ◽  
Active Site ◽  
Dextran Sulfate ◽  
Catalytic Domain ◽  
Emission Peak ◽  
Wild Type ◽  
Zymogen Activation ◽  
A1 Domain

Abstract Abstract 19 Coagulation factor XI (FXI) is a uniquely dimeric coagulation protein, which in its activated form (FXIa) activates FIX to FIXa. We have previously shown that the dimeric structure of FXI is essential for normal autoactivation and activation by thrombin and FXIIa, but not for the expression of enzymatic activity against FIX (Wu W, et al J. Biol. Chem. 283:18655-18664, 2008). A comparison of three separate structures of FXI/XIa from our laboratory (i.e., the crystal structure of the catalytic domain of FXIa in complex with the kunitz protease inhibitor domain of protease nexin-2; the crystal structure of full-length, dimeric FXI; and the NMR structure of the FXI A4 domain) predicts a major conformational change accompanying the conversion of FXI to FXIa. We now show that when FXI binds to the negatively charged polymer, dextran sulfate and is autoactivated to generate FXIa, changes of intrinsic fluorescence are observed, i.e, a decrease in fluorescence intensity and a red shift of emission wavelength, which also suggests that a conformational change accompanies FXI activation. To investigate the mechanism of FXI zymogen activation and the allosteric transition accompanying the conversion of FXI to FXIa, which exposes binding sites for FXIa ligands, we have carried out fluorescence resonance energy transfer (FRET) studies to characterize the conformational changes accompanying zymogen activation. Using a sensitive free thiol quantitation assay, we confirmed the presence of a single free cysteine residue (Cys11) per subunit of recombinant FXI, which was quantitatively labeled with the thiol reactive fluorescence dye IAEDANS (5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid). Fluorescence excitation of AEDANS-labeled FXI at 280 nm shows a prominent dansyl emission peak (∼450 nm) in addition to the Trp emission peak (∼325 nm) indicative of efficient FRET from Trp donors to the AEDANS acceptor. Controls using a C11S mutant of FXI showed ∼10-fold lower levels of AEDANS labeling, confirming that Cys11 is the predominant labeling site. Autoactivation of FXI in the presence of dextran sulfate results in a major decrease in donor emission, but has little effect on acceptor emission. This indicates that, for wild-type FXI, FRET is dominated by transfer within the A1 domain originating from Trp55, which is located at a distance of 18 Å from Cys11, far closer than any other tryptophan. The changes in Trp emission, which are similar in the presence and abence of AEDANS, allow us to follow the kinetics of zymogen activation. The S557A active-site mutant of FXI, which cannot undergo autoactivation, showed no fluorescence changes upon addition of dextran sulfate, confirming that the observed decrease in Trp fluorescence is due to formation of active FXIa enzyme. In an effort to observe specific inter-domain FRET, we prepared an AEDANS labeled W55H mutant of FXI, which eliminates the Trp donor in the A1 domain that dominates energy transfer in wild-type FXI. Our data show that autoactivation of the W55H mutant is accompanied by a significant increase in AEDANS emission that can be attributed to the movement of the labeled Cys11 (in A1) relative to Trp228 in the A3 domain of the opposite dimer subunit. In the crystal structure of FXI, the distance for this donor-acceptor pair is 29 Å (compared to a distance of 40 Å for the second closest Trp, Trp407 in the catalytic domain), making it a sensitive and specific FRET probe for monitoring changes in domain arrangement associated with enzyme activation and ligand interactions. A comparison of the FXI crystal structure with our model of FXIa showed that the distance between the active site serines (Ser557) of each catalytic triad is shortened from ∼118 Å in the zymogen to 40–75 Å in the enzyme. Since the distance between the two scissile bonds of each subunit of FXI is also ∼75 Å, we propose that during autoactivation, either the active site of each catalytic domain of FXIa is positioned to cleave the Arg369-Ile370 bond of the opposite subunit (intersubunit transactivation) or a FXIa dimer positions its two active sites adjacent to the two scissile bonds of a separate FXI dimer (intermolecular activation). These studies support a model in which the autoactivating transition from zymogen to enzyme is accompanied by the movement of each catalytic domain of the dimer to facilitate efficient autoactivation of FXI. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 1.12 Å resolution crystal structure of the catalytic domain of the plasmid-mediated colistin resistance determinant MCR-2

2017 ◽  
Vol 73 (8) ◽  
pp. 443-449 ◽  
Author(s):  
Katie Coates ◽  
Timothy R. Walsh ◽  
James Spencer ◽  
Philip Hinchliffe
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
High Resolution ◽  
Active Site ◽  
Catalytic Domain ◽  
Resistance Determinant ◽  
Mechanistic Studies ◽  
Colistin Resistance ◽  
Gram Negative ◽  
Starting Model

MCR-2 confers resistance to colistin, a `last-line' antibiotic against extensively resistant Gram-negative pathogens. It is a plasmid-encoded phosphoethanolamine transferase that is closely related to MCR-1. To understand the diversity in the MCR family, the 1.12 Å resolution crystal structure of the catalytic domain of MCR-2 was determined. Variable amino acids are located distant from both the di-zinc active site and the membrane-proximal face. The exceptionally high resolution will provide an accurate starting model for further mechanistic studies.

Homology modelling and structural analysis of human arylamine N-acetyltransferase NAT1: evidence for the conservation of a cysteine protease catalytic domain and an active-site loop

2001 ◽  
Vol 356 (2) ◽  
pp. 327-334 ◽  
Author(s):  
Fernando RODRIGUES-LIMA ◽  
Claudine DELOMÉNIE ◽  
Geoffrey H. GOODFELLOW ◽  
Denis M. GRANT ◽  
Jean-Marie DUPRET
Keyword(s):  
Crystal Structure ◽  
Cysteine Protease ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Homology Modelling ◽  
Metabolic Activation ◽  
Cysteine Proteases ◽  
Catalytic Triad ◽  
Quality Model

Arylamine N-acetyltransferases (EC 2.3.1.5) (NATs) catalyse the biotransformation of many primary arylamines, hydrazines and their N-hydroxylated metabolites, thereby playing an important role in both the detoxification and metabolic activation of numerous xenobiotics. The recently published crystal structure of the Salmonella typhimurium NAT (StNAT) revealed the existence of a cysteine protease-like (Cys-His-Asp) catalytic triad. In the present study, a three-dimensional homology model of human NAT1, based upon the crystal structure of StNAT [Sinclair, Sandy, Delgoda, Sim and Noble (2000) Nat. Struct. Biol. 7, 560–564], is demonstrated. Alignment of StNAT and NAT1, together with secondary structure predictions, have defined a consensus region (residues 29–131) in which 37% of the residues are conserved. Homology modelling provided a good quality model of the corresponding region in human NAT1. The location of the catalytic triad was found to be identical in StNAT and NAT1. Comparison of active-site structural elements revealed that a similar length loop is conserved in both species (residues 122–131 in NAT1 model and residues 122–133 in StNAT). This observation may explain the involvement of residues 125, 127 and 129 in human NAT substrate selectivity. Our model, and the fact that cysteine protease inhibitors do not affect the activity of NAT1, suggests that human NATs may have adapted a common catalytic mechanism from cysteine proteases to accommodate it for acetyl-transfer reactions.

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