scholarly journals N-Acetylanthranilate Amidase from Arthrobacter nitroguajacolicus Rü61a, an α/β-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant

2006 ◽  
Vol 188 (24) ◽  
pp. 8430-8440 ◽  
Author(s):  
Stephan Kolkenbrock ◽  
Katja Parschat ◽  
Bernd Beermann ◽  
Hans-Jürgen Hinz ◽  
Susanne Fetzner
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Catalytic Properties ◽  
Catalytic Efficiency ◽  
Catalytic Triad ◽  
Kinetic Properties ◽  
Conserved Residues ◽  
Hydrolase Fold ◽  
Secondary Structure Predictions ◽  
Amide Hydrolase

ABSTRACT N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the α/β-hydrolase-fold superfamily of enzymes; inactivation of (His6-tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (k cat/Km = 208 mM−1 s−1), while among the aryl-acetylamides, o-carboxy- or o-nitro-substituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His6Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His6AmqC22A/C63A markedly differed from those of His6Amq. The replacements effected some changes in Km s of the enzyme and increased k cats for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing k cat for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in α-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.

P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency

2020 ◽  
Vol 168 (5) ◽  
pp. 557-567
Author(s):  
Wanitcha Rachadech ◽  
Yusuke Kato ◽  
Rabab M Abou El-Magd ◽  
Yuji Shishido ◽  
Soo Hyeon Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Active Site ◽  
Structural Changes ◽  
Catalytic Efficiency ◽  
Acid Oxidase ◽  
Wild Type ◽  
Amino Acid Oxidase ◽  
Adenine Ring ◽  
The Impact

Abstract Human D-amino acid oxidase (DAO) is a flavoenzyme that is implicated in neurodegenerative diseases. We investigated the impact of replacement of proline with leucine at Position 219 (P219L) in the active site lid of human DAO on the structural and enzymatic properties, because porcine DAO contains leucine at the corresponding position. The turnover numbers (kcat) of P219L were unchanged, but its Km values decreased compared with wild-type, leading to an increase in the catalytic efficiency (kcat/Km). Moreover, benzoate inhibits P219L with lower Ki value (0.7–0.9 µM) compared with wild-type (1.2–2.0 µM). Crystal structure of P219L in complex with flavin adenine dinucleotide (FAD) and benzoate at 2.25 Å resolution displayed conformational changes of the active site and lid. The distances between the H-bond-forming atoms of arginine 283 and benzoate and the relative position between the aromatic rings of tyrosine 224 and benzoate were changed in the P219L complex. Taken together, the P219L substitution leads to an increase in the catalytic efficiency and binding affinity for substrates/inhibitors due to these structural changes. Furthermore, an acetic acid was located near the adenine ring of FAD in the P219L complex. This study provides new insights into the structure–function relationship of human DAO.

scholarly journals Cofactor and Cofactor Mimetics

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-17-SCI-17
Author(s):  
Peter J. Lenting
Keyword(s):  
Proteolytic Activity ◽  
Light Chain ◽  
Active Site ◽  
Membrane Surface ◽  
Catalytic Efficiency ◽  
Catalytic Triad ◽  
Two Dimensions ◽  
Coagulation Cascade ◽  
Protease Domain ◽  
A2 Domain

Many natural enzymes need the assistance of protein cofactors to catalyze chemical reactions at a physiologically relevant speed and several of the enzymes that make up for the coagulation cascade are no exception in this regard. Notably, activated factors VII, IX and X display relatively poor enzymatic activity towards their respective macromolecular substrates. The reason for their low proteolytic activity originates from a number of structural restrictions. For instance, not all enzymes are capable to efficiently fold their new amino-terminus into the active site pocket, leaving the catalytic triad immature. Furthermore, serine protease activation is often associated with a reduced plasticity of the protease domain, which improves their proteolytic activity. Nevertheless, some enzymes still require additional stabilization to reduce flexibility of their protease domain. Protein cofactors are designed to optimize the proteolytic activity of such serine proteases, and can improve the catalytic efficiency of these enzymes by one-thousand to one-million fold. The allosteric changes induced by these protein cofactors are specific to each cofactor/enzyme pair. When focusing on the cofactor role of Factor VIIIa (FVIIIa; which stimulates the catalytic activity of factor IXa; FIXa), several aspects are of importance. First, FVIIIa has high affinity for phosphatidylserine-containing phospholipid-membranes, favoring formation of the FVIIIa/FIXa complex at the membrane surface. Being assembled at the membrane surface limits their movements to two dimensions, and enforces the affinity between both proteins. Second, the interactions between FVIIIa and FIXa involve an extended protein surface, which includes interactions between the FVIIIa light chain and FIXa light chain as well as between the FVIIIa A2 domain and the FIXa protease domain. Due to this extended interactive surface, the complex mimics a staked tree, in which FVIIIa orients the FIXa active site at the appropriate distance from the membrane surface. Moreover, binding of the FVIIIa A2 domain to FIXa surface loops reduces flexibility of the protease domain, and it is likely that allosteric changes induced by the A2-domain optimize the conformation of the active site region. Finally, FVIIIa provides also a binding site for the substrate FX. This not only allows FVIIa to function as a molecular bridge between enzyme and substrate, but also helps to align the FX activation peptide with the FIXa active site. This multistep process by which FVIII acts as a cofactor for FIXa may help us to understand how other non-FVIII molecules can be used to stimulate FIXa activity. Several molecular entities have been reported that are enhancing FIXa activity, including short synthetic peptides, monoclonal antibodies and, perhaps best known at this moment, bispecific antibodies that bind both FIXa and FX. Given the complex molecular structure that FVIIIa has and needs to stimulate FIXa activity, it is of interest to reflect on how this translates to the non-FVIII molecules in terms of regulation and potential cofactor activity. Differences in regulation and activity are of particular relevance for laboratory monitoring of these molecules and in the therapeutic setting. Knowing these limitations will help us to optimize the therapeutic application of non-FVIII molecules. Disclosures Lenting: Spark Therapeutics: Honoraria; Catalyst Biosciences: Honoraria; Sobi: Honoraria; Shire/Takeda: Honoraria; NovoNordisk: Honoraria; Biotest: Honoraria; LFB: Honoraria; Roche: Honoraria; laelaps therapeutics: Equity Ownership.

scholarly journals Multiple Activation Loop Conformations and Their Regulatory Properties in the Insulin Receptor's Kinase Domain

2001 ◽  
Vol 276 (50) ◽  
pp. 46933-46940 ◽  
Author(s):  
Ararat J. Ablooglu ◽  
Mark Frankel ◽  
Elena Rusinova ◽  
John B. Alexander Ross ◽  
Ronald A. Kohanski
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Kinase Domain ◽  
Kinetic Properties ◽  
Activation Loop ◽  
Steady State Kinetic ◽  
Tryptic Cleavage ◽  
Loop Conformations ◽  
Steric Accessibility ◽  
Regulatory Properties

Low catalytic efficiency of protein kinases often results from intrasteric inhibition caused by the activation loop blocking the active site. In the insulin receptor's kinase domain, Asp-1161 and Tyr-1162 in the peptide substrate-like sequence of the unphosphorylated activation loop can interact with four invariant residues in the active site: Lys-1085, Asp-1132, Arg-1136, and Gln-1208. Contributions of these six residues to intrasteric inhibition were tested by mutagenesis, and the unphosphorylated kinase domains were characterized. The mutations Q1208S, K1085N, and Y1162F each relieved intrasteric inhibition, increasing catalytic efficiency but without changing the rate-limiting step of the reaction. The mutants R1136Q and D1132N were virtually inactive. Steric accessibility of the active site was ranked by relative changes in iodide quenching of intrinsic fluorescence, and A-loop conformation was ranked by limited tryptic cleavage. Together these ranked the openness of the active site cleft as R1136Q ≈ D1132N ≥ D1161A > Y1162F ≈ K1085N > Q1208S ≥ wild-type. These findings demonstrate the importance of specific invariant residues for intrasteric inhibition and show that diverse activation loop conformations can produce similar steady-state kinetic properties. This suggests a broader range of regulatory properties for the activation loop than expected from a simple off-versus-on switch for kinase activation.

scholarly journals The Enigma of Substrate Recognition and Catalytic Efficiency of APE1-Like Enzymes

2021 ◽  
Vol 9 ◽  
Author(s):  
Anastasiia T. Davletgildeeva ◽  
Alexander A. Ishchenko ◽  
Murat Saparbaev ◽  
Olga S. Fedorova ◽  
Nikita A. Kuznetsov
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Substrate Recognition ◽  
Catalytic Efficiency ◽  
Dna Glycosylases ◽  
Enzyme Substrate ◽  
Damage Recognition ◽  
Key Factor ◽  
Dna Substrates ◽  
Time Courses

Despite significant achievements in the elucidation of the nature of protein-DNA contacts that control the specificity of nucleotide incision repair (NIR) by apurinic/apyrimidinic (AP) endonucleases, the question on how a given nucleotide is accommodated by the active site of the enzyme remains unanswered. Therefore, the main purpose of our study was to compare kinetics of conformational changes of three homologous APE1-like endonucleases (insect Drosophila melanogaster Rrp1, amphibian Xenopus laevis xAPE1, and fish Danio rerio zAPE1) during their interaction with various damaged DNA substrates, i.e., DNA containing an F-site (an uncleavable by DNA-glycosylases analog of an AP-site), 1,N6-ethenoadenosine (εA), 5,6-dihydrouridine (DHU), uridine (U), or the α-anomer of adenosine (αA). Pre-steady-state analysis of fluorescence time courses obtained for the interaction of the APE1-like enzymes with DNA substrates containing various lesions allowed us to outline a model of substrate recognition by this class of enzymes. It was found that the differences in rates of DNA substrates’ binding do not lead to significant differences in the cleavage efficiency of DNA containing a damaged base. The results suggest that the formation of enzyme–substrate complexes is not the key factor that limits enzyme turnover; the mechanisms of damage recognition and cleavage efficacy are related to fine conformational tuning inside the active site.

scholarly journals Mechanism of the CBM35 domain in assisting catalysis by Ape1, a Campylobacter jejuni O-acetyl esterase

2021 ◽  
Author(s):  
Chang Sheng-Huei Lin ◽  
Ian Y. Yen ◽  
Anson C. K. Chan ◽  
Michael E. P. Murphy
Keyword(s):  
Campylobacter Jejuni ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Binding Mode ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Acetyl Esterase ◽  
Lytic Transglycosylases ◽  
Active Site Cleft ◽  
And Function

AbstractPeptidoglycan (PG) is O-acetylated by bacteria to resist killing by host lysozyme. During PG turnover, however, deacetylation is a prerequisite for glycan strand hydrolysis by lytic transglycosylases. Ape1, a de-O-acetylase from Campylobacter jejuni, is a bi-modular protein composed of an SGNH hydrolase domain and a CBM35 domain. The conserved Asp-His-Ser catalytic triad in the SGNH hydrolase domain confers enzymatic activity. The PG binding mode and function of the CBM35 domain in de-O-acetylation remained unclear. In this paper, we present a 1.8 Å resolution crystal structure of a complex between acetate and Ape1. An active site cleft is formed at the interface of the two domains and two large loops from the CBM35 domain form part of the active site. Site-directed mutagenesis of residues in these loops coupled with activity assays using p-nitrophenol acetate indicate the CBM35 loops are required for full catalytic efficiency. Molecular docking of a model O-acetylated hexasaccharide PG substrate to Ape1 using HADDOCK suggests the interaction is formed by the active cleft and the saccharide motif of PG. Together, we propose that the active cleft of Ape1 diverges from other SGNH hydrolase members by using the CBM35 loops to assist catalysis. The concave Ape1 active cleft may accommodate the long glycan strands for selecting PG substrates to regulate subsequent biological events.

scholarly journals Anti-CUB1 or Anti-Spacer Antibodies That Increase ADAMTS13 Activity Act By Allosterically Enhancing Metalloprotease Domain Function

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 23-23
Author(s):  
An-Sofie Schelpe ◽  
Anastasis Petri ◽  
Nele Vandeputte ◽  
Hans Deckmyn ◽  
Simon F De Meyer ◽  
...  
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Active Site ◽  
Current Model ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Activation Mechanism ◽  
Rich Domain ◽  
Cryptic Epitope

Abstract Background ADAMTS13 circulates in a folded conformation, which is mediated by interactions between the C-terminal CUB domains and its central Spacer domain. Binding of ADAMTS13 to the VWF D4-CK domains disrupts the CUB-Spacer interaction, inducing a structural change that extends ADAMTS13 into an open conformation that enhances catalytic efficiency ~2-fold. This mechanism supports a model in which ADAMTS13 unfolding induces exposure of an exosite in the Spacer domain that interacts with the VWF A2 domain, increasing the affinity between the two molecules, and, therefore, the rate of proteolysis. The D4-CK-mediated conformational activation of ADAMTS13 can be mimicked in vitro with the use of antibodies that disrupt the CUB-Spacer interaction, such as the previously published anti-CUB antibody, Ab17G2. We recently generated a novel, activating antibody against the Spacer domain (Ab3E4). Aim To characterize the mechanism by which the Ab17G2 and Ab3E4 enhance the catalytic efficiency of ADAMTS13. Methods The effects of the Ab17G2 and Ab3E4 on the activity of ADAMTS13 were studied using FRETS-VWF73. The effects of the Ab17G2 and Ab3E4 on the kinetics of VWF96 (VWF G1573-R1668) proteolysis were characterized using an in-house assay. ELISA was used to investigate conformational changes in ADAMTS13 induced by the Ab17G2 and Ab3E4. Results Both Ab17G2 and Ab3E4 enhanced FRETS-VWF73 proteolysis by ~1.7-fold. This result was reproduced using the VWF96 substrate; the Ab17G2 and Ab3E4 enhanced the catalytic efficiency (kcat/Km) of ADAMTS13 by ~1.8- and ~2.0-fold, respectively. The activation was dependent on the conformational extension of ADAMTS13, since the antibodies could not enhance the activity of an ADAMTS13 variant that lacks the TSP2-CUB2 domains (MDTCS). Surprisingly, ADAMTS13 activation was not mediated through exposure of the Spacer or Cys-rich domain exosites as previously proposed, as the Ab17G2 and Ab3E4 efficiently enhanced proteolysis of VWF96 variants in which the Spacer/Cys-rich exosite binding sites were disrupted. Kinetic analysis of VWF96 proteolysis showed that the Ab17G2- and Ab3E4-induced activation of ADAMTS13 is primarily manifest through a ~1.5- to ~2-fold increase in enzyme turnover (kcat). Thus, contrary to the current model, this suggests that the conformational extension of ADAMTS13 influences the functionality of the active site, and not substrate binding affinity (Km). Incubating ADAMTS13 with either Ab17G2 or Ab3E4 exposed a cryptic epitope in the metalloprotease domain that was specifically detected by ELISA, further corroborating that the antibodies induce a conformational change in ADAMTS13 affecting the M domain. Conclusion Antibodies can be used as tools for understanding the structure/function of enzymes. Using activating antibodies against the Spacer and CUB1 domains of ADAMTS13, we show for the first time that the activation of ADAMTS13 following its unfolding is not a result of exposure of a functional exosite in Spacer/Cys-rich domain that increases affinity to VWF. Rather, our data are consistent with an allosteric activation mechanism upon the metalloprotease domain. We propose that ADAMTS13 unfolding causes a conformational change in the active site that further activates the enzyme. We are currently investigating whether the D4-CK-induced enhancement of ADAMTS13 proteolytic activity is also mediated by conformational changes in the active site. Disclosures Vanhoorelbeke: Ablynx: Consultancy; Shire: Consultancy.

scholarly journals Synergistic effects of functionally distinct substitutions in β-lactamase variants shed light on the evolution of bacterial drug resistance

2018 ◽  
Vol 293 (46) ◽  
pp. 17971-17984 ◽  
Author(s):  
Meha P. Patel ◽  
Liya Hu ◽  
Cameron A. Brown ◽  
Zhizeng Sun ◽  
Carolyn J. Adamski ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Conformational Changes ◽  
Active Site ◽  
Enzyme Stability ◽  
Synergistic Effects ◽  
Catalytic Efficiency ◽  
Bacterial Drug Resistance ◽  
Natural Variant ◽  
Loop Conformation ◽  
Shed Light

The CTX-M β-lactamases have emerged as the most widespread extended-spectrum β-lactamases (ESBLs) in Gram-negative bacteria. These enzymes rapidly hydrolyze cefotaxime, but not the related cephalosporin, ceftazidime. ESBL variants have evolved, however, that provide enhanced ceftazidime resistance. We show here that a natural variant at a nonactive site, i.e. second-shell residue N106S, enhances enzyme stability but reduces catalytic efficiency for cefotaxime and ceftazidime and decreases resistance levels. However, when the N106S variant was combined with an active-site variant, D240G, that enhances enzyme catalytic efficiency, but decreases stability, the resultant double mutant exhibited higher resistance levels than predicted on the basis of the phenotypes of each variant. We found that this epistasis is due to compensatory effects, whereby increased stability provided by N106S overrides its cost of decreased catalytic activity. X-ray structures of the variant enzymes in complex with cefotaxime revealed conformational changes in the active-site loop spanning residues 103–106 that were caused by the N106S substitution and relieve steric strain to stabilize the enzyme, but also alter contacts with cefotaxime and thereby reduce catalytic activity. We noted that the 103–106 loop conformation in the N106S-containing variants is different from that of WT CTX-M but nearly identical to that of the non-ESBL, TEM-1 β-lactamase, having a serine at the 106 position. Therefore, residue 106 may serve as a “switch” that toggles the conformations of the 103–106 loop. When it is serine, the loop is in the non-ESBL, TEM-like conformation, and when it is asparagine, the loop is in a CTX-M–like, cefotaximase-favorable conformation.

scholarly journals GII.4 Norovirus Protease Shows pH-Sensitive Proteolysis with a Unique Arg-His Pairing in the Catalytic Site

2019 ◽  
Vol 93 (6) ◽  
Author(s):  
Mariya A. Viskovska ◽  
Boyang Zhao ◽  
Sreejesh Shanker ◽  
Jae-Mun Choi ◽  
Lisheng Deng ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Structural Information ◽  
Catalytic Triad ◽  
Ph Sensitive ◽  
Structural Basis ◽  
Genotype 4 ◽  
Structure Based Drug Design ◽  
Human Noroviruses ◽  
Substrate Inhibitor

ABSTRACTHuman noroviruses (NoVs) are the main cause of epidemic and sporadic gastroenteritis. Phylogenetically, noroviruses are divided into seven genogroups, with each divided into multiple genotypes. NoVs belonging to genogroup II and genotype 4 (GII.4) are globally most prevalent. Genetic diversity among the NoVs and the periodic emergence of novel strains present a challenge for the development of vaccines and antivirals to treat NoV infection. NoV protease is essential for viral replication and is an attractive target for the development of antivirals. The available structure of GI.1 protease provided a basis for the design of inhibitors targeting the active site of the protease. These inhibitors, although potent against the GI proteases, poorly inhibit the GII proteases, for which structural information is lacking. To elucidate the structural basis for this difference in the inhibitor efficiency, we determined the crystal structure of a GII.4 protease. The structure revealed significant changes in the S2 substrate-binding pocket, making it noticeably smaller, and in the active site, with the catalytic triad residues showing conformational changes. Furthermore, a conserved arginine is found inserted into the active site, interacting with the catalytic histidine and restricting substrate/inhibitor access to the S2 pocket. This interaction alters the relationships between the catalytic residues and may allow for a pH-dependent regulation of protease activity. The changes we observed in the GII.4 protease structure may explain the reduced potency of the GI-specific inhibitors against the GII protease and therefore must be taken into account when designing broadly cross-reactive antivirals against NoVs.IMPORTANCEHuman noroviruses (NoVs) cause sporadic and epidemic gastroenteritis worldwide. They are divided into seven genogroups (GI to GVII), with each genogroup further divided into several genotypes. Human NoVs belonging to genogroup II and genotype 4 (GII.4) are the most prevalent. Currently, there are no vaccines or antiviral drugs available for NoV infection. The protease encoded by NoV is considered a valuable target because of its essential role in replication. NoV protease structures have only been determined for the GI genogroup. We show here that the structure of the GII.4 protease exhibits several significant changes from GI proteases, including a unique pairing of an arginine with the catalytic histidine that makes the proteolytic activity of GII.4 protease pH sensitive. A comparative analysis of NoV protease structures may provide a rational framework for structure-based drug design of broadly cross-reactive inhibitors targeting NoVs.

scholarly journals Structural Insights into the Active Site Formation of DUSP22 in N-loop-containing Protein Tyrosine Phosphatases

2020 ◽  
Vol 21 (20) ◽  
pp. 7515
Author(s):  
Chih-Hsuan Lai ◽  
Co-Chih Chang ◽  
Huai-Chia Chuang ◽  
Tse-Hua Tan ◽  
Ping-Chiang Lyu
Keyword(s):  
Hydrogen Bonding ◽  
Phosphatase Activity ◽  
Conformational Changes ◽  
Active Site ◽  
Protein Tyrosine Phosphatases ◽  
Site Formation ◽  
Tyrosine Phosphatases ◽  
Conserved Residues ◽  
Protein Tyrosine ◽  
Hydrogen Bonding Network

Cysteine-based protein tyrosine phosphatases (Cys-based PTPs) perform dephosphorylation to regulate signaling pathways in cellular responses. The hydrogen bonding network in their active site plays an important conformational role and supports the phosphatase activity. Nearly half of dual-specificity phosphatases (DUSPs) use three conserved residues, including aspartate in the D-loop, serine in the P-loop, and asparagine in the N-loop, to form the hydrogen bonding network, the D-, P-, N-triloop interaction (DPN–triloop interaction). In this study, DUSP22 is used to investigate the importance of the DPN–triloop interaction in active site formation. Alanine mutations and somatic mutations of the conserved residues, D57, S93, and N128 substantially decrease catalytic efficiency (kcat/KM) by more than 102-fold. Structural studies by NMR and crystallography reveal that each residue can perturb the three loops and induce conformational changes, indicating that the hydrogen bonding network aligns the residues in the correct positions for substrate interaction and catalysis. Studying the DPN–triloop interaction reveals the mechanism maintaining phosphatase activity in N-loop-containing PTPs and provides a foundation for further investigation of active site formation in different members of this protein class.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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