scholarly journals Crystal structure of archaeal phosphopantothenate synthetase

2014 ◽  
Vol 70 (a1) ◽  
pp. C455-C455
Author(s):  
Akiko Kita ◽  
Asako Kishimoto ◽  
Takuya Ishibashi ◽  
Hiroya Tomita ◽  
Yuusuke Yokooji ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Rotation Axis ◽  
Structural Features ◽  
Common Pathway ◽  
Hyperthermophilic Archaeon ◽  
Pantothenate Synthetase ◽  
Almost All ◽  
Coenzyme A Biosynthesis

Bacteria/eukaryotes share a common pathway for coenzyme A biosynthesis which involves two enzymes, pantothenate synthetase and pantothenate kinase, to convert pantoate to 4'-phosphopantothenate. These two enzymes are absent in almost all archaea. Recently, it was reported that two novel enzymes, pantoate kinase (PoK) and phosphopantothenate synthetase (PPS), are responsible for this conversion in archaea[1]. In archaea, pantoate is phosphorylated by PoK to produce 4-phosphopantoate (PPo), and then condensation of PPo and β-alanine is catalyzed by PPS, generating 4'-phosphopantothenate. Here, we report the crystal structure of PPS from the hyperthermophilic archaeon, Thermococcus kodakarensis and its complexes with ATP, and ATP and 4-phosphopantoate (PPo). PPS forms an asymmetric homodimer, in which two monomers composing a dimer, deviated from the exact 2-fold symmetry, displaying 40-130distortion. Two active sites in PPS dimer are located near the rotation axis. Due to the asymmetricity of PPS dimer molecule, two active sites in PPS dimer are not equivalent. The structural features are consistent with the mutagenesis data and the results of biochemical experiments previously reported. Based on the structures of PPS, PPS/ATP complex, and PPS/ATP/PPo complex, we discuss the catalytic mechanism by which PPS produces phosphopantoyl adenylate (PPA), which is thought to be a reaction intermediate.

scholarly journals Crystal structure of glycosyltrehalose synthase fromSulfolobus shibataeDSM5389

2018 ◽  
Vol 74 (11) ◽  
pp. 741-746
Author(s):  
Nobuo Okazaki ◽  
Michael Blaber ◽  
Ryota Kuroki ◽  
Taro Tamada
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Structural Basis ◽  
Hyperthermophilic Archaeon ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sulfolobus Shibatae ◽  
Glucosidic Bond

Glycosyltrehalose synthase (GTSase) converts the glucosidic bond between the last two glucose residues of amylose from an α-1,4 bond to an α-1,1 bond, generating a nonreducing glycosyl trehaloside, in the first step of the biosynthesis of trehalose. To better understand the structural basis of the catalytic mechanism, the crystal structure of GTSase from the hyperthermophilic archaeonSulfolobus shibataeDSM5389 (5389-GTSase) has been determined to 2.4 Å resolution by X-ray crystallography. The structure of 5389-GTSase can be divided into five domains. The central domain contains the (β/α)8-barrel fold that is conserved as the catalytic domain in the α-amylase family. Three invariant catalytic carboxylic amino acids in the α-amylase family are also found in GTSase at positions Asp241, Glu269 and Asp460 in the catalytic domain. The shape of the catalytic cavity and the pocket size at the bottom of the cavity correspond to the intramolecular transglycosylation mechanism proposed from previous enzymatic studies.

scholarly journals The Reaction Mechanism of Metallo-β-Lactamases Is Tuned by the Conformation of an Active-Site Mobile Loop

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Antonela R. Palacios ◽  
María F. Mojica ◽  
Estefanía Giannini ◽  
Magdalena A. Taracila ◽  
Christopher R. Bethel ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Structural Changes ◽  
Catalytic Mechanism ◽  
Multidrug Resistant ◽  
Loop Sequence ◽  
Life Saving ◽  
Life Threatening ◽  
A Minor ◽  
Almost All

ABSTRACTCarbapenems are “last resort” β-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-β-lactamases (MβLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all β-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MβLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MβLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different β-lactams in all MβLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MβLs.

scholarly journals Crystal Structure of Complete Rhinovirus RNA Polymerase Suggests Front Loading of Protein Primer

2005 ◽  
Vol 79 (1) ◽  
pp. 277-288 ◽  
Author(s):  
Todd C. Appleby ◽  
Hartmut Luecke ◽  
Jae Hoon Shim ◽  
Jim Z. Wu ◽  
I. Wayne Cheney ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Rna Polymerase ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Structural Information ◽  
Structural Features ◽  
Replication Initiation ◽  
Front Face ◽  
Large Cleft ◽  
Active Site Cavity

ABSTRACT Picornaviruses utilize virally encoded RNA polymerase and a uridylylated protein primer to ensure replication of the entire viral genome. The molecular details of this mechanism are not well understood due to the lack of structural information. We report the crystal structure of human rhinovirus 16 3D RNA-dependent RNA polymerase (HRV16 3Dpol) at a 2.4-Å resolution, representing the first complete polymerase structure from the Picornaviridae family. HRV16 3Dpol shares the canonical features of other known polymerase structures and contains an N-terminal region that tethers the fingers and thumb subdomains, forming a completely encircled active site cavity which is accessible through a small tunnel on the backside of the molecule. The small thumb subdomain contributes to the formation of a large cleft on the front face of the polymerase which also leads to the active site. The cleft appears large enough to accommodate a template:primer duplex during RNA elongation or a protein primer during the uridylylation stage of replication initiation. Based on the structural features of HRV16 3Dpo1 and the catalytic mechanism known for all polymerases, a front-loading model for uridylylation is proposed.

scholarly journals Roles of the Conserved Aspartate and Arginine in the Catalytic Mechanism of an Archaeal β-Class Carbonic Anhydrase

2002 ◽  
Vol 184 (15) ◽  
pp. 4240-4245 ◽  
Author(s):  
Kerry S. Smith ◽  
Cheryl Ingram-Smith ◽  
James G. Ferry
Keyword(s):  
Crystal Structure ◽  
Proton Transfer ◽  
Rate Constants ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Zinc Hydroxide ◽  
Carbonic Anhydrases ◽  
Wild Type ◽  
Transfer Step ◽  
Mechanism Of Catalysis

ABSTRACT The roles of an aspartate and an arginine, which are completely conserved in the active sites of β-class carbonic anhydrases, were investigated by steady-state kinetic analyses of replacement variants of the β-class enzyme (Cab) from the archaeon Methanobacterium thermoautotrophicum. Previous kinetic analyses of wild-type Cab indicated a two-step zinc-hydroxide mechanism of catalysis in which the k cat/Km value depends only on the rate constants for the CO2 hydration step, whereas k cat also depends on rate constants from the proton transfer step (K. S. Smith, N. J. Cosper, C. Stalhandske, R. A. Scott, and J. G. Ferry, J. Bacteriol. 182:6605-6613, 2000). The recently solved crystal structure of Cab shows the presence of a buffer molecule within hydrogen bonding distance of Asp-34, implying a role for this residue in the proton transport step (P. Strop, K. S. Smith, T. M. Iverson, J. G. Ferry, and D. C. Rees, J. Biol. Chem. 276:10299-10305, 2001). The k cat/Km values of Asp-34 variants were decreased relative to those of the wild type, although not to an extent which supports an essential role for this residue in the CO2 hydration step. Parallel decreases in k cat and k cat/Km values for the variants precluded any conclusions regarding a role for Asp-34 in the proton transfer step; however, the k cat of the D34A variant was chemically rescued by replacement of 2-(N-morpholino)propanesulfonic acid buffer with imidazole at pH 7.2, supporting a role for the conserved aspartate in the proton transfer step. The crystal structure of Cab also shows Arg-36 with two hydrogen bonds to Asp-34. Arg-36 variants had both k cat and k cat/Km values that were decreased at least 250-fold relative to those of the wild type, establishing an essential function for this residue. Imidazole was unable to rescue the k cat of the R36A variant; however, partial rescue of the kinetic parameter was obtained with guanidine-HCl indicating that the guanido group of this residue is important.

Structural and functional characterization of a glycoside hydrolase family 3 β-N-acetylglucosaminidase from Paenibacillus sp. str. FPU-7

2019 ◽  
Vol 166 (6) ◽  
pp. 503-515
Author(s):  
Takafumi Itoh ◽  
Tomomitsu Araki ◽  
Tomohiro Nishiyama ◽  
Takao Hibi ◽  
Hisashi Kimoto
Keyword(s):  
Crystal Structure ◽  
Glycoside Hydrolase ◽  
Catalytic Mechanism ◽  
Functional Characterization ◽  
Structural Features ◽  
Expression Cloning ◽  
Glycoside Hydrolase Family ◽  
Paenibacillus Sp ◽  
Hydrolase Family

Abstract Chitin, a β-1,4-linked homopolysaccharide of N-acetyl-d-glucosamine (GlcNAc), is one of the most abundant biopolymers on Earth. Paenibacillus sp. str. FPU-7 produces several different chitinases and converts chitin into N,N′-diacetylchitobiose ((GlcNAc)2) in the culture medium. However, the mechanism by which the Paenibacillus species imports (GlcNAc)2 into the cytoplasm and divides it into the monomer GlcNAc remains unclear. The gene encoding Paenibacillus β-N-acetyl-d-glucosaminidase (PsNagA) was identified in the Paenibacillus sp. str. FPU-7 genome using an expression cloning system. The deduced amino acid sequence of PsNagA suggests that the enzyme is a part of the glycoside hydrolase family 3 (GH3). Recombinant PsNagA was successfully overexpressed in Escherichia coli and purified to homogeneity. As assessed by gel permeation chromatography, the enzyme exists as a 57-kDa monomer. PsNagA specifically hydrolyses chitin oligosaccharides, (GlcNAc)2–4, 4-nitrophenyl N-acetyl β-d-glucosamine (pNP-GlcNAc) and pNP-(GlcNAc)2–6, but has no detectable activity against 4-nitrophenyl β-d-glucose, 4-nitrophenyl β-d-galactosamine and colloidal chitin. In this study, we present a 1.9 Å crystal structure of PsNagA bound to GlcNAc. The crystal structure reveals structural features related to substrate recognition and the catalytic mechanism of PsNagA. This is the first study on the structural and functional characterization of a GH3 β-N-acetyl-d-glucosaminidase from Paenibacillus sp.

scholarly journals A novel dimeric active site and regulation mechanism revealed by the crystal structure of iPLA2β

2017 ◽  
Author(s):  
Konstantin R. Malley ◽  
Olga Koroleva ◽  
Ian Miller ◽  
Ruslan Sanishvili ◽  
Christopher M. Jenkins ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Sites ◽  
Cellular Localization ◽  
Structural Features ◽  
Regulation Mechanism ◽  
Catalytic Domains ◽  
Physiological Processes ◽  
The Brain ◽  
Cooperative Activation

AbstractCalcium-independent phospholipase A2β (iPLA2β) regulates several physiological processes including inflammation, calcium homeostasis and apoptosis. It is linked genetically to neurodegenerative disorders including Parkinson’s disease. Despite its known enzymatic activity, the mechanisms underlying pathologic phenotypes remain unknown. Here, we present the first crystal structure of iPLA2β that significantly revises existing mechanistic models. The catalytic domains form a tight dimer. The ankyrin repeat domains wrap around the catalytic domains in an outwardly flared orientation, poised to interact with membrane proteins. The closely integrated active sites are positioned for cooperative activation and internal transacylation. A single calmodulin binds and allosterically inhibits both catalytic domains. These unique structural features identify the molecular interactions that can regulate iPLA2β activity and its cellular localization, which can be targeted to identify novel inhibitors for therapeutic purposes. The structure provides a well-defined framework to investigate the role of neurodegenerative mutations and the function of iPLA2β in the brain.

scholarly journals Allosteric activation of CwlD amidase activity by the GerS lipoprotein during Clostridioides difficile spore formation

2021 ◽  
Author(s):  
Carolina Alves Feliciano ◽  
Brian E Eckenroth ◽  
Oscar R Diaz ◽  
Syvlie Doublie ◽  
Aimee Shen
Keyword(s):  
Crystal Structure ◽  
Spore Germination ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Allosteric Activation ◽  
Amidase Activity ◽  
Clostridioides Difficile ◽  
Binding Partners ◽  
Insight Into ◽  
Peptidoglycan Layer

Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. Spore germination depends on the degradation of the protective spore peptidoglycan layer known as the spore cortex. Cortex degradation is mediated by enzymes that recognize the spore-specific peptidoglycan modification, muramic-∂-lactam (MAL). In C. difficile, MAL synthesis depends on the activity of the CwlD amidase and the GerS lipoprotein, which directly binds CwlD. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind zinc stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to zinc, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of zinc co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought.

scholarly journals A lipoprotein allosterically activates the CwlD amidase during Clostridioides difficile spore formation

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009791
Author(s):  
Carolina Alves Feliciano ◽  
Brian E. Eckenroth ◽  
Oscar R. Diaz ◽  
Sylvie Doublié ◽  
Aimee Shen
Keyword(s):  
Crystal Structure ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Protective Layer ◽  
Lytic Enzymes ◽  
Amidase Activity ◽  
Binding Partner ◽  
Clostridioides Difficile ◽  
Binding Partners ◽  
Insight Into

Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. During gemination, spores must degrade their cortex layer, which is a thick, protective layer of modified peptidoglycan. Cortex degradation depends on the presence of the spore-specific peptidoglycan modification, muramic-∂-lactam (MAL), which is specifically recognized by cortex lytic enzymes. In C. difficile, MAL production depends on the CwlD amidase and its binding partner, the GerS lipoprotein. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind Zn2+ stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to Zn2+, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of Zn2+ co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought.

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