scholarly journals Crystal structure of the dopamine N-acetyltransferase–acetyl-CoA complex provides insights into the catalytic mechanism

2012 ◽  
Vol 446 (3) ◽  
pp. 395-404 ◽  
Author(s):  
Kuo-Chang Cheng ◽  
Jhen-Ni Liao ◽  
Ping-Chiang Lyu
Keyword(s):  
Crystal Structure ◽  
Catalytic Mechanism ◽  
Complex Structure ◽  
Kinetic Studies ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Acetyl Coa ◽  
Acetyl Transfer

The daily cycle of melatonin biosynthesis in mammals is regulated by AANAT (arylalkylamine N-acetyltransferase; EC 2.3.1.87), making it an attractive target for therapeutic control of abnormal melatonin production in mood and sleep disorders. Drosophila melanogaster Dat (dopamine N-acetyltransferase) is an AANAT. Until the present study, no insect Dat structure had been solved, and, consequently, the structural basis for its acetyl-transfer activity was not well understood. We report in the present paper the high-resolution crystal structure for a D. melanogaster Dat–AcCoA (acetyl-CoA) complex obtained using one-edge (selenium) single-wavelength anomalous diffraction. A binding study using isothermal titration calorimetry suggested that the cofactor bound to Dat first before substrate. Examination of the complex structure and a substrate-docked model indicated that Dat contains a novel AANAT catalytic triad. Site-directed mutagenesis, kinetic studies and pH-rate profiles confirmed that Glu47, Ser182 and Ser186 were critical for catalysis. Collectively, the results of the present study suggest that Dat possesses a specialized active site structure dedicated to a catalytic mechanism.

scholarly journals Structural basis of Naa20 activity towards a canonical NatB substrate

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Dominik Layer ◽  
Jürgen Kopp ◽  
Miriam Fontanillo ◽  
Maja Köhn ◽  
Karine Lapouge ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Drug Development ◽  
Catalytic Mechanism ◽  
Peptide Binding ◽  
Competitive Inhibitor ◽  
Structural Basis ◽  
Substrate Affinity ◽  
Protein Homeostasis ◽  
Auxiliary Subunit

AbstractN-terminal acetylation is one of the most common protein modifications in eukaryotes and is carried out by N-terminal acetyltransferases (NATs). It plays important roles in protein homeostasis, localization, and interactions and is linked to various human diseases. NatB, one of the major co-translationally active NATs, is composed of the catalytic subunit Naa20 and the auxiliary subunit Naa25, and acetylates about 20% of the proteome. Here we show that NatB substrate specificity and catalytic mechanism are conserved among eukaryotes, and that Naa20 alone is able to acetylate NatB substrates in vitro. We show that Naa25 increases the Naa20 substrate affinity, and identify residues important for peptide binding and acetylation activity. We present the first Naa20 crystal structure in complex with the competitive inhibitor CoA-Ac-MDEL. Our findings demonstrate how Naa20 binds its substrates in the absence of Naa25 and support prospective endeavors to derive specific NAT inhibitors for drug development.

Structural insights into the substrate specificity of a glycoside hydrolase family 5 lichenase from Caldicellulosiruptor sp. F32

2017 ◽  
Vol 474 (20) ◽  
pp. 3373-3389 ◽  
Author(s):  
Dong-Dong Meng ◽  
Xi Liu ◽  
Sheng Dong ◽  
Ye-Fei Wang ◽  
Xiao-Qing Ma ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Glycoside Hydrolase ◽  
Complex Structure ◽  
Dynamics Simulation ◽  
Structural Basis ◽  
Glycoside Hydrolase Family ◽  
Substrate Selectivity ◽  
Glucose Residue ◽  
Structural Insights

Glycoside hydrolase (GH) family 5 is one of the largest GH families with various GH activities including lichenase, but the structural basis of the GH5 lichenase activity is still unknown. A novel thermostable lichenase F32EG5 belonging to GH5 was identified from an extremely thermophilic bacterium Caldicellulosiruptor sp. F32. F32EG5 is a bi-functional cellulose and a lichenan-degrading enzyme, and exhibited a high activity on β-1,3-1,4-glucan but side activity on cellulose. Thin-layer chromatography and NMR analyses indicated that F32EG5 cleaved the β-1,4 linkage or the β-1,3 linkage while a 4-O-substitued glucose residue linked to a glucose residue through a β-1,3 linkage, which is completely different from extensively studied GH16 lichenase that catalyses strict endo-hydrolysis of the β-1,4-glycosidic linkage adjacent to a 3-O-substitued glucose residue in the mixed-linked β-glucans. The crystal structure of F32EG5 was determined to 2.8 Å resolution, and the crystal structure of the complex of F32EG5 E193Q mutant and cellotetraose was determined to 1.7 Å resolution, which revealed that the exit subsites of substrate-binding sites contribute to both thermostability and substrate specificity of F32EG5. The sugar chain showed a sharp bend in the complex structure, suggesting that a substrate cleft fitting to the bent sugar chains in lichenan is a common feature of GH5 lichenases. The mechanism of thermostability and substrate selectivity of F32EG5 was further demonstrated by molecular dynamics simulation and site-directed mutagenesis. These results provide biochemical and structural insights into thermostability and substrate selectivity of GH5 lichenases, which have potential in industrial processes.

scholarly journals Structural and biochemical characterization of bifunctional XynA

2020 ◽  
Author(s):  
Wei Xie ◽  
Qi Yu ◽  
Yun Liu ◽  
Ruoting Cao ◽  
Ruiqing Zhang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Enzyme Activity ◽  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Cellulase Activity ◽  
Cost Savings ◽  
Site Directed Mutagenesis ◽  
Enzyme Activity Assay ◽  
Great Cost ◽  
Terminal Domain

AbstractXylan and cellulose are the two major constituents in numerous types of lignocellulosic biomass, representing a promising resource for biofuels and other biobased industries. The efficient degradation of lignocellulose requires the synergistic actions of cellulase and xylanase. Thus, bifunctional enzyme incorporated xylanase/cellulase activity has attracted considerable attention since it has great cost savings potential. Recently, a novel GH10 family enzyme XynA identified from Bacillus sp. is found to degrade both cellulose and xylan. To understand its molecular catalytic mechanism, here we first solve the crystal structure of XynA at 2.3 Å. XynA is characterized with a classic (α/β)8 TIM-barrel fold (GH10 domain) flanked by the flexible N-terminal domain and C-terminal domain. Circular dichroism, protein thermal shift and enzyme activity assays reveal that conserved residues Glu182 and Glu280 are both important for catalytic activities of XynA, which is verified by the crystal structure of XynA with E182A/E280A double mutant. Molecular docking studies of XynA with xylohexaose and cellohexaose as well as site-directed mutagenesis and enzyme activity assay demonstrat that Gln250 and His252 are indispensible to cellulase and bifunctional activity, separately. These results elucidate the structural and biochemical features of XynA, providing clues for further modification of XynA for industrial application.

scholarly journals Structural Basis of Ubiquitin Recognition by the Ubiquitin-associated (UBA) Domain of the Ubiquitin Ligase EDD

2007 ◽  
Vol 282 (49) ◽  
pp. 35787-35795 ◽  
Author(s):  
Guennadi Kozlov ◽  
Long Nguyen ◽  
Tong Lin ◽  
Gregory De Crescenzo ◽  
Morag Park ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Polyubiquitin Chains ◽  
C Terminus ◽  
Damage Repair ◽  
The Family ◽  
Uba Domain ◽  
Ordered Water

EDD (or HYD) is an E3 ubiquitin ligase in the family of HECT (homologous to E6-AP C terminus) ligases. EDD contains an N-terminal ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated processes. Here, we use isothermal titration calorimetry (ITC), NMR titrations, and pull-down assays to show that the EDD UBA domain binds ubiquitin. The 1.85Å crystal structure of the complex with ubiquitin reveals the structural basis of ubiquitin recognition by UBA helices α1 and α3. The structure shows a larger number of intermolecular hydrogen bonds than observed in previous UBA/ubiquitin complexes. Two of these involve ordered water molecules. The functional importance of residues at the UBA/ubiquitin interface was confirmed using site-directed mutagenesis. Surface plasmon resonance (SPR) measurements show that the EDD UBA domain does not have a strong preference for polyubiquitin chains over monoubiquitin. This suggests that EDD binds to monoubiquitinated proteins, which is consistent with its involvement in DNA damage repair pathways.

scholarly journals Catalytic mechanism of the glutathione peroxidase-type tryparedoxin peroxidase of Trypanosoma brucei

2007 ◽  
Vol 405 (3) ◽  
pp. 445-454 ◽  
Author(s):  
Tanja Schlecker ◽  
Marcelo A. Comini ◽  
Johannes Melchers ◽  
Thomas Ruppert ◽  
R. Luise Krauth-Siegel
Keyword(s):  
Glutathione Peroxidase ◽  
Trypanosoma Brucei ◽  
Catalytic Mechanism ◽  
Disulfide Bridge ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Glutathione Peroxidases ◽  
Thiolate Anion ◽  
In The Wild

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three nearly identical genes for cysteine-homologues of the selenocysteine-containing glutathione peroxidases. The enzymes, which are essential for the parasites, lack glutathione peroxidase activity but catalyse the trypanothione/Tpx (tryparedoxin)-dependent reduction of hydroperoxides. Cys47, Gln82 and Trp137 correspond to the selenocysteine, glutamine and tryptophan catalytic triad of the mammalian selenoenzymes. Site-directed mutagenesis revealed that Cys47 and Gln82 are essential. A glycine mutant of Trp137 had 13% of wild-type activity, which suggests that the aromatic residue may play a structural role but is not directly involved in catalysis. Cys95, which is conserved in related yeast and plant proteins but not in the mammalian selenoenzymes, proved to be essential as well. In contrast, replacement of the highly conserved Cys76 by a serine residue resulted in a fully active enzyme species and its role remains unknown. Thr50, proposed to stabilize the thiolate anion at Cys47, is also not essential for catalysis. Treatment of the C76S/C95S but not of the C47S/C76S double mutant with H2O2 induced formation of a sulfinic acid and covalent homodimers in accordance with Cys47 being the peroxidative active site thiol. In the wild-type peroxidase, these oxidations are prevented by formation of an intramolecular disulfide bridge between Cys47 and Cys95. As shown by MS, regeneration of the reduced enzyme by Tpx involves a transient mixed disulfide between Cys95 of the peroxidase and Cys40 of Tpx. The catalytic mechanism of the Tpx peroxidase resembles that of atypical 2-Cys-peroxiredoxins but is distinct from that of the selenoenzymes.

scholarly journals Family D DNA polymerase interacts with GINS to promote CMG-helicase in the archaeal replisome

2021 ◽  
Author(s):  
Keisuke Oki ◽  
Mariko Nagata ◽  
Takeshi Yamagami ◽  
Tomoyuki Numata ◽  
Sonoko Ishino ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dna Polymerase ◽  
Direct Interaction ◽  
Crystal Structure Analysis ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Functional Connection ◽  
Terminal Domain ◽  
The Family ◽  
Lagging Strand

Abstract Genomic DNA replication requires replisome assembly. We show here the molecular mechanism by which CMG (GAN–MCM–GINS)-like helicase cooperates with the family D DNA polymerase (PolD) in Thermococcus kodakarensis. The archaeal GINS contains two Gins51 subunits, the C-terminal domain of which (Gins51C) interacts with GAN. We discovered that Gins51C also interacts with the N-terminal domain of PolD’s DP1 subunit (DP1N) to connect two PolDs in GINS. The two replicases in the replisome should be responsible for leading- and lagging-strand synthesis, respectively. Crystal structure analysis of the DP1N–Gins51C–GAN ternary complex was provided to understand the structural basis of the connection between the helicase and DNA polymerase. Site-directed mutagenesis analysis supported the interaction mode obtained from the crystal structure. Furthermore, the assembly of helicase and replicase identified in this study is also conserved in Eukarya. PolD enhances the parental strand unwinding via stimulation of ATPase activity of the CMG-complex. This is the first evidence of the functional connection between replicase and helicase in Archaea. These results suggest that the direct interaction of PolD with CMG-helicase is critical for synchronizing strand unwinding and nascent strand synthesis and possibly provide a functional machinery for the effective progression of the replication fork.

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 Structural basis for the O-acetyltransferase function of the extracytoplasmic domain of OatA from Staphylococcus aureus

2020 ◽  
Vol 295 (24) ◽  
pp. 8204-8213 ◽  
Author(s):  
Carys S. Jones ◽  
David Sychantha ◽  
P. Lynne Howell ◽  
Anthony J. Clarke
Keyword(s):  
Staphylococcus Aureus ◽  
Biochemical Characterization ◽  
Multidrug Resistant ◽  
Catalytic Triad ◽  
Structural Basis ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Hydrolase Fold ◽  
Acetyl Transfer ◽  
Human Infections

Many bacteria possess enzymes that modify the essential cell-wall polymer peptidoglycan by O-acetylation. This modification occurs in numerous Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, a common cause of human infections. O-Acetylation of peptidoglycan protects bacteria from the lytic activity of lysozyme, a mammalian innate immune enzyme, and as such is important for bacterial virulence. The O-acetylating enzyme in Gram-positive bacteria, O-acetyltransferase A (OatA), is a two-domain protein consisting of an N-terminal integral membrane domain and a C-terminal extracytoplasmic domain. Here, we present the X-ray crystal structure at 1.71 Å resolution and the biochemical characterization of the C-terminal domain of S. aureus OatA. The structure revealed that this OatA domain adopts an SGNH-hydrolase fold and possesses a canonical catalytic triad. Site-specific replacement of active-site amino acids revealed the presence of a water-coordinating aspartate residue that limits esterase activity. This residue, although conserved in staphyloccocal OatA and most other homologs, is not present in the previously characterized streptococcal OatA. These results provide insights into the mechanism of acetyl transfer in the SGNH/GDSL hydrolase family and highlight important evolutionary differences between homologous OatA enzymes. Furthermore, this study enhances our understanding of PG O-acetyltransferases, which could guide the development of novel antibacterial drugs to combat infections with multidrug-resistant bacterial pathogens.

scholarly journals Crystal Structure of TDP-Fucosamine Acetyltransferase (WecD) from Escherichia coli, an Enzyme Required for Enterobacterial Common Antigen Synthesis

2006 ◽  
Vol 188 (15) ◽  
pp. 5606-5617 ◽  
Author(s):  
Ming-Ni Hung ◽  
Erumbi Rangarajan ◽  
Christine Munger ◽  
Guy Nadeau ◽  
Traian Sulea ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Coenzyme A ◽  
Structural Data ◽  
Enteric Bacteria ◽  
Common Antigen ◽  
Acetyl Coa ◽  
Enterobacterial Common Antigen ◽  
Thiolate Anion ◽  
Acetyl Transfer

ABSTRACT Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose, organized into trisaccharide repeating units having the sequence →3)-α-d-Fuc4NAc-(1→4)-β-d-ManNAcA-(1→4)-α-d-GlcNAc-(1→. While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin-producing Escherichia coli (STEC) O157:H7 to organic acids and the resistance of Salmonella enterica to bile salts. The final step in the synthesis of 4-acetamido-4,6-dideoxy-d-galactose, the acetyl-coenzyme A (CoA)-dependent acetylation of the 4-amino group, is carried out by TDP-fucosamine acetyltransferase (WecD). We have determined the crystal structure of WecD in apo form at a 1.95-Å resolution and bound to acetyl-CoA at a 1.66-Å resolution. WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars. The crystal structure of WecD, however, represents the first structure of a GNAT family member that acts on nucleotide sugars. Based on this cocrystal structure, we have used flexible docking to generate a WecD-bound model of the acetyl-CoA-TDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. Our structural data show that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of coenzyme A.

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