scholarly journals Molecular mechanism of N-terminal acetylation by the ternary NatC complex

2021 ◽  
Author(s):  
Sunbin Deng ◽  
Leah Gottlieb ◽  
Buyan Pan ◽  
Julianna Supplee ◽  
Xuepeng Wei ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Molecular Mechanism ◽  
Molecular Basis ◽  
Catalytic Subunit ◽  
Inositol Hexaphosphate ◽  
Auxiliary Subunits

AbstractProtein N-terminal acetylation is predominantly a ribosome-associated modification, with NatA-E serving as the major enzymes. NatC is the most unusual of these enzymes, containing one Naa30 catalytic subunit and two auxiliary subunits, Naa35 and Naa38; and substrate specificity profile that overlaps with NatE. Here, we report the Cryo-EM structure of S. pombe NatC with a NatE/C-type bisubstrate analogue and inositol hexaphosphate (IP6), and associated biochemistry studies. We find that the presence of three subunits is a prerequisite for normal NatC acetylation activity in yeast and that IP6 binds tightly to NatC to stabilize the complex. We also describe the molecular basis for IP6-mediated NatC complex stabilization and the overlapping yet distinct substrate profiles of NatC and NatE.

Human Intestinal Maltase–Glucoamylase: Crystal Structure of the N-Terminal Catalytic Subunit and Basis of Inhibition and Substrate Specificity

2008 ◽  
Vol 375 (3) ◽  
pp. 782-792 ◽  
Author(s):  
Lyann Sim ◽  
Roberto Quezada-Calvillo ◽  
Erwin E. Sterchi ◽  
Buford L. Nichols ◽  
David R. Rose
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Catalytic Subunit ◽  
Human Intestinal Maltase

scholarly journals The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation

2019 ◽  
Author(s):  
Natalie S. Al-Otaibi ◽  
Aidan J. Taylor ◽  
Daniel P. Farrell ◽  
Svetomir B. Tzokov ◽  
Frank DiMaio ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Campylobacter Jejuni ◽  
Bacterial Cell ◽  
Molecular Basis ◽  
Molecular Model ◽  
Molecular Motor ◽  
Terminal Region ◽  
Bacterial Flagellum ◽  
Filament Assembly ◽  
Oligomeric Assembly

AbstractThe bacterial flagellum is a remarkable molecular motor, present at the surface of many bacteria, whose primary function is to allow motility through the rotation of a long filament protruding from the bacterial cell. A cap complex, consisting of an oligomeric assembly of the protein FliD, is localized at the tip of the flagellum, and is essential for filament assembly, as well as adherence to surfaces in some bacteria. However, the structure of the intact cap complex, and the molecular basis for its interaction with the filament, remains elusive. Here we report the cryo-EM structure of the Campylobacter jejuni cap complex. This structure reveals that FliD is pentameric, with the N-terminal region of the protomer forming an unexpected extensive set of contacts across several subunits, that contribute to FliD oligomerization. We also demonstrate that the native C. jejuni flagellum filament is 11-stranded and propose a molecular model for the filament-cap interaction.

scholarly journals Molecular Basis of Substrate Recognition of Endonuclease Q from the Euryarchaeon Pyrococcus furiosus

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Miyako Shiraishi ◽  
Shigenori Iwai
Keyword(s):  
Dna Repair ◽  
Bacillus Subtilis ◽  
Substrate Specificity ◽  
Molecular Basis ◽  
Pyrococcus Furiosus ◽  
Substrate Recognition ◽  
Base Flipping ◽  
Content Type ◽  
Cleavage Activity

ABSTRACT Endonuclease Q (EndoQ), a DNA repair endonuclease, was originally identified in the hyperthermophilic euryarchaeon Pyrococcus furiosus in 2015. EndoQ initiates DNA repair by generating a nick on DNA strands containing deaminated bases and an abasic site. Although EndoQ is thought to be important for maintaining genome integrity in certain bacteria and archaea, the underlying mechanism catalyzed by EndoQ remains unclear. Here, we provide insights into the molecular basis of substrate recognition by EndoQ from P. furiosus (PfuEndoQ) using biochemical approaches. Our results of the substrate specificity range and the kinetic properties of PfuEndoQ demonstrate that PfuEndoQ prefers the imide structure in nucleobases along with the discovery of its cleavage activity toward 5,6-dihydrouracil, 5-hydroxyuracil, 5-hydroxycytosine, and uridine in DNA. The combined results for EndoQ substrate binding and cleavage activity analyses indicated that PfuEndoQ flips the target base from the DNA duplex, and the cleavage activity is highly dependent on spontaneous base flipping of the target base. Furthermore, we find that PfuEndoQ has a relatively relaxed substrate specificity; therefore, the role of EndoQ in restriction modification systems was explored. The activity of the EndoQ homolog from Bacillus subtilis was found not to be inhibited by the uracil glycosylase inhibitor from B. subtilis bacteriophage PBS1, whose genome is completely replaced by uracil instead of thymine. Our findings suggest that EndoQ not only has additional functions in DNA repair but also could act as an antiviral enzyme in organisms with EndoQ. IMPORTANCE Endonuclease Q (EndoQ) is a lesion-specific DNA repair enzyme present in certain bacteria and archaea. To date, it remains unclear how EndoQ recognizes damaged bases. Understanding the mechanism of substrate recognition by EndoQ is important to grasp genome maintenance systems in organisms with EndoQ. Here, we find that EndoQ from the euryarchaeon Pyrococcus furiosus recognizes the imide structure in nucleobases by base flipping, and the cleavage activity is enhanced by the base pair instability of the target base, along with the discovery of its cleavage activity toward 5,6-dihydrouracil, 5-hydroxyuracil, 5-hydroxycytosine, and uridine in DNA. Furthermore, a potential role of EndoQ in Bacillus subtilis as an antiviral enzyme by digesting viral genome is demonstrated.

Probing the Molecular Basis of Substrate Specificity, Stereospecificity, and Catalysis in the Class II Pyruvate Aldolase, BphI

Biochemistry ◽  
2011 ◽  
Vol 50 (17) ◽  
pp. 3559-3569 ◽  
Author(s):  
Perrin Baker ◽  
Jason Carere ◽  
Stephen Y. K. Seah
Keyword(s):  
Substrate Specificity ◽  
Molecular Basis ◽  
Class Ii

scholarly journals Molecular basis for acceptor substrate specificity of the human β1,3-glucuronosyltransferases GlcAT-I and GlcAT-P involved in glycosaminoglycan and HNK-1 carbohydrate epitope biosynthesis, respectively

Glycobiology ◽  
2007 ◽  
Vol 17 (8) ◽  
pp. 857-867 ◽  
Author(s):  
Magali Fondeur-Gelinotte ◽  
Virginie Lattard ◽  
Sandrine Gulberti ◽  
Rafael Oriol ◽  
Guillermo Mulliert ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Molecular Basis ◽  
Acceptor Substrate ◽  
Carbohydrate Epitope

scholarly journals Molecular Basis for the Relative Substrate Specificity of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Proteases

2001 ◽  
Vol 75 (19) ◽  
pp. 9458-9469 ◽  
Author(s):  
Zachary Q. Beck ◽  
Ying-Chuan Lin ◽  
John H. Elder
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Feline Immunodeficiency Virus ◽  
Molecular Basis ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3′ region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF↓VVNGLVK-NH2 (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2′ position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1′, FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2′ subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1′ subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF↓VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVFΨ(CH2NH)VVNGL-NH2. This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.

scholarly journals Unique ATP-cone-driven allosteric regulation of ribonucleotide reductase via the radical-generating subunit

2017 ◽  
Author(s):  
Inna Rozman Grinberg ◽  
Daniel Lundin ◽  
Mahmudul Hasan ◽  
Mikael Crona ◽  
Venkateswara Rao Jonna ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Quaternary Structure ◽  
Catalytic Subunit ◽  
Present Evidence ◽  
Key Enzymes ◽  
Ribonucleotide Reductases ◽  
Allosteric Mechanisms

AbstractRibonucleotide reductases (RNRs) are key enzymes in DNA synthesis and repair, with sophisticated allosteric mechanisms controlling both substrate specificity and overall activity. In RNRs, the activity master-switch, the ATP-cone, has been found exclusively in the catalytic subunit. In two class I RNR subclasses whose catalytic subunit lacks the ATP-cone, we discovered ATP-cones in the radical-generating subunit. The ATP-cone in the Leewenhoekiella blandensis radical-generating subunit regulates activity via modifications of quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas dATP induces non-productive tetramers, resulting in different holoenzyme complexes. The tetramer forms solely by interactions between ATP-cones, as evidenced by a 2.45 Å crystal structure. We also present evidence for an MnIIIMnIV metal center. In summary, lack of an ATP-cone domain in the catalytic subunit was compensated by evolutionary capture of the domain by the radical-generating subunit. Our findings present a novel opportunity for dATP-regulation of engineered proteins.

scholarly journals Structure of the RZZ complex and molecular basis of Spindly-driven corona assembly at human kinetochores

2021 ◽  
Author(s):  
Tobias Raisch ◽  
Giuseppe Ciossani ◽  
Ennio d’Amico ◽  
Verena Cmentowski ◽  
Sara Carmignani ◽  
...  
Keyword(s):  
High Resolution ◽  
Binding Site ◽  
Molecular Mechanism ◽  
Building Block ◽  
Molecular Basis ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Structural Requirements ◽  
Necessary And Sufficient

In metazoans, a ≍1 megadalton (MDa) super-complex comprising the Dynein-Dynactin adaptor Spindly and the ROD-Zwilch-ZW10 (RZZ) complex is the building block of a fibrous biopolymer, the kinetochore fibrous corona. The corona assembles on mitotic kinetochores to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. We report here a high-resolution cryo-EM structure that captures the essential features of the RZZ complex, including a farnesyl binding site required for Spindly binding. Using a highly predictive in vitro assay, we demonstrate that the SAC kinase MPS1 is necessary and sufficient for corona assembly at supercritical concentrations of the RZZ-Spindly (RZZS) complex, and describe the molecular mechanism of phosphorylation-dependent filament nucleation. We identify several structural requirements for RZZS polymerization in rings and sheets. Finally, we identify determinants of kinetochore localization and corona assembly of Spindly. Our results describe a framework for the long-sought-for molecular basis of corona assembly on metazoan kinetochores.

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