scholarly journals Decision letter: Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

2020 ◽  
Author(s):  
Tony Hunter ◽  
David Barford
Keyword(s):  
Substrate Specificity ◽  
Molecular Basis

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 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.

scholarly journals Molecular Basis for Ser/Thr Specificity in PKA Signaling

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1548 ◽  
Author(s):  
Matthias J. Knape ◽  
Maximilian Wallbott ◽  
Nicole C. G. Burghardt ◽  
Daniela Bertinetti ◽  
Jan Hornung ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Substrate Specificity ◽  
Metal Ions ◽  
Protein Kinases ◽  
Molecular Basis ◽  
Kinase Inhibitor ◽  
Adenine Nucleotides ◽  
Catalytic Efficiency ◽  
Specific Protein ◽  
Divalent Metal Ions

cAMP-dependent protein kinase (PKA) is the major receptor of the second messenger cAMP and a prototype for Ser/Thr-specific protein kinases. Although PKA strongly prefers serine over threonine substrates, little is known about the molecular basis of this substrate specificity. We employ classical enzyme kinetics and a surface plasmon resonance (SPR)-based method to analyze each step of the kinase reaction. In the absence of divalent metal ions and nucleotides, PKA binds serine (PKS) and threonine (PKT) substrates, derived from the heat-stable protein kinase inhibitor (PKI), with similar affinities. However, in the presence of metal ions and adenine nucleotides, the Michaelis complex for PKT is unstable. PKA phosphorylates PKT with a higher turnover due to a faster dissociation of the product complex. Thus, threonine substrates are not necessarily poor substrates of PKA. Mutation of the DFG+1 phenylalanine to β-branched amino acids increases the catalytic efficiency of PKA for a threonine peptide substrate up to 200-fold. The PKA Cα mutant F187V forms a stable Michaelis complex with PKT and shows no preference for serine versus threonine substrates. Disease-associated mutations of the DFG+1 position in other protein kinases underline the importance of substrate specificity for keeping signaling pathways segregated and precisely regulated.

scholarly journals Structural and mutational analyses of dipeptidyl peptidase 11 from Porphyromonas gingivalis reveal the molecular basis for strict substrate specificity

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Yasumitsu Sakamoto ◽  
Yoshiyuki Suzuki ◽  
Ippei Iizuka ◽  
Chika Tateoka ◽  
Saori Roppongi ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Porphyromonas Gingivalis ◽  
Molecular Basis ◽  
Dipeptidyl Peptidase

scholarly journals The Molecular Basis of K+ Exclusion by the Escherichia coli Ammonium Channel AmtB

2013 ◽  
Vol 288 (20) ◽  
pp. 14080-14086 ◽  
Author(s):  
Jason A. Hall ◽  
Dalai Yan
Keyword(s):  
Escherichia Coli ◽  
Membrane Potential ◽  
Substrate Specificity ◽  
Concentration Gradient ◽  
Molecular Basis ◽  
Subsequent Work ◽  
Mutant Proteins ◽  
Gas Molecules

Members of the Amt family of channels mediate the transport of ammonium. The form of ammonium, NH3 or NH4+, carried by these proteins remains controversial, and the mechanism by which they select against K+ ions is unclear. We describe here a set of Escherichia coli AmtB proteins carrying mutations at the conserved twin-histidine site within the conduction pore that have altered substrate specificity and now transport K+. Subsequent work established that AmtB-mediated K+ uptake occurred against a concentration gradient and was membrane potential-dependent. These findings indicate that the twin-histidine element serves as a filter to prevent K+ conduction and strongly support the notion that Amt proteins transport cations (NH4+ or, in mutant proteins, K+) rather than NH3 gas molecules through their conduction pores.

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