Contributions of a unique β-clamp to substrate recognition illuminates the molecular basis of exolysis in ferulic acid esterases

2016 ◽  
Vol 473 (7) ◽  
pp. 839-849 ◽  
Author(s):  
Robert J. Gruninger ◽  
Chris Cote ◽  
Tim A. McAllister ◽  
D. Wade Abbott
Keyword(s):  
Ferulic Acid ◽  
Molecular Basis ◽  
Structural Model ◽  
Substrate Recognition ◽  
Hydroxycinnamic Acids ◽  
Flexible Loop

Ferulic acid esterases (FAEs) hydrolyse bonds between hemicellulose and hydroxycinnamic acids. We characterize an FAE that utilizes a flexible loop to bind substrate and that is essential for catalysis. We propose a structural model for endolytic compared with exolytic FAE activity.

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.

scholarly journals Mechanism of Substrate Recognition by Drug-Resistant Human Immunodeficiency Virus Type 1 Protease Variants Revealed by a Novel Structural Intermediate

2006 ◽  
Vol 80 (7) ◽  
pp. 3607-3616 ◽  
Author(s):  
Moses Prabu-Jeyabalan ◽  
Ellen A. Nalivaika ◽  
Keith Romano ◽  
Celia A. Schiffer
Keyword(s):  
Human Immunodeficiency Virus ◽  
Crystal Structures ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Structural Model ◽  
Substrate Recognition ◽  
Drug Resistant ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) protease processes and cleaves the Gag and Gag-Pol polyproteins, allowing viral maturation, and therefore is an important target for antiviral therapy. Ligand binding occurs when the flaps open, allowing access to the active site. This flexibility in flap geometry makes trapping and crystallizing structural intermediates in substrate binding challenging. In this study, we report two crystal structures of two HIV-1 protease variants bound with their corresponding nucleocapsid-p1 variant. One of the flaps in each of these structures exhibits an unusual “intermediate” conformation. Analysis of the flap-intermediate and flap-closed crystal structures reveals that the intermonomer flap movements may be asynchronous and that the flap which wraps over the P3 to P1 (P3-P1) residues of the substrate might close first. This is consistent with our hypothesis that the P3-P1 region is crucial for substrate recognition. The intermediate conformation is conserved in both the wild-type and drug-resistant variants. The structural differences between the variants are evident only when the flaps are closed. Thus, a plausible structural model for the adaptability of HIV-1 protease to recognize substrates in the presence of drug-resistant mutations has been proposed.

scholarly journals Molecular Basis of Sphingosine Kinase 1 Substrate Recognition and Catalysis

Structure ◽  
2013 ◽  
Vol 21 (5) ◽  
pp. 798-809 ◽  
Author(s):  
Zhulun Wang ◽  
Xiaoshan Min ◽  
Shou-Hua Xiao ◽  
Sheree Johnstone ◽  
William Romanow ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Substrate Recognition ◽  
Sphingosine Kinase ◽  
Sphingosine Kinase 1

scholarly journals Insights into substrate-mediated assembly of the chloroplast TAT receptor complex

2020 ◽  
Author(s):  
Qianqian Ma ◽  
Christopher Paul New ◽  
Carole Dabney-Smith
Keyword(s):  
Structural Model ◽  
Substrate Recognition ◽  
Transmembrane Helix ◽  
Complex Function ◽  
Transmembrane Helices ◽  
Translocation Event ◽  
Active Substrate ◽  
Tat System ◽  
Folded Proteins ◽  
Transport Complex

AbstractThe Twin Arginine Transport (TAT) system translocates fully folded proteins across the thylakoid membrane in the chloroplast (cp) and the cytoplasmic membrane of bacteria. In chloroplasts, cpTAT transport is achieved by three components: Tha4, Hcf106, and cpTatC. Hcf106 and cpTatC function as the substrate recognition/binding complex while Tha4 is thought to play a significant role in forming the translocation pore. Recent studies challenged this idea by suggesting that cpTatC-Hcf106-Tha4 function together in the active translocase. Here, we have mapped the inter-subunit contacts of cpTatC-Hcf106 during the resting state and built a cpTatC-Hcf106 structural model based on our crosslinking data. In addition, we have identified a substrate-mediated reorganization of cpTatC-Hcf106 contact sites during active substrate translocation. The proximity of Tha4 to the cpTatC-Hcf106 complex was also identified. Our data suggest a model for cpTAT function in which the transmembrane helices of Hcf106 and Tha4 may each contact the fifth transmembrane helix of cpTatC while the insertion of the substrate signal peptide may rearrange the cpTatC-Hcf106-Tha4 complex and initiate the translocation event.One sentence summaryProtein subunits of the thylakoidal twin arginine transport complex function together during substrate recognition and translocase assembly.

scholarly journals Insight into the molecular basis of substrate recognition by the wall teichoic acid glycosyltransferase TagA

2021 ◽  
pp. 101464
Author(s):  
Orlando E. Martinez ◽  
Brendan J. Mahoney ◽  
Andrew K. Goring ◽  
Sung-Wook Yi ◽  
Denise P. Tran ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Substrate Recognition ◽  
Teichoic Acid ◽  
Wall Teichoic Acid ◽  
Insight Into

The Role of the Flexible Loop in Substrate Recognition and Catalysis in Malate Dehydrogenase

2020 ◽  
Vol 34 (S1) ◽  
pp. 1-1
Author(s):  
Alexandra Roessling ◽  
Ellis Bell ◽  
Jessica Bell
Keyword(s):  
Malate Dehydrogenase ◽  
Substrate Recognition ◽  
Flexible Loop

scholarly journals Divergent Evolution of Eukaryotic CC- and A-Adding Enzymes

2020 ◽  
Vol 21 (2) ◽  
pp. 462 ◽  
Author(s):  
Lieselotte Erber ◽  
Paul Franz ◽  
Heike Betat ◽  
Sonja Prohaska ◽  
Mario Mörl
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Molecular Basis ◽  
Divergent Evolution ◽  
Full Activity ◽  
Flexible Loop ◽  
Evolutionary Origins ◽  
Loop Region ◽  
Restricted Activity ◽  
Salpingoeca Rosetta

Synthesis of the CCA end of essential tRNAs is performed either by CCA-adding enzymes or as a collaboration between enzymes restricted to CC- and A-incorporation. While the occurrence of such tRNA nucleotidyltransferases with partial activities seemed to be restricted to Bacteria, the first example of such split CCA-adding activities was reported in Schizosaccharomyces pombe. Here, we demonstrate that the choanoflagellate Salpingoeca rosetta also carries CC- and A-adding enzymes. However, these enzymes have distinct evolutionary origins. Furthermore, the restricted activity of the eukaryotic CC-adding enzymes has evolved in a different way compared to their bacterial counterparts. Yet, the molecular basis is very similar, as highly conserved positions within a catalytically important flexible loop region are missing in the CC-adding enzymes. For both the CC-adding enzymes from S. rosetta as well as S. pombe, introduction of the loop elements from closely related enzymes with full activity was able to restore CCA-addition, corroborating the significance of this loop in the evolution of bacterial as well as eukaryotic tRNA nucleotidyltransferases. Our data demonstrate that partial CC- and A-adding activities in Bacteria and Eukaryotes are based on the same mechanistic principles but, surprisingly, originate from different evolutionary events.

Sign in / Sign up

Export Citation Format

Share Document