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

Characterization of class II fumarase from Schistosoma mansoni provides the molecular basis for selective inhibition

2021 ◽  
Vol 175 ◽  
pp. 406-421
Author(s):  
Iara Aimê Cardoso ◽  
Aline Kusumota Luiz de Souza ◽  
Adam Muslem George Burgess ◽  
Iain Wyllie Chalmers ◽  
Karl Francis Hoffmann ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Molecular Basis ◽  
Selective Inhibition ◽  
Class Ii

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 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 Characterisation and modelling of a polyhydroxyalkanoate synthase fromAquitaleasp. USM4 reveals a mechanism for polymer elongation

2017 ◽  
Author(s):  
Aik-Hong Teh ◽  
Nyet-Cheng Chiam ◽  
Go Furusawa ◽  
Kumar Sudesh
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Substrate Specificity ◽  
Class Ii ◽  
Class I ◽  
Common Mechanism ◽  
The Third ◽  
Dimeric Interface ◽  
Key Enzyme ◽  
Polyhydroxyalkanoate Synthase ◽  
Key Residues

AbstractPolyhydroxyalkanoate synthase, PhaC, is a key enzyme in the biosynthesis of PHA, a type of bioplastics with huge potential to replace conventional petroleum-based plastics. While two PhaC structures have been determined recently, the exact mechanism remains unclear partly due to the absence of a tunnel for product passage. The PhaC fromAquitaleasp. USM4, PhaCAq, was characterised and showed aKmof 394 µM and akcatof 476.4 s−1on the 3HB-CoA substrate. A model based on the structure of the closely related PhaC fromCupriavidus necator, PhaCCnrevealed a three-branched tunnel at the dimeric interface. Two of the branches open to the solvent and serve as the putative routes for substrate entrance and product exit, while the third branch is elongated in a PhaC1 model fromPseudomonas aeruginosa, indicating a function of accommodating the hydroxyalkanoate (HA) moiety of the HA-CoA substrate. Docking of the two tetrahedral intermediates formed during catalysis suggests a PHA elongation mechanism that requires the HA moiety of the ligand to rotate ~180°. Both classes I and II PhaCs share a common mechanism for polymer elongation, and substrate specificity is determined in part by a bulky Phe/Tyr/Trp residue in the third branch in class I, which is conserved as Ala in class II to create room for longer substrates. The PhaCAqmodel provides fresh insights into a general PhaC mechanism, pinpointing key residues for potential engineering of PhaCs with desirable characteristics.

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