Role of the N-terminus in human 4-hydroxyphenylpyruvate dioxygenase activity

2019 ◽  
Vol 167 (3) ◽  
pp. 315-322
Author(s):  
An-Ning Feng ◽  
Chih-Wei Huang ◽  
Chi-Huei Lin ◽  
Yung-Lung Chang ◽  
Meng-Yuan Ni ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Dynamics Simulation ◽  
Wild Type ◽  
Catalytic Function ◽  
C Terminus ◽  
N Terminus ◽  
Key Enzyme ◽  
The Stability

Abstract 4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in tyrosine catabolism, catalysing the oxidation of 4-hydroxyphenylpyruvate to homogentisate. Genetic deficiency of this enzyme causes type III tyrosinaemia. The enzyme comprises two barrel-shaped domains formed by the N- and C-termini, with the active site located in the C-terminus. This study investigated the role of the N-terminus, located at the domain interface, in HPPD activity. We observed that the kcat/Km decreased ∼8-fold compared with wild type upon removal of the 12 N-terminal residues (ΔR13). Interestingly, the wild-type level of activity was retained in a mutant missing the 17 N-terminal residues, with a kcat/Km 11-fold higher than that of the ΔR13 mutant; however, the structural stability of this mutant was lower than that of wild type. A 2-fold decrease in catalytic efficiency was observed for the K10A and E12A mutants, indicating synergism between these residues in the enzyme catalytic function. A molecular dynamics simulation showed large RMS fluctuations in ΔR13 suggesting that conformational flexibility at the domain interface leads to lower activity in this mutant. These results demonstrate that the N-terminus maintains the stability of the domain interface to allow for catalysis at the active site of HPPD.

scholarly journals The role of an evolutionarily conserved cis-proline in the thioredoxin-like domain of human class Alpha glutathione transferase A1-1

2003 ◽  
Vol 372 (1) ◽  
pp. 241-246 ◽  
Author(s):  
Chris NATHANIEL ◽  
Louise A. WALLACE ◽  
Jonathan BURKE ◽  
Heini W. DIRR
Keyword(s):  
Glutathione Transferase ◽  
Conformational Stability ◽  
Wild Type ◽  
Catalytic Function ◽  
Evolutionarily Conserved ◽  
Glutathione S Transferases ◽  
Equilibrium Unfolding ◽  
The Stability ◽  
Unfolding Kinetics

The thioredoxin-like fold has a βαβαββα topology, and most proteins/domains with this fold have a topologically conserved cis-proline residue at the N-terminus of β-strand 3. This residue plays an important role in the catalytic function and stability of thioredoxin-like proteins, but is reported not to contribute towards the stability of glutathione S-transferases (GSTs) [Allocati, Casalone, Masulli, Caccarelli, Carletti, Parker and Di Ilio (1999) FEBS Lett. 445, 347–350]. In order to further address the role of the cis-proline in the structure, function and stability of GSTs, cis-Pro-56 in human GST (hGST) A1-1 was replaced with a glycine, and the properties of the P56G mutant were compared with those of the wild-type protein. Not only was the catalytic function of the mutant dramatically reduced, so was its conformational stability, as indicated by equilibrium unfolding and unfolding kinetics experiments with urea as denaturant. These findings are discussed in the context of other thioredoxin-like proteins.

Molecular Modeling and Virtual Screening of Molecular Inhibitors for Leptospiral Collagenase

2021 ◽  
Author(s):  
Vikram Kumar ◽  
Nagesh Srikaku ◽  
Veeranarayanan Surya Aathmanathan ◽  
Padikara K Satheeshkumar ◽  
Madanan Gopalakrishnan Madathiparambil ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Modeling ◽  
Active Site ◽  
Binding Sites ◽  
Catalytic Site ◽  
Simulation Studies ◽  
C Terminus ◽  
Ligand Binding Sites ◽  
N Terminus ◽  
The Stability

Abstract Collagenase is a virulence factor which facilitates the invasion of pathogenic Leptospira into the host. In the present study, the model of Leptopsiral collagenase was constructed by employing threading method with the crystal structure of collagenase G. Three ligand binding sites at N- terminus, catalytic site and C-terminus were predicted by Metapocket server. Among sixty seven inhibitors from the ChEBI and Zinc databases, Protohypericin is predicted as the best inhibitor since it binds at the catalytic site of Leptopsiral collagenase. Molecular dynamic simulation studies validated the stability of interaction between the active site of Leptospiral collagenase and Protohypericin. The docking and molecular simulation studies corroborated the potential of the ligand to curb leptospiral infection.

scholarly journals Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

2016 ◽  
Vol 60 (5) ◽  
pp. 3123-3126 ◽  
Author(s):  
Carlo Bottoni ◽  
Mariagrazia Perilli ◽  
Francesca Marcoccia ◽  
Alessandra Piccirilli ◽  
Cristina Pellegrini ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

Distinct functional roles of the two terminal halves of eukaryotic phosphofructokinase

2012 ◽  
Vol 445 (2) ◽  
pp. 213-218 ◽  
Author(s):  
Oscar H. Martínez-Costa ◽  
Valentina Sánchez ◽  
Antonio Lázaro ◽  
Eloy D. Hernández ◽  
Keith Tornheim ◽  
...  
Keyword(s):  
Active Site ◽  
Kinetic Properties ◽  
Wild Type ◽  
Allosteric Site ◽  
Functional Roles ◽  
Allosteric Interactions ◽  
C Terminus ◽  
N Terminus ◽  
Metabolite Binding

Eukaryotic PFK (phosphofructokinase), a key regulatory enzyme in glycolysis, has homologous N- and C-terminal domains thought to result from duplication, fusion and divergence of an ancestral prokaryotic gene. It has been suggested that both the active site and the Fru-2,6-P2 (fructose 2,6-bisphosphate) allosteric site are formed by opposing N- and C-termini of subunits orientated antiparallel in a dimer. In contrast, we show in the present study that in fact the N-terminal halves form the active site, since expression of the N-terminal half of the enzymes from Dictyostelium discoideum and human muscle in PFK-deficient yeast restored growth on glucose. However, the N-terminus alone was not stable in vitro. The C-terminus is not catalytic, but is needed for stability of the enzyme, as is the connecting peptide that normally joins the two domains (here included in the N-terminus). Co-expression of homologous, but not heterologous, N- and C-termini yielded stable fully active enzymes in vitro with sizes and kinetic properties similar to those of the wild-type tetrameric enzymes. This indicates that the separately translated domains can fold sufficiently well to bind to each other, that such binding of complementary domains is stable and that the alignment is sufficiently accurate and tight as to preserve metabolite binding sites and allosteric interactions.

scholarly journals Significant role of Asn-247 and Arg-64 residues in close proximity of the active site in maintaining the catalytic function of CTX-M-15 type β-lactamase

RSC Advances ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 5325-5337 ◽  
Author(s):  
Lubna Maryam ◽  
Shamsi Khalid ◽  
Abid Ali ◽  
Asad U. Khan
Keyword(s):  
Amino Acid ◽  
Significant Role ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Beta Lactamase ◽  
Amino Acid Residues ◽  
Catalytic Function ◽  
Close Proximity

Mutations of amino acid residues present near active site decrease the catalytic efficiency of beta lactamase enzymes.

Contribution of the 60 Loop To Substrate and Inhibitor Specificity of Thrombin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1734-1734 ◽  
Author(s):  
Alireza R. Rezaie
Keyword(s):  
Active Site ◽  
Protein C ◽  
Activated Protein C ◽  
Catalytic Efficiency ◽  
Inhibitor Specificity ◽  
Active Site Pocket ◽  
Catalytic Function ◽  
Target Molecules

Relative to chymotrypsin, the 60-loop of thrombin contains 8–9 insertion residues which are believed to be partly responsible for the restricted substrate and inhibitor specificity of thrombin. Previous deletion of 3–4 residues of this loop (des-PPW and des-YPPW) dramatically impaired the activity of thrombin toward antithrombin, protein C and fibrinogen, implicating a key role for the productive interaction of these residues with the target macromolecules. To further investigate the role of this loop, we expressed a mutant of thrombin in which all 8 insertion residues (Tyr-Pro-Pro-Trp-Asp-Lys-Asn-Phe) of the 60-loop were deleted (des-60-loop). In contrast to the partially deleted loop mutants, we discovered that des-60-loop thrombin cleaved small synthetic substrates, clotted purified fibrinogen, and activated protein C with a near normal catalytic efficiency; however, its activity toward cofactors V and VIII was impaired ~2–4-fold. Further studies revealed that the reactivity of des-60-loop with antithrombin is not impaired, but rather improved ~2-fold. Remarkably, the mutant could also activate prothrombin to thrombin. These results suggest that the 60-loop plays a key role in regulating the specificity of thrombin by shielding the active-site pocket; however, its productive interaction with the target molecules may not be as critical for the catalytic function of thrombin as has been speculated in previous reports.

scholarly journals Role of Flexibility in Protein-DNA-Drug Recognition: The Case of Asp677Gly-Val703Ile Topoisomerase Mutant Hypersensitive to Camptothecin

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Ilda D'Annessa ◽  
Cinzia Tesauro ◽  
Paola Fiorani ◽  
Giovanni Chillemi ◽  
Silvia Castelli ◽  
...  
Keyword(s):  
Active Site ◽  
Drug Sensitivity ◽  
Dynamics Simulation ◽  
Wild Type ◽  
Dna Topoisomerase ◽  
Dynamical Properties ◽  
Dna Substrate ◽  
Covalent Complex ◽  
Linker Flexibility

Topoisomerases I are ubiquitous enzymes that control DNA topology within the cell. They are the unique target of the antitumor drug camptothecin that selectively recognizes the DNA-topoisomerase covalent complex and reversibly stabilizes it. The biochemical and structural-dynamical properties of the Asp677Gly-Val703Ile double mutant with enhanced CPT sensitivity have been investigated. The mutant displays a lower religation rate of the DNA substrate when compared to the wild-type protein. Analyses of the structural dynamical properties by molecular dynamics simulation show that the mutant has reduced flexibility and an active site partially destructured at the level of the Lys532 residue. These results demonstrate long-range communication mechanism where reduction of the linker flexibility alters the active site geometry with the consequent lowering of the religation rate and increase in drug sensitivity.

Human cytomegalovirus UL48 deubiquitinase primarily targets innermost tegument proteins pp150 and itself to regulate their stability and protects virions from inclusion of ubiquitin conjugates

2021 ◽  
Author(s):  
Ki Mun Kwon ◽  
Young Eui Kim ◽  
Myoung Kyu Lee ◽  
Woo-Chang Chung ◽  
Seokhwan Hyeon ◽  
...  
Keyword(s):  
Immune Responses ◽  
Active Site ◽  
Viral Proteins ◽  
Innate Immune ◽  
Wild Type Virus ◽  
Innate Immune Responses ◽  
Wild Type ◽  
Tegument Proteins ◽  
The Stability

Viral deubiquitinases (DUBs) regulate cellular innate immunity to benefit viral replication. In human cytomegalovirus (HCMV), the UL48-encoded DUB regulates innate immune responses including NF-κB signaling. Although UL48 DUB is known to regulate its stability via auto-deubiquitination, its impact on other viral proteins is not well understood. In this study, we investigated the role of UL48 DUB in regulating the ubiquitination of viral proteins by comparing the levels of ubiquitinated viral peptides in wild-type and DUB active-site mutant virus-infected cells using mass spectrometry. We found that ubiquitinated peptides were increased in DUB-mutant virus infection for 90% of viral proteins with the innermost tegument proteins pp150 (encoded by UL32) and pUL48 itself being most significantly affected. The highly deubiquitinated lysine residues of pUL48 were mapped within its N-terminal DUB domain and the nuclear localization signal. Among them, the arginine substitution of lysine 2 (K2R) increased pUL48 stability and enhanced viral growth at low multiplicity of infection, indicating that K2 auto-deubiquitination has a role in regulating pUL48 stability. pUL48 also interacted with pp150 and increased pp150 expression by downregulating its ubiquitination. Furthermore, we found that, unlike the wild-type virus, mutant viruses expressing the UL48 protein with the DUB-domain deleted or DUB active-site mutated contain higher levels of ubiquitin conjugates, including the ubiquitinated forms of pp150, in their virions. Collectively, our results demonstrate that UL48 DUB mainly acts on the innermost tegument proteins pp150 and pUL48 itself during HCMV infection and may play a role in protecting virions from the inclusion of ubiquitin conjugates. Importance Herpesviruses encode highly conserved tegument proteins that contain deubiquitinase (DUB) activity. Although the role of viral DUBs in the regulation of host innate immune responses has been established, their roles in the stability or function of viral proteins are not well understood. In this study, we performed a comparative analysis of the levels of ubiquitinated viral peptides between wild-type and DUB-inactive HCMV infections and demonstrate that the innermost tegument proteins pp150 and pUL48 (DUB itself) are major targets of viral DUB. We also show that ubiquitinated viral proteins are effectively incorporated into the virions of DUB mutant viruses but not the wild-type virus. Our study demonstrates that viral DUBs may play important roles in promoting the stability of viral proteins and inhibiting the inclusion of ubiquitin conjugates into virions.

scholarly journals Integrating Terminal Truncation and Oligopeptide Fusion for a Novel Protein Engineering Strategy To Improve Specific Activity and Catalytic Efficiency: Alkaline α-Amylase as a Case Study

2013 ◽  
Vol 79 (20) ◽  
pp. 6429-6438 ◽  
Author(s):  
Haiquan Yang ◽  
Long Liu ◽  
Hyun-dong Shin ◽  
Rachel R. Chen ◽  
Jianghua Li ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Active Site ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
C Terminus ◽  
N Terminus ◽  
Engineering Strategy ◽  
Specific Activities ◽  
Novel Protein

ABSTRACTIn this work, we integrated terminal truncation and N-terminal oligopeptide fusion as a novel protein engineering strategy to improve specific activity and catalytic efficiency of alkaline α-amylase (AmyK) fromAlkalimonas amylolytica. First, the C terminus or N terminus of AmyK was partially truncated, yielding 12 truncated mutants, and then an oligopeptide (AEAEAKAKAEAEAKAK) was fused at the N terminus of the truncated AmyK, yielding another 12 truncation-fusion mutants. The specific activities of the truncation-fusion mutants AmyKΔC500-587::OP and AmyKΔC492-587::OP were 25.5- and 18.5-fold that of AmyK, respectively. Thekcat/Kmwas increased from 1.0 × 105liters · mol−1· s−1for AmyK to 30.6 × and 23.2 × 105liters · mol−1· s−1for AmyKΔC500-587::OP and AmyKΔC492-587::OP, respectively. Comparative analysis of structure models indicated that the higher flexibility around the active site may be the main reason for the improved catalytic efficiency. The proposed terminal truncation and oligopeptide fusion strategy may be effective to engineer other enzymes to improve specific activity and catalytic efficiency.

scholarly journals Effects of N-Terminal and C-Terminal Polyhistidine Tag on the Stability and Function of the Thermophilic P450 CYP119

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Yaprak Aslantas ◽  
Nur Basak Surmeli
Keyword(s):  
Active Site ◽  
High Efficiency ◽  
Industrial Applications ◽  
Heme Iron ◽  
Mechanistic Studies ◽  
Wild Type ◽  
His Tag ◽  
C Terminus ◽  
The Stability ◽  
And Function

Biocatalysts are sought-after in synthesis of pharmaceuticals and agrochemicals due to their high regioselectivity and enantioselectivity. Among biocatalysts, heme-containing cytochrome P450 (P450) oxygenases are an attractive target since they catalyze oxidation of “unactivated” carbon-hydrogen bonds with high efficiency. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius, which has the potential to be widely used as a biocatalyst since it shows activity at high temperatures and low pH. Polyhistidine tags (His-tags) are widely used to simplify purification of proteins. However, His-tags can cause changes to protein structure and function. Here, we demonstrate the effects of His-tags on CYP119. To this end, the His-tags were cloned at the N-terminus or C-terminus of the CYP119, and His-tagged proteins were expressed and isolated. The thermostability and peroxidase activity of His-tagged CYP119s were tested and compared to wild type CYP119. Results indicated that while addition of His-tags increased the yield and simplified isolation of CYP119, they also influenced the electronic structure of active site and the activity of the protein. We show that N-terminal His-tagged CYP119 has desirable properties and potential to be used in industrial applications, but mechanistic studies using this protein need careful interpretation since the His-tag affects electronic properties of the active site heme iron.

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