scholarly journals Nucleotide specificity of HIV-1 reverse transcriptases with amino acid substitutions affecting Ala-114

2005 ◽  
Vol 387 (1) ◽  
pp. 221-229 ◽  
Author(s):  
Clara E. CASES-GONZÁLEZ ◽  
Luis MENÉNDEZ-ARIAS
Keyword(s):  
Catalytic Efficiency ◽  
Fold Increase ◽  
Polymerase Activity ◽  
Site Directed Mutagenesis ◽  
Van Der Waals Interactions ◽  
Reverse Transcriptases ◽  
Nucleotide Incorporation ◽  
Steady State Kinetic ◽  
Nucleotide Specificity ◽  
Hiv 1

Ala-114, together with Asp-113, Tyr-115 and Gln-151, form the pocket that accommodates the 3′-OH of the incoming dNTP in the HIV-1 RT (reverse transcriptase). Four mutant RTs having serine, glycine, threonine or valine instead of Ala-114 were obtained by site-directed mutagenesis. While mutants A114S and A114G retained significant DNA polymerase activity, A114T and A114V showed very low catalytic efficiency in nucleotide incorporation assays, due to their high apparent Km values for dNTP. Discrimination between AZTTP (3′-azido-3′-deoxythymidine triphosphate) and dTTP was not significantly affected by mutations A114S and A114G in assays carried out with heteropolymeric template/primers. However, both mutants showed decreased susceptibility to AZTTP when poly(rA)/(dT)16 was used as substrate. Steady-state kinetic analysis of the incorporation of ddNTPs compared with dNTPs showed that substituting glycine for Ala-114 produced a 5–6-fold increase in the RT's ability to discriminate against ddNTPs (including the physiologically relevant metabolites of zalcitabine and didanosine), a result that was confirmed in primer-extension assays. In contrast, A114S and A114V showed wild-type ddNTP/dNTP discrimination efficiencies. Discrimination against ribonucleotides was not affected by mutations at position 114. Misinsertion and mispair extension fidelity assays as well as determinations of G→A mutation frequencies using a lacZ complementation assay showed that, unlike Tyr-115 or Gln-151 mutants, the fidelity of HIV-1 RT was not largely affected by substitutions of Ala-114. The role of the side-chain of Ala-114 in ddNTP/dNTP discrimination appears to be determined by its participation in van der Waals interactions with the ribose moiety of the incoming nucleotide.

scholarly journals Electrostatic control of oxidative deamination catalysed by bovine serum amine oxidase

1994 ◽  
Vol 299 (1) ◽  
pp. 317-320 ◽  
Author(s):  
R Stevanato ◽  
B Mondovi ◽  
O Befani ◽  
M Scarpa ◽  
A Rigo
Keyword(s):  
Ionic Strength ◽  
Bovine Serum ◽  
Amine Oxidase ◽  
Relative Dielectric Constant ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Ionic Strength Dependence ◽  
Catalytic Rate Constant ◽  
Steady State Kinetic ◽  
Strength Dependence

The ionic-strength-dependence of steady-state kinetic parameters (kc and Km') for non-biogenic (benzylamine, butylamine) and biogenic (spermine, spermidine) amines has been measured in the bovine serum amine oxidase reaction. The catalytic rate constant (kc) values are similar (0.9-2.5 s-1) for all the substrates studied and are almost constant over the experimental ionic strength range (24-155 mM). In contrast, Km' values are in the range 6-2300 microM and undergo a 4-12-fold increase with increasing ionic strength, parallelled by a decrease in catalytic efficiency. From an analysis of the kc and Km' values and their dependence on ionic strength, we conclude that more than one negative site is involved in the binding of these amines and that the relative dielectric constant of the binding site is lower than that of aqueous solutions.

scholarly journals Mutational analysis of Lys65 of HIV-1 reverse transcriptase

2000 ◽  
Vol 348 (1) ◽  
pp. 77-82 ◽  
Author(s):  
Nicolas SLUIS-CREMER ◽  
Dominique ARION ◽  
Neerja KAUSHIK ◽  
Henry LIM ◽  
Michael A. PARNIAK
Keyword(s):  
Amino Acid ◽  
Reverse Transcriptase ◽  
Mutational Analysis ◽  
Catalytic Efficiency ◽  
Salt Bridge ◽  
Polymerase Activity ◽  
Ph Optimum ◽  
Modelling Studies ◽  
Dntp Binding ◽  
Hiv 1

Amino acid Lys65 is part of the highly flexible β3-β4 loop in the fingers domain of the 66 kDa subunit of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Recent crystal data show that the ϵ-amino group of Lys65 interacts with the γ-phosphate of the bound deoxynucleoside triphosphate (‘dNTP’) substrate [Huang, Chopra, Verdine and Harrison (1998) Science 282, 1669-1675]. In order to biochemically define the function of RT Lys65, we have used site-specific mutagenesis to generate RT with a variety of substitutions at this position, including K65E, K65Q, K65A and K65R. Kinetic analyses demonstrate that if Lys65 in RT is substituted with an amino acid other than arginine the enzyme exhibits dramatic decreases in the binding affinity (Km) for all dNTP substrates, in RT catalytic efficiency (kcat/Km) and in the mutant enzyme's ability to carry out pyrophosphorolysis, the reverse reaction of DNA synthesis. The pH optimum for the DNA polymerase activity of K65E RT was 6.5, compared to 7.5 for the wild-type enzyme, and 8.0 for the K65R, K65A and K65Q mutants. Molecular modelling studies show that mutations of Lys65 do not affect the geometry of the loop's α-carbon backbone, but rather lead to changes in positioning of the side chains of residues Lys70 and Arg72. In particular, Glu in K65E can form a salt bridge with Arg72, leading to the diminution of the latter residue's interaction with the α-phosphate of the dNTP residue. This alteration in dNTP-binding may explain the large pH-dependent changes in both dNTP-binding and catalytic efficiency noted with the enzyme. Furthermore, the K65A, K65Q and K65E mutant enzymes are 100-fold less sensitive to all dideoxynucleoside triphosphate (‘ddNTP’) inhibitors, whereas the K65R mutation results in a selective 10-fold decrease in binding of ddCTP and ddATP only. This implies that mutations at position 65 in HIV-1 RT influence the nucleotide-binding specificity of the enzyme.

scholarly journals Physiological Magnesium Concentrations Increase Fidelity of Diverse Reverse Transcriptases from HIV-1, HIV-2, and Foamy Virus, but not MuLV or AMV

2021 ◽  
Author(s):  
Ruofan Wang ◽  
Ashton T. Belew ◽  
Vasudevan Achuthan ◽  
Najib M. El-Sayed ◽  
Jeffrey J DeStefano
Keyword(s):  
Leukemia Virus ◽  
Foamy Virus ◽  
Fold Increase ◽  
Common Mutation ◽  
Avian Myeloblastosis Virus ◽  
Reverse Transcriptases ◽  
Subtype B ◽  
Substitution Mutations ◽  
Hiv 1

Reverse transcriptases (RTs) are typically assayed in vitro using optimized Mg2+ concentrations (~ 5-10 mM) that are several-fold higher than physiological cellular free Mg2+ (~ 0.5 mM). Analysis of fidelity using lacZα-based α-complementation assays showed that tested HIV RTs, including HIV-1 from subtype B (HXB2-derived), HIV-2, subtype A/E, and several drug-resistant HXB2 derivatives all showed significantly higher fidelity using physiological Mg2+. This also occurred with prototype foamy virus (PFV) RT. In contrast, Moloney murine leukemia virus (MuLV) and avian myeloblastosis virus (AMV) RTs demonstrated equivalent fidelity in both low and high Mg2+. In 0.5 mM Mg2+, all RTs demonstrated ≈ equal fidelity, except for PFV RT which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to accurately determine mutation rates and profiles was used to examine the types of mutations made by HIV-1 (subtype B, wild type) in low (0.5 mM) and high (6 mM) Mg2+ with DNA or RNA that coded for lacZα. Unlike the α-complementation assay, which is dependent on LacZα activity, the NGS assay scores mutations at all positions and of every type. A ~ 4-fold increase in substitution mutations was observed in high Mg2+. The general trend was an exacerbation in high Mg2+ of more common mutation in low Mg2+, rather than the creation of new mutation hotspots. These findings help explain why HIV RT displays lower fidelity in vitro (with high Mg2+ concentrations) than other RTs (e.g., MuLV and AMV), yet cellular fidelity for these viruses is comparable.

scholarly journals Physiological magnesium concentrations increase fidelity of diverse reverse transcriptases from HIV-1, HIV-2, and foamy virus, but not MuLV or AMV

2021 ◽  
Vol 102 (12) ◽  
Author(s):  
Ruofan Wang ◽  
Ashton T. Belew ◽  
Vasudevan Achuthan ◽  
Najib El Sayed ◽  
Jeffrey J. DeStefano
Keyword(s):  
Foamy Virus ◽  
Fold Increase ◽  
Reverse Transcriptases ◽  
Subtype B ◽  
Mutation Profile ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Hiv 1 ◽  
Current Report

Reverse transcriptases (RTs) are typically assayed using optimized Mg2+ concentrations (~5–10 mM) several-fold higher than physiological cellular free Mg2+ (~0.5 mM). Recent analyses demonstrated that HIV-1, but not Moloney murine leukaemia (MuLV) or avain myeloblastosis (AMV) virus RTs has higher fidelity in low Mg2+. In the current report, lacZα-based α-complementation assays were used to measure the fidelity of several RTs including HIV-1 (subtype B and A/E), several drug-resistant HIV-1 derivatives, HIV-2, and prototype foamy virus (PFV), all which showed higher fidelity using physiological Mg2+, while MuLV and AMV RTs demonstrated equivalent fidelity in low and high Mg2+. In 0.5 mM Mg2+, all RTs demonstrated approximately equal fidelity, except for PFV which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to determine mutation profiles was used to examine the types of mutations made by HIV-1 RT (type B) in low (0.5 mM) and high (6 mM) Mg2+ on a lacZα template. Unlike α-complementation assays which are dependent on LacZα activity, the NGS assay scores mutations at all positions and of every type. Consistent with α-complementation assays, a ~four-fold increase in mutations was observed in high Mg2+. These findings help explain why HIV-1 RT displays lower fidelity in vitro (with high Mg2+ concentrations) than other RTs (e.g. MuLV and AMV), yet cellular fidelity for these viruses is comparable. Establishing in vitro conditions that accurately represent RT’s activity in cells is pivotal to determining the contribution of RT and other factors to the mutation profile observed with HIV-1.

scholarly journals Targeting HIV-1 RNase H: N’-(2-Hydroxy-benzylidene)-3,4,5-Trihydroxybenzoylhydrazone as Selective Inhibitor Active against NNRTIs-Resistant Variants

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 729 ◽  
Author(s):  
Angela Corona ◽  
Ester Ballana ◽  
Simona Distinto ◽  
Dominga Rogolino ◽  
Claudia Del Vecchio ◽  
...  
Keyword(s):  
Polymerase Activity ◽  
Rnase H ◽  
Site Directed Mutagenesis ◽  
Drug Resistant ◽  
Biochemical Assays ◽  
Promising Target ◽  
Starting Point ◽  
Reverse Transcription Step ◽  
Docking Calculations ◽  
Hiv 1

HIV-1 infection requires life-long treatment and with 2.1 million new infections/year, faces the challenge of an increased rate of transmitted drug-resistant mutations. Therefore, a constant and timely effort is needed to identify new HIV-1 inhibitors active against drug-resistant variants. The ribonuclease H (RNase H) activity of HIV-1 reverse transcriptase (RT) is a very promising target, but to date, still lacks an efficient inhibitor. Here, we characterize the mode of action of N’-(2-hydroxy-benzylidene)-3,4,5-trihydroxybenzoylhydrazone (compound 13), an N-acylhydrazone derivative that inhibited viral replication (EC50 = 10 µM), while retaining full potency against the NNRTI-resistant double mutant K103N-Y181C virus. Time-of-addition and biochemical assays showed that compound 13 targeted the reverse-transcription step in cell-based assays and inhibited the RT-associated RNase H function, being >20-fold less potent against the RT polymerase activity. Docking calculations revealed that compound 13 binds within the RNase H domain in a position different from other selective RNase H inhibitors; site-directed mutagenesis studies revealed interactions with conserved amino acid within the RNase H domain, suggesting that compound 13 can be taken as starting point to generate a new series of more potent RNase H selective inhibitors active against circulating drug-resistant variants.

scholarly journals Base pairing, structural and functional insights into N4-methylcytidine (m4C) and N4,N4-dimethylcytidine (m42C) modified RNA

2020 ◽  
Vol 48 (18) ◽  
pp. 10087-10100
Author(s):  
Song Mao ◽  
Bartosz Sekula ◽  
Milosz Ruszkowski ◽  
Srivathsan V Ranganathan ◽  
Phensinee Haruehanroengra ◽  
...  
Keyword(s):  
Base Pairing ◽  
Reverse Transcriptases ◽  
X Ray Crystallography ◽  
Coding Efficiency ◽  
Gene Replication ◽  
Nucleotide Incorporation ◽  
Duplex Stability ◽  
Rna Duplexes ◽  
Fine Tune ◽  
Hiv 1

Abstract The N4-methylation of cytidine (m4C and m42C) in RNA plays important roles in both bacterial and eukaryotic cells. In this work, we synthesized a series of m4C and m42C modified RNA oligonucleotides, conducted their base pairing and bioactivity studies, and solved three new crystal structures of the RNA duplexes containing these two modifications. Our thermostability and X-ray crystallography studies, together with the molecular dynamic simulation studies, demonstrated that m4C retains a regular C:G base pairing pattern in RNA duplex and has a relatively small effect on its base pairing stability and specificity. By contrast, the m42C modification disrupts the C:G pair and significantly decreases the duplex stability through a conformational shift of native Watson-Crick pair to a wobble-like pattern with the formation of two hydrogen bonds. This double-methylated m42C also results in the loss of base pairing discrimination between C:G and other mismatched pairs like C:A, C:T and C:C. The biochemical investigation of these two modified residues in the reverse transcription model shows that both mono- or di-methylated cytosine bases could specify the C:T pair and induce the G to T mutation using HIV-1 RT. In the presence of other reverse transcriptases with higher fidelity like AMV-RT, the methylation could either retain the normal nucleotide incorporation or completely inhibit the DNA synthesis. These results indicate the methylation at N4-position of cytidine is a molecular mechanism to fine tune base pairing specificity and affect the coding efficiency and fidelity during gene replication.

scholarly journals Noncatalytic aspartate at the exonuclease domain of proofreading DNA polymerases regulates both degradative and synthetic activities

2018 ◽  
Vol 115 (13) ◽  
pp. E2921-E2929 ◽  
Author(s):  
Alicia del Prado ◽  
Elsa Franco-Echevarría ◽  
Beatriz González ◽  
Luis Blanco ◽  
Margarita Salas ◽  
...  
Keyword(s):  
Structural Alignment ◽  
Dna Polymerases ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Exonuclease Activity ◽  
Phosphodiester Bond ◽  
Functional Conservation ◽  
Nucleotide Incorporation ◽  
Exonuclease Domain ◽  
Exo Iii

Most replicative DNA polymerases (DNAPs) are endowed with a 3′-5′ exonuclease activity to proofread the polymerization errors, governed by four universally conserved aspartate residues belonging to the Exo I, Exo II, and Exo III motifs. These residues coordinate the two metal ions responsible for the hydrolysis of the last phosphodiester bond of the primer strand. Structural alignment of the conserved exonuclease domain of DNAPs from families A, B, and C has allowed us to identify an additional and invariant aspartate, located between motifs Exo II and Exo III. The importance of this aspartate has been assessed by site-directed mutagenesis at the corresponding Asp121 of the family B ϕ29 DNAP. Substitution of this residue by either glutamate or alanine severely impaired the catalytic efficiency of the 3′-5′ exonuclease activity, both on ssDNA and dsDNA. The polymerization activity of these mutants was also affected due to a defective translocation following nucleotide incorporation. Alanine substitution for the homologous Asp90 in family A T7 DNAP showed essentially the same phenotype as ϕ29 DNAP mutant D121A. This functional conservation, together with a close inspection of ϕ29 DNAP/DNA complexes, led us to conclude a pivotal role for this aspartate in orchestrating the network of interactions required during internal proofreading of misinserted nucleotides.

scholarly journals Enhancing the thermostability and activity of uronate dehydrogenase from Agrobacterium tumefaciens LBA4404 by semi-rational engineering

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Hui-Hui Su ◽  
Fei Peng ◽  
Pei Xu ◽  
Xiao-Ling Wu ◽  
Min-Hua Zong ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Rational Design ◽  
Glucuronic Acid ◽  
Specific Activity ◽  
Value Added ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Glucaric Acid ◽  
Triple Mutant ◽  
Pharmaceutical Industries

Abstract Background Glucaric acid, one of the aldaric acids, has been declared a “top value-added chemical from biomass”, and is especially important in the food and pharmaceutical industries. Biocatalytic production of glucaric acid from glucuronic acid is more environmentally friendly, efficient and economical than chemical synthesis. Uronate dehydrogenases (UDHs) are the key enzymes for the preparation of glucaric acid in this way, but the poor thermostability and low activity of UDH limit its industrial application. Therefore, improving the thermostability and activity of UDH, for example by semi-rational design, is a major research goal. Results In the present work, three UDHs were obtained from different Agrobacterium tumefaciens strains. The three UDHs have an approximate molecular weight of 32 kDa and all contain typically conserved UDH motifs. All three UDHs showed optimal activity within a pH range of 6.0–8.5 and at a temperature of 30 °C, but the UDH from A. tumefaciens (At) LBA4404 had a better catalytic efficiency than the other two UDHs (800 vs 600 and 530 s−1 mM−1). To further boost the catalytic performance of the UDH from AtLBA4404, site-directed mutagenesis based on semi-rational design was carried out. An A39P/H99Y/H234K triple mutant showed a 400-fold improvement in half-life at 59 °C, a 5 °C improvement in $$ {\text{T}}_{ 5 0}^{ 1 0} $$ T 50 10 value and a 2.5-fold improvement in specific activity at 30 °C compared to wild-type UDH. Conclusions In this study, we successfully obtained a triple mutant (A39P/H99Y/H234K) with simultaneously enhanced activity and thermostability, which provides a novel alternative for the industrial production of glucaric acid from glucuronic acid.

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