The role of conserved residues in the catalytic activity of NDM-1: an approach involving site directed mutagenesis and molecular dynamics

2019 ◽  
Vol 21 (32) ◽  
pp. 17821-17835 ◽  
Author(s):  
Abid Ali ◽  
Rakesh Kumar ◽  
Mir Asif Iquebal ◽  
Sarika Jaiswal ◽  
Dinesh Kumar ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Catalytic Activity ◽  
Cell Survival ◽  
Conformational Changes ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Loss Of Function ◽  
Conserved Residues

Drug degraded by enzyme and hence not targeted on to the cell leading to cell survival. After mutation leading to conformational changes and loss of function hence drug was not degraded and remained available for the target to lyse the cell.

Role of Glu51 for Cofactor Binding and Catalytic Activity in Pyruvate Decarboxylase from Yeast Studied by Site-Directed Mutagenesis†

Biochemistry ◽  
1997 ◽  
Vol 36 (7) ◽  
pp. 1900-1905 ◽  
Author(s):  
Margrit Killenberg-Jabs ◽  
Stephan König ◽  
Ines Eberhardt ◽  
Stefan Hohmann ◽  
Gerhard Hübner
Keyword(s):  
Catalytic Activity ◽  
Pyruvate Decarboxylase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Cofactor Binding

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.

scholarly journals Role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1

2000 ◽  
Vol 350 (3) ◽  
pp. 677-683 ◽  
Author(s):  
Ana M. PAJOR ◽  
Esther S. KAHN ◽  
Rama GANGULA
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Xenopus Oocytes ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Conserved Residues ◽  
In The Wild ◽  
Cationic Amino Acids

The role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1 was investigated by site-directed mutagenesis and subsequent expression of mutant transporters in Xenopus oocytes. Of the ten residues chosen for mutagenesis, eight (Lys-34, Lys-107, Arg-108, Lys-333, Lys-390, Arg-368, Lys-414 and Arg-541) were found to be non-essential for function or targeting. Only two conserved residues, Lys-84 (at the cytoplasmic end of helix 3) and Arg-349 (at the extracellular end of helix 7), were found to be important for transport. Both mutant transporters were expressed at the plasma membrane. The mutation of Lys-84 to Ala resulted in an increased Km for succinate of 1.8mM, compared with 0.3mM in the wild-type NaDC-1. The R349A mutant had Na+ and citrate kinetics that were similar to those of the wild type. However, succinate handling in the R349A mutant was altered, with evidence of inhibition at high succinate concentrations. In conclusion, charge neutralization of Lys-84 and Arg-349 in NaDC-1 affects succinate handling, suggesting that these residues might have roles in substrate binding.

scholarly journals A conserved motif in the yeast nucleolar protein Nop2p contains an essential cysteine residue

1998 ◽  
Vol 337 (1) ◽  
pp. 29-35 ◽  
Author(s):  
Michelle KING ◽  
Duy TON ◽  
Kent L. REDMAN
Keyword(s):  
Ribosomal Subunit ◽  
Cysteine Residue ◽  
Nucleolar Protein ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Large Ribosomal Subunit ◽  
Conserved Residues ◽  
Conserved Motif ◽  
Yeast Viability

Nop2p is an essential nucleolar protein in Saccharomyces cerevisiae that is involved in large ribosomal subunit assembly. It has substantial homology with human p120, the proliferation-associated nucleolar antigen that is overexpressed in many human cancers. A motif containing an invariant Pro–Cys dipeptide is found in Nop2p, p120 and the bacterial Fmu proteins. A total of nine conserved residues, including Pro423 and Cys424, were individually altered in Nop2p by site-directed mutagenesis. Nop2p function was abolished by conversion of Cys424 into either alanine or serine. All of the other Nop2p mutations tested sustained yeast viability, including glycine replacement of Pro423 and the conversion of a second conserved cysteine into alanine. The crucial role of Cys424 in Nop2p is intriguing, due to the critical roles that cysteine residues adjacent to a proline have in a number of nucleotide-modifying enzymes.

Exploring the role of histidines in the catalytic activity of duck δ-Crystallins using site-directed mutagenesis

1995 ◽  
Vol 61 (2) ◽  
pp. 151-154 ◽  
Author(s):  
Gerald Patejunas ◽  
Peter Barbosa ◽  
Michelle Lacombe ◽  
William E. o'Brien
Keyword(s):  
Catalytic Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Sodium and proton coupling in the conformational cycle of a MATE antiporter from Vibrio cholerae

2018 ◽  
Vol 115 (27) ◽  
pp. E6182-E6190 ◽  
Author(s):  
Derek P. Claxton ◽  
Kevin L. Jagessar ◽  
P. Ryan Steed ◽  
Richard A. Stein ◽  
Hassane S. Mchaourab
Keyword(s):  
Vibrio Cholerae ◽  
Conformational Changes ◽  
Conformational Dynamics ◽  
Site Directed Mutagenesis ◽  
Spin Labels ◽  
Release Mechanism ◽  
Conserved Residues ◽  
Terminal Domain ◽  
Mate Transporter

Secondary active transporters belonging to the multidrug and toxic compound extrusion (MATE) family harness the potential energy of electrochemical ion gradients to export a broad spectrum of cytotoxic compounds, thus contributing to multidrug resistance. The current mechanistic understanding of ion-coupled substrate transport has been informed by a limited set of MATE transporter crystal structures from multiple organisms that capture a 12-transmembrane helix topology adopting similar outward-facing conformations. Although these structures mapped conserved residues important for function, the mechanistic role of these residues in shaping the conformational cycle has not been investigated. Here, we use double-electron electron resonance (DEER) spectroscopy to explore ligand-dependent conformational changes of NorM from Vibrio cholerae (NorM-Vc), a MATE transporter proposed to be coupled to both Na+ and H+ gradients. Distance measurements between spin labels on the periplasmic side of NorM-Vc identified unique structural intermediates induced by binding of Na+, H+, or the substrate doxorubicin. The Na+- and H+-dependent intermediates were associated with distinct conformations of TM1. Site-directed mutagenesis of conserved residues revealed that Na+- and H+-driven conformational changes are facilitated by a network of polar residues in the N-terminal domain cavity, whereas conserved carboxylates buried in the C-terminal domain are critical for stabilizing the drug-bound state. Interpreted in conjunction with doxorubicin binding of mutant NorM-Vc and cell toxicity assays, these results establish the role of ion-coupled conformational dynamics in the functional cycle and implicate H+ in the doxorubicin release mechanism.

Role of Highly Conserved Residues in the Reaction Catalyzed by Recombinant Δ7-Sterol-C5(6)-Desaturase Studied by Site-Directed Mutagenesis

Biochemistry ◽  
2000 ◽  
Vol 39 (4) ◽  
pp. 701-711 ◽  
Author(s):  
Maryse Taton ◽  
Tania Husselstein ◽  
Pierre Benveniste ◽  
Alain Rahier
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Conserved Residues

Molecular Basis of Glucose Transport Mechanism in Plants

2019 ◽  
Author(s):  
Balaji Selvam ◽  
Ya-Chi Yu ◽  
Liqing Chen ◽  
Diwakar Shukla
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Molecular Dynamics Simulations ◽  
Conformational Changes ◽  
Transport Mechanism ◽  
Conformational Dynamics ◽  
Site Directed Mutagenesis ◽  
Free Energy Barrier ◽  
Transport Cycle ◽  
Dynamics Simulations

<p>The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.</p>

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