Probing the Mechanism of Enzyme Action Through Specific Amino Acid Substitutions

Comparative studies of orthologous proteins of species evolved at different temperatures have revealed consistent patterns of temperature-related variation in thermal stabilities of structure and function. However, the precise mechanisms by which interspecific variations in sequence foster these adaptive changes remain largely unknown. Here, we compare orthologs of cytosolic malate dehydrogenase (cMDH) from marine molluscs adapted to temperatures ranging from −1.9 °C (Antarctica) to ∼55 °C (South China coast) and show how amino acid usage in different regions of the enzyme (surface, intermediate depth, and protein core) varies with adaptation temperature. This eukaryotic enzyme follows some but not all of the rules established in comparisons of archaeal and bacterial proteins. To link the effects of specific amino acid substitutions with adaptive variations in enzyme thermal stability, we combined site-directed mutagenesis (SDM) and in vitro protein experimentation with in silico mutagenesis using molecular dynamics simulation (MDS) techniques. SDM and MDS methods generally but not invariably yielded common effects on protein stability. MDS analysis is shown to provide insights into how specific amino acid substitutions affect the conformational flexibilities of mobile regions (MRs) of the enzyme that are essential for binding and catalysis. Whereas these substitutions invariably lie outside of the MRs, they effectively transmit their flexibility-modulating effects to the MRs through linked interactions among surface residues. This discovery illustrates that regions of the protein surface lying outside of the site of catalysis can help establish an enzyme’s thermal responses and foster evolutionary adaptation of function.

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Contribution of Specific Amino Acid Changes in Penicillin Binding Protein 1 to Amoxicillin Resistance in ClinicalHelicobacter pyloriisolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00545-10 ◽

2010 ◽

Vol 55 (1) ◽

pp. 101-109 ◽

Cited By ~ 18

Author(s):

Nadia N. Qureshi ◽

Dimitrios Morikis ◽

Neal L. Schiller

Keyword(s):

Amino Acid ◽

Homology Modeling ◽

Binding Protein ◽

Site Directed Mutagenesis ◽

Amino Acid Substitutions ◽

Peptic Ulcers ◽

Penicillin Binding Protein ◽

Specific Amino Acid ◽

Binding Cleft ◽

H Pylori

ABSTRACTAmoxicillin is commonly used to treatHelicobacter pylori, a major cause of peptic ulcers, stomach cancer, and B-cell mucosa-associated lymphoid tissue lymphoma. Amoxicillin resistance inH. pyloriis increasing steadily, especially in developing countries, leading to treatment failures. In this study, we characterize the mechanism of amoxicillin resistance in the U.S. clinical isolate B258. Transformation of amoxicillin-susceptible strain 26695 with the penicillin binding protein 1 gene (pbp1) from B258 increased the amoxicillin resistance of 26695 to equal that of B258, while studies using biotinylated amoxicillin showed a decrease in the binding of amoxicillin to the PBP1 of B258. Transformation with 4pbp1fragments, each encompassing several amino acid substitutions, combined with site-directed mutagenesis studies, identified 3 amino acid substitutions in PBP1 of B258 which affected amoxicillin susceptibility (Val 469 Met, Phe 473 Leu, and Ser 543 Arg). Homology modeling showed the spatial orientation of these specific amino acid changes in PBP1 from 26695 and B258. The results of these studies demonstrate that amoxicillin resistance in the clinical U.S. isolate B258 is due solely to an altered PBP1 protein with a lower binding affinity for amoxicillin. Homology modeling analyses using previously identified amino acid substitutions of amoxicillin-resistant PBP1s demonstrate the importance of specific amino acid substitutions in PBP1 that affect the binding of amoxicillin in the putative binding cleft, defining those substitutions deemed most important in amoxicillin resistance.

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Amino Acid Substitutions Analysis of the Putative Epitopes of Neuraminidase Protein from Influenza A H1N1 Virus

Revista Unimontes Científica ◽

10.46551/ruc.v22n1a04 ◽

2021 ◽

Vol 22 (1) ◽

pp. 1-16

Author(s):

Ludmila Alves Dias Souto ◽

Alessandra Rejane Ericsson de Oliveira Xavier ◽

Mauro Aparecido de Sousa Xavier

Keyword(s):

Amino Acid ◽

Influenza A ◽

H1n1 Virus ◽

Epitope Prediction ◽

Viral Population ◽

Amino Acid Substitutions ◽

Specific Amino Acid ◽

Influenza A H1n1 ◽

Virus Sequence ◽

Clustal Omega

Objective: This study verified whether the neuraminidase protein of Influenza A H1N1 virus sequence has modified from 2009–2017 and its impact on the 2018 Brazilian vaccine. Method: The reference neuraminidase protein sequence from H1N1 Puerto Rico/1934 strain was subjected to three different methods of epitope prediction and the top five from each method were aligned using Clustal omega, resulting in eight putative epitopes. These epitopes were aligned to 7,438 neuraminidase sequences spanning from 2009–2017 and analyzed for specific amino acid substitutions and counted. The resultant neuraminidase protein was aligned against the 2015 and 2018 neuraminidase proteins, from Influenza A H1N1 virus subtypes, used for vaccine production. Result: Twenty-one main substitutions were detected, of which 16/21 (76.2%) substitutions points remained stable and 1/21 (4.8%) returned to the original amino acid residue in the viral population from 2009–2017. Additionally, 19% (4/21) substitutions occurred in Brazil and worldwide in this period, indicating that changes in the neuraminidase viral population profile is time-dependent rather than geographical. Conclusion: The neuraminidase protein containing these amino acid substitutions is more closely related to the neuraminidase protein from influenza A/Michigan/45/2015 than A/California/7/2009, supporting the replacement of this virus subtype in the Brazilian vaccine in 2018.

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Specific Amino Acid Substitutions in the Proline-Rich Motif of the Rhizobium meliloti ExoP Protein Result in Enhanced Production of Low-Molecular-Weight Succinoglycan at the Expense of High-Molecular-Weight Succinoglycan

Journal of Bacteriology ◽

10.1128/jb.180.2.395-399.1998 ◽

1998 ◽

Vol 180 (2) ◽

pp. 395-399 ◽

Cited By ~ 45

Author(s):

Anke Becker ◽

Alfred Pühler

Keyword(s):

Molecular Weight ◽

Amino Acid ◽

High Molecular Weight ◽

Rhizobium Meliloti ◽

Low Molecular Weight ◽

Amino Acid Substitutions ◽

Wild Type ◽

Specific Amino Acid ◽

Enhanced Production ◽

Mutant Strains

ABSTRACT The production of the acidic exopolysaccharide succinoglycan (EPS I) by Rhizobium meliloti exoP* mutants expressing an ExoP protein lacking its C-terminal cytoplasmic domain and by mutants characterized by specific amino acid substitutions in the proline-rich motif (RX4PX2PX4SPKX9IXGXMXGXG) located from positions 443 to 476 of the ExoP protein was analyzed. The absence of the C-terminal cytoplasmic ExoP domain (positions 484 to 786) and the substitution of both arginine443 by isoleucine443 and proline457 by serine457 within the proline-rich motif resulted in enhanced production of low-molecular-weight (LMW) EPS I at the expense of high-molecular-weight (HMW) EPS I. The ratios of HMW to LMW EPS I of the wild type and mutant strains increased with osmolarity.

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KRAS-specific Amino Acid Substitutions are Associated With Different Responses to Chemotherapy in Advanced Non–small-cell Lung Cancer

Clinical Lung Cancer ◽

10.1016/j.cllc.2018.08.005 ◽

2018 ◽

Vol 19 (6) ◽

pp. e919-e931 ◽

Cited By ~ 5

Author(s):

Stéphane Renaud ◽

Francesco Guerrera ◽

Joseph Seitlinger ◽

Jérémie Reeb ◽

Anne-Claire Voegeli ◽

...

Keyword(s):

Lung Cancer ◽

Amino Acid ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Small Cell ◽

Amino Acid Substitutions ◽

Small Cell Lung ◽

Specific Amino Acid

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Probing Structural Features of Alzheimer’s Amyloid-β Pores in Bilayers Using Site-Specific Amino Acid Substitutions

Biochemistry ◽

10.1021/bi2017427 ◽

2012 ◽

Vol 51 (3) ◽

pp. 776-785 ◽

Cited By ~ 39

Author(s):

Ricardo Capone ◽

Hyunbum Jang ◽

Samuel A. Kotler ◽

Bruce L. Kagan ◽

Ruth Nussinov ◽

...

Keyword(s):

Amino Acid ◽

Amyloid Β ◽

Structural Features ◽

Amino Acid Substitutions ◽

Site Specific ◽

Specific Amino Acid

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Lineage-Specific Amino Acid Substitutions in Region 2 of the RNA Polymerase σ Subunit Affect the Temperature of Promoter Opening

Journal of Bacteriology ◽

10.1128/jb.00008-08 ◽

2008 ◽

Vol 190 (8) ◽

pp. 3088-3092 ◽

Cited By ~ 4

Author(s):

N. Barinova ◽

E. Zhilina ◽

I. Bass ◽

V. Nikiforov ◽

A. Kulbachinskiy

Keyword(s):

Amino Acid ◽

Rna Polymerase ◽

Amino Acid Substitutions ◽

Amino Acid Residues ◽

Specific Amino Acid ◽

Promoter Recognition

ABSTRACT Highly conserved amino acid residues in region 2 of the RNA polymerase σ subunit are known to participate in promoter recognition and opening. We demonstrated that nonconserved residues in this region collectively determine lineage-specific differences in the temperature of promoter opening.

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