An integrated screening system for the selection of exemplary substrates for natural and engineered cytochrome P450s

AbstractInformation about substrate and product selectivity is critical for understanding the function of cytochrome P450 monooxygenases. In addition, comprehensive understanding of changes in substrate selectivity of P450 upon amino acid mutation would enable the design and creation of engineered P450s with desired selectivities. Therefore, systematic methods for obtaining such information are required. Herein, we developed an integrated P450 substrate screening system for the selection of “exemplary” substrates for a P450 of interest. The established screening system accurately selected the known exemplary substrates and also identified previously unknown exemplary substrates for microbial-derived P450s from a library containing sp3-rich synthetic small molecules. Synthetically potent transformations were also found by analyzing the reactions and oxidation products. The screening system was applied to analyze the substrate selectivity of the P450 BM3 mutants F87A and F87A/A330W, which acquired an ability to hydroxylate non-natural substrate steroids regio- and stereoselectively by two amino acid mutations. The distinct transition of exemplary substrates due to each single amino acid mutation was revealed, demonstrating the utility of the established system.

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Mutations within the P2 Domain of Norovirus Capsid Affect Binding to Human Histo-Blood Group Antigens: Evidence for a Binding Pocket

Journal of Virology ◽

10.1128/jvi.77.23.12562-12571.2003 ◽

2003 ◽

Vol 77 (23) ◽

pp. 12562-12571 ◽

Cited By ~ 139

Author(s):

Ming Tan ◽

Pengwei Huang ◽

Jaroslaw Meller ◽

Weiming Zhong ◽

Tibor Farkas ◽

...

Keyword(s):

Amino Acid ◽

Blood Group ◽

Binding Pocket ◽

Single Amino Acid ◽

Blood Group Antigens ◽

Sequence Alignments ◽

Amino Acid Mutation ◽

Multiple Sequence ◽

Amino Acid Mutations ◽

Function Relationship

ABSTRACT Noroviruses (NORs) are an important cause of acute gastroenteritis. Recent studies of NOR receptors showed that different NORs bind to different histo-blood group antigens (HBGAs), and at least four distinct binding patterns were observed. To determine the structure-function relationship for NORs and their receptors, two strains representing two of the four binding patterns were studied. Strain VA387 binds to HBGAs of A, B, and O secretors, whereas strain MOH binds to HBGAs of A and B secretors only. Using multiple sequence alignments, homology modeling, and structural analysis of NOR capsids, we identified a plausible “pocket” in the P2 domain that may be responsible for binding to HBGA receptors. This pocket consists of a conserved RGD/K motif surrounded by three strain-specific hot spots (N302, T337, and Q375 for VA387 and N302, N338, and E378 for MOH). Subsequent mutagenesis experiments demonstrated that all four sites played important roles in binding. A single amino acid mutation at T337 (to A) in VA387 or a double amino acid mutation at RN338 (to TT) in MOH abolished binding completely. Change of the entire RGD motif to SAS abolished binding in case of VA387, whereas single amino acid mutations in that motif did not have an apparent effect on binding to A and B antigens but decreased binding to H antigen. Multiple mutations at the RGK motif of MOH (SIRGK to TFRGD) completely knocked out the binding. Mutation of N302 or Q375 in VA387 affected binding to type O HBGA only, while switch mutants with three amino acid changes at either site from MOH to VA387 resulted in a weak binding to type O HBGAs. A further switch mutant with three amino acid changes at E378 from MOH to VA387 diminished the binding to type A HBGA only. Taken together, our data indicate that the binding pocket likely exists on NOR capsids. Direct evidence of this hypothesis requires crystallography studies.

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Genetic Variation and Evolution of the 2019 Novel Coronavirus

Public Health Genomics ◽

10.1159/000513530 ◽

2021 ◽

pp. 1-13

Author(s):

Salvatore Dimonte ◽

Muhammed Babakir-Mina ◽

Taib Hama-Soor ◽

Salar Ali

Keyword(s):

Amino Acid ◽

Receptor Binding ◽

Rapid Evolution ◽

Single Amino Acid ◽

Angiotensin Converting Enzyme 2 ◽

New Type ◽

Amino Acid Mutations ◽

Host Infection ◽

Human Coronaviruses ◽

Novel Coronavirus

Introduction: SARS-CoV-2 is a new type of coronavirus causing a pandemic severe acute respiratory syndrome (SARS-2). Coronaviruses are very diverting genetically and mutate so often periodically. The natural selection of viral mutations may cause host infection selectivity and infectivity. Methods: This study was aimed to indicate the diversity between human and animal coronaviruses through finding the rate of mutation in each of the spike, nucleocapsid, envelope, and membrane proteins. Results: The mutation rate is abundant in all 4 structural proteins. The most number of statistically significant amino acid mutations were found in spike receptor-binding domain (RBD) which may be because it is responsible for a corresponding receptor binding in a broad range of hosts and host selectivity to infect. Among 17 previously known amino acids which are important for binding of spike to angiotensin-converting enzyme 2 (ACE2) receptor, all of them are conservative among human coronaviruses, but only 3 of them significantly are mutated in animal coronaviruses. A single amino acid aspartate-454, that causes dissociation of the RBD of the spike and ACE2, and F486 which gives the strength of binding with ACE2 remain intact in all coronaviruses. Discussion/Conclusion: Observations of this study provided evidence of the genetic diversity and rapid evolution of SARS-CoV-2 as well as other human and animal coronaviruses.

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A single amino acid mutation (Ser180----Cys) determines the polymorphism in cytochrome P450g (P4502C13) by altering protein stability.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)46049-8 ◽

1992 ◽

Vol 267 (3) ◽

pp. 2032-2037

Author(s):

M B Faletto ◽

P Linko ◽

J A Goldstein

Keyword(s):

Amino Acid ◽

Protein Stability ◽

Single Amino Acid ◽

Amino Acid Mutation ◽

Single Amino Acid Mutation

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Prospective mapping of viral mutations that escape antibodies used to treat COVID-19

Science ◽

10.1126/science.abf9302 ◽

2021 ◽

Vol 371 (6531) ◽

pp. 850-854 ◽

Cited By ~ 10

Author(s):

Tyler N. Starr ◽

Allison J. Greaney ◽

Amin Addetia ◽

William W. Hannon ◽

Manish C. Choudhary ◽

...

Keyword(s):

Amino Acid ◽

Infected Patient ◽

Viral Escape ◽

Single Amino Acid ◽

Receptor Binding Domain ◽

Amino Acid Mutation ◽

The Individual ◽

Viral Surveillance ◽

Single Amino Acid Mutation

Antibodies are a potential therapy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the risk of the virus evolving to escape them remains unclear. Here we map how all mutations to the receptor binding domain (RBD) of SARS-CoV-2 affect binding by the antibodies in the REGN-COV2 cocktail and the antibody LY-CoV016. These complete maps uncover a single amino acid mutation that fully escapes the REGN-COV2 cocktail, which consists of two antibodies, REGN10933 and REGN10987, targeting distinct structural epitopes. The maps also identify viral mutations that are selected in a persistently infected patient treated with REGN-COV2 and during in vitro viral escape selections. Finally, the maps reveal that mutations escaping the individual antibodies are already present in circulating SARS-CoV-2 strains. These complete escape maps enable interpretation of the consequences of mutations observed during viral surveillance.

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