QM/MM Molecular Modeling Reveals Mechanism Insights into Flavin Peroxide Formation in Bacterial Luciferase

Narin Lawan; Ruchanok Tinikul; Panida Surawatanawong; Adrian J Mulholland; Pimchai Chaiyen

doi:10.1021/acs.jcim.1c01187

QM/MM Molecular Modeling Reveals Mechanism Insights into Flavin Peroxide Formation in Bacterial Luciferase

Narin Lawan
, Ruchanok Tinikul
, Panida Surawatanawong
, Adrian J Mulholland
, Pimchai Chaiyen

Research output: Contribution to journal › Article (Academic Journal) › peer-review

12 Citations (Scopus)

Abstract

Bacterial luciferase (Lux) catalyzes oxidation of reduced flavin mononucleotide (FMN) and aldehyde to form oxidized FMN and carboxylic acid via molecular oxygen with concomitant light generation. The enzyme is useful for various detection applications in biomedical experiments. Upon reacting with oxygen, the reduced FMN generates C4a-peroxy-FMN (FMNH-C4a-OO -) as a reactive intermediate, which is required for light generation. However, the mechanism and control of FMNH-C4a-OO - formation are not clear. This work investigated the reaction of FMNH-C4a-OO - formation in Lux using QM/MM methods. The B3LYP/6-31G*/CHARMM27 calculations indicate that Lux controls the formation of FMNH-C4a-OO - via the conserved His44 residue. The steps in intermediate formation are found to be as follows: (i) H + reacts with O 2 to generate +OOH. (ii) +OOH attacks C4a of FMNH - to generate FMNH-C4a-OOH. (iii) H + is transferred from FMNH-C4a-OOH to His44 to generate FMNH-C4a-OO - while His44 stabilizes FMNH-C4a-OO - by forming a hydrogen bond to an oxygen atom. This controlling key mechanism for driving the change from FMNH-C4a-OOH to the FMNH-C4a-OO - adduct is confirmed because FMNH-C4a-OO - is more stable than FMNH-C4a-OOH in the luciferase active site.

Original language	English
Pages (from-to)	399-411
Number of pages	13
Journal	Journal of Chemical Information and Modeling
Volume	62
Issue number	2
Early online date	6 Jan 2022
DOIs	https://doi.org/10.1021/acs.jcim.1c01187
Publication status	Published - 24 Jan 2022

Bibliographical note

Funding Information:
This research project is supported by Thailand Science Research and Innovation (TSRI) and Chiang Mai University to N.L. P.S. is thankful for grant no. RSA6280041. P.C. is thankful for research support from the Vidyasirimedhi Institute of Science and Technology (VISTEC), Thailand Science Research Innovation, and the NSRF via the Program Management Unit for Human Resources & Institutional Development, Research and Innovation (grant number B05F640089), and the Global Partnership program. A.J.M. thanks EPSRC (Engineering and Physical Sciences Research Council, UK) for funding (grant number EP/M022609/1). R.T. thanks the Central Instrument Facility (CIF), Faculty of Science, Mahidol University. CHARMM topology and additional parameter files for FMNH-C4a–OO or FMNH-C4a–OOH, structure files, input and output files, and data are available at https://github.com/N-Lawan/Luciferase.git . –

Publisher Copyright:
© 2022 American Chemical Society.

Research Groups and Themes

Physical & Theoretical

Keywords

Flavins/chemistry
Kinetics
Luciferases/metabolism
Luciferases, Bacterial/chemistry
Oxidation-Reduction
Peroxides

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Cite this

@article{383be5e868454ecf9624f1c66a8819a6,

title = "QM/MM Molecular Modeling Reveals Mechanism Insights into Flavin Peroxide Formation in Bacterial Luciferase",

abstract = "Bacterial luciferase (Lux) catalyzes oxidation of reduced flavin mononucleotide (FMN) and aldehyde to form oxidized FMN and carboxylic acid via molecular oxygen with concomitant light generation. The enzyme is useful for various detection applications in biomedical experiments. Upon reacting with oxygen, the reduced FMN generates C4a-peroxy-FMN (FMNH-C4a-OO -) as a reactive intermediate, which is required for light generation. However, the mechanism and control of FMNH-C4a-OO - formation are not clear. This work investigated the reaction of FMNH-C4a-OO - formation in Lux using QM/MM methods. The B3LYP/6-31G*/CHARMM27 calculations indicate that Lux controls the formation of FMNH-C4a-OO - via the conserved His44 residue. The steps in intermediate formation are found to be as follows: (i) H + reacts with O 2 to generate +OOH. (ii) +OOH attacks C4a of FMNH - to generate FMNH-C4a-OOH. (iii) H + is transferred from FMNH-C4a-OOH to His44 to generate FMNH-C4a-OO - while His44 stabilizes FMNH-C4a-OO - by forming a hydrogen bond to an oxygen atom. This controlling key mechanism for driving the change from FMNH-C4a-OOH to the FMNH-C4a-OO - adduct is confirmed because FMNH-C4a-OO - is more stable than FMNH-C4a-OOH in the luciferase active site. ",

keywords = "Flavins/chemistry, Kinetics, Luciferases/metabolism, Luciferases, Bacterial/chemistry, Oxidation-Reduction, Peroxides",

author = "Narin Lawan and Ruchanok Tinikul and Panida Surawatanawong and Mulholland, \{Adrian J\} and Pimchai Chaiyen",

note = "Funding Information: This research project is supported by Thailand Science Research and Innovation (TSRI) and Chiang Mai University to N.L. P.S. is thankful for grant no. RSA6280041. P.C. is thankful for research support from the Vidyasirimedhi Institute of Science and Technology (VISTEC), Thailand Science Research Innovation, and the NSRF via the Program Management Unit for Human Resources \& Institutional Development, Research and Innovation (grant number B05F640089), and the Global Partnership program. A.J.M. thanks EPSRC (Engineering and Physical Sciences Research Council, UK) for funding (grant number EP/M022609/1). R.T. thanks the Central Instrument Facility (CIF), Faculty of Science, Mahidol University. CHARMM topology and additional parameter files for FMNH-C4a{\textendash}OO or FMNH-C4a{\textendash}OOH, structure files, input and output files, and data are available at https://github.com/N-Lawan/Luciferase.git . {\textendash} Publisher Copyright: {\textcopyright} 2022 American Chemical Society.",

year = "2022",

month = jan,

day = "24",

doi = "10.1021/acs.jcim.1c01187",

language = "English",

volume = "62",

pages = "399--411",

journal = "Journal of Chemical Information and Modeling",

issn = "1549-9596",

publisher = "American Chemical Society",

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TY - JOUR

T1 - QM/MM Molecular Modeling Reveals Mechanism Insights into Flavin Peroxide Formation in Bacterial Luciferase

AU - Lawan, Narin

AU - Tinikul, Ruchanok

AU - Surawatanawong, Panida

AU - Mulholland, Adrian J

AU - Chaiyen, Pimchai

N1 - Funding Information: This research project is supported by Thailand Science Research and Innovation (TSRI) and Chiang Mai University to N.L. P.S. is thankful for grant no. RSA6280041. P.C. is thankful for research support from the Vidyasirimedhi Institute of Science and Technology (VISTEC), Thailand Science Research Innovation, and the NSRF via the Program Management Unit for Human Resources & Institutional Development, Research and Innovation (grant number B05F640089), and the Global Partnership program. A.J.M. thanks EPSRC (Engineering and Physical Sciences Research Council, UK) for funding (grant number EP/M022609/1). R.T. thanks the Central Instrument Facility (CIF), Faculty of Science, Mahidol University. CHARMM topology and additional parameter files for FMNH-C4a–OO or FMNH-C4a–OOH, structure files, input and output files, and data are available at https://github.com/N-Lawan/Luciferase.git . – Publisher Copyright: © 2022 American Chemical Society.

PY - 2022/1/24

Y1 - 2022/1/24

N2 - Bacterial luciferase (Lux) catalyzes oxidation of reduced flavin mononucleotide (FMN) and aldehyde to form oxidized FMN and carboxylic acid via molecular oxygen with concomitant light generation. The enzyme is useful for various detection applications in biomedical experiments. Upon reacting with oxygen, the reduced FMN generates C4a-peroxy-FMN (FMNH-C4a-OO -) as a reactive intermediate, which is required for light generation. However, the mechanism and control of FMNH-C4a-OO - formation are not clear. This work investigated the reaction of FMNH-C4a-OO - formation in Lux using QM/MM methods. The B3LYP/6-31G*/CHARMM27 calculations indicate that Lux controls the formation of FMNH-C4a-OO - via the conserved His44 residue. The steps in intermediate formation are found to be as follows: (i) H + reacts with O 2 to generate +OOH. (ii) +OOH attacks C4a of FMNH - to generate FMNH-C4a-OOH. (iii) H + is transferred from FMNH-C4a-OOH to His44 to generate FMNH-C4a-OO - while His44 stabilizes FMNH-C4a-OO - by forming a hydrogen bond to an oxygen atom. This controlling key mechanism for driving the change from FMNH-C4a-OOH to the FMNH-C4a-OO - adduct is confirmed because FMNH-C4a-OO - is more stable than FMNH-C4a-OOH in the luciferase active site.

AB - Bacterial luciferase (Lux) catalyzes oxidation of reduced flavin mononucleotide (FMN) and aldehyde to form oxidized FMN and carboxylic acid via molecular oxygen with concomitant light generation. The enzyme is useful for various detection applications in biomedical experiments. Upon reacting with oxygen, the reduced FMN generates C4a-peroxy-FMN (FMNH-C4a-OO -) as a reactive intermediate, which is required for light generation. However, the mechanism and control of FMNH-C4a-OO - formation are not clear. This work investigated the reaction of FMNH-C4a-OO - formation in Lux using QM/MM methods. The B3LYP/6-31G*/CHARMM27 calculations indicate that Lux controls the formation of FMNH-C4a-OO - via the conserved His44 residue. The steps in intermediate formation are found to be as follows: (i) H + reacts with O 2 to generate +OOH. (ii) +OOH attacks C4a of FMNH - to generate FMNH-C4a-OOH. (iii) H + is transferred from FMNH-C4a-OOH to His44 to generate FMNH-C4a-OO - while His44 stabilizes FMNH-C4a-OO - by forming a hydrogen bond to an oxygen atom. This controlling key mechanism for driving the change from FMNH-C4a-OOH to the FMNH-C4a-OO - adduct is confirmed because FMNH-C4a-OO - is more stable than FMNH-C4a-OOH in the luciferase active site.

KW - Flavins/chemistry

KW - Kinetics

KW - Luciferases/metabolism

KW - Luciferases, Bacterial/chemistry

KW - Oxidation-Reduction

KW - Peroxides

U2 - 10.1021/acs.jcim.1c01187

DO - 10.1021/acs.jcim.1c01187

M3 - Article (Academic Journal)

C2 - 34989561

SN - 1549-9596

VL - 62

SP - 399

EP - 411

JO - Journal of Chemical Information and Modeling

JF - Journal of Chemical Information and Modeling

IS - 2

ER -

QM/MM Molecular Modeling Reveals Mechanism Insights into Flavin Peroxide Formation in Bacterial Luciferase

Abstract

Bibliographical note

Research Groups and Themes

Keywords

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