Crystal structures of NADH:FMN oxidoreductase (EmoB) at different stages of catalysis

Mark S Nissen; Buhyun Youn; Benjamin D Knowles; Jordan W Ballinger; Se-Young Jun; Sara M Belchik; Luying Xun; ChulHee Kang

doi:10.1074/jbc.M804535200

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Crystal structures of NADH:FMN oxidoreductase (EmoB) at different stages of catalysis

Journal article

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Crystal structures of NADH:FMN oxidoreductase (EmoB) at different stages of catalysis

Mark S Nissen, Buhyun Youn, Benjamin D Knowles, Jordan W Ballinger, Se-Young Jun, Sara M Belchik, Luying Xun and ChulHee Kang

The Journal of biological chemistry, Vol.283(42), pp.28710-28720

10/17/2008

DOI: https://doi.org/10.1074/jbc.M804535200

Handle:

https://hdl.handle.net/2376/107951

PMID: 18701448

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Abstract

Protein Structure, Tertiary

Amino Acid Sequence

Models, Chemical

Molecular Conformation

Bacterial Proteins - chemistry

Models, Molecular

Molecular Sequence Data

Phylogeny

Bacterial Proteins - physiology

Crystallography, X-Ray - methods

Sequence Homology, Amino Acid

NADH, NADPH Oxidoreductases - physiology

Edetic Acid - chemistry

Models, Biological

Protein Structure, Quaternary

NADH, NADPH Oxidoreductases - chemistry

Catalysis

Dimerization

EDTA has become a major organic pollutant in the environment because of its extreme usage and resistance to biodegradation. Recently, two critical enzymes, EDTA monooxygenase (EmoA) and NADH:FMN oxidoreductase (EmoB), belonging to the newly established two-component flavin-diffusible monooxygenase family, were identified in the EDTA degradation pathway in Mesorhizobium sp. BNC1. EmoA is an FMNH2-dependent enzyme that requires EmoB to provide FMNH2 for the conversion of EDTA to ethylenediaminediacetate. To understand the molecular basis of this FMN-mediated reaction, the crystal structures of the apo-form, FMN.FMN complex, and FMN.NADH complex of EmoB were determined at 2.5 angstroms resolution. The structure of EmoB is a homotetramer consisting of four alpha/beta-single-domain monomers of five parallel beta-strands flanked by five alpha-helices, which is quite different from those of other known two-component flavin-diffusible monooxygenase family members, such as PheA2 and HpaC, in terms of both tertiary and quaternary structures. For the first time, the crystal structures of both the FMN.FMN and FMN.NADH complexes of an NADH:FMN oxidoreductase were determined. Two stacked isoalloxazine rings and nicotinamide/isoalloxazine rings were at a proper distance for hydride transfer. The structures indicated a ping-pong reaction mechanism, which was confirmed by activity assays. Thus, the structural data offer detailed mechanistic information for hydride transfer between NADH to an enzyme-bound FMN and between the bound FMNH2 and a diffusible FMN.

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Title: Crystal structures of NADH:FMN oxidoreductase (EmoB) at different stages of catalysis
Creators: Mark S Nissen - School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660, USA
Buhyun Youn
Benjamin D Knowles
Jordan W Ballinger
Se-Young Jun
Sara M Belchik
Luying Xun
ChulHee Kang
Publication Details: The Journal of biological chemistry, Vol.283(42), pp.28710-28720
Academic Unit: Chemistry, Department of; Molecular Biosciences, School of
Publisher: United States
Identifiers: 99900547501001842
Language: English
Resource Type: Journal article