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

Abstract

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.

Highlights

Crystal Structures and Reaction Mechanisms of EmoBDiscovered, including 4-hydroxyphenylacetate monooxygenase of Escherichia coli strain W (19), 2,4,6-trichlorophenol 4-monooxygenase of Cupriavidus necator JMP134 (20), and 2,4,5trichlorophenol 4-monooxygenase of Burkholderia cepacia AC1100 (21)
The crystal structure of the FMN1⁄7FMN complex of EmoB provides the first direct observation for two FMN molecules stacked in opposite directions, making a proper distance for hydride transfer
The first FMN molecule is tightly bound through several hydrogen bonds in a shallow pocket, and its N-1 atom of the isoalloxazine ring is hydrogen-bonded to Gly112, which potentially catalyzes the hydride transfer reaction

Summary

Crystal Structures and Reaction Mechanisms of EmoB

Discovered, including 4-hydroxyphenylacetate monooxygenase of Escherichia coli strain W (19), 2,4,6-trichlorophenol 4-monooxygenase of Cupriavidus necator JMP134 (20), and 2,4,5trichlorophenol 4-monooxygenase of Burkholderia cepacia AC1100 (21). PheA2 has a tightly bound FAD prosthetic group (Kd ϭ 10 nM), and its reaction follows a Ping Pong Bi Bi mechanism. HpaCSt, the small-component reductase for 4-hydroxyphenylacetate 3-monooxygenase from Sulfolobus tokodaii, has a tightly bound FMN, probably following a Ping Pong Bi Bi mechanism. Another HpaC from Thermus thermophilus HB8 and EmoB from Mesorhizobium sp. Crystal structures for some of these smallcomponent reductases have been reported, such as PheA2 (26), HpaCSt (27), and HpaCTt (25) In addition to their structural similarities, the crystal structures of HpaCSt and PheA2 reveal tightly bound FAD and FMN, respectively. Systematic investigations using light scattering and isothermal calorimetry offer critical information about EmoB in the EDTA degradation pathway

EXPERIMENTAL PROCEDURES

RESULTS

Protein and ligand