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Dynamic interactions in the l-lactate oxidase active site facilitate substrate binding at pH4.5

Furubayashi, Naoki Inaka, Koji Kamo, Masayuki Umena, Yasufumi Matsuoka, Takeshi Morimoto, Yukio 京都大学 DOI:10.1016/j.bbrc.2021.06.078

2021.09

概要

The crystal structure of l-lactate oxidase in complex with l-lactate was solved at a 1.33 Å resolution. The electron density of the bound l-lactate was clearly shown and comparisons of the free form and substrate bound complexes demonstrated that l-lactate was bound to the FMN and an additional active site within the enzyme complex. l-lactate interacted with the related side chains, which play an important role in enzymatic catalysis and especially the coupled movement of H265 and D174, which may be essential to activity. These observations not only reveal the enzymatic mechanism for l-lactate binding but also demonstrate the dynamic motion of these enzyme structures in response to substrate binding and enzymatic reaction progression.

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Figure Legends

Figure 1. Structures of the active site in FMN and the surrounding amino acid residues of

the L-lactate oxidase (LOX) in the presence of (a) acetic acid, (b) L-lactate, (c) D-lactate

or (d) pyruvate represented as stick models. Electron density maps are shown as a 2mFoDFc omit map at 3.0 s levels. Images were prepared using the PyMOL Molecular Graphic

System program.

Figure 2. Distances between the L-lactate and surrounding amino acid residues. Values

are designated using the angstrom unit and shown by the dotted line.

Figure 3. Molecular surfaces in the LOX as seen from the invading substrate; (a) substrate

FREE open form (b). In the magnified square in image (a) FMN can be seen in red and

acetic acid in cyan. (c) closed form of the L-lactate bound complex, areas highlighted in

magenta represent those in a new position when compared to the open (a) form. Images

represent a LOX dimer with the opposite molecule being the B-mol.

Figure 4. Coupled movements of the H265 and D174 residues near the FMN. (a) Substrate

FREE form, two amino acids far from the FMN si-side, (b) substrate bound closed form,

two coupled residues move toward FMN but retain a distance of 2.8 Å.

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(a)

(c)

Fig.1 morimoto

(b)

(d)

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Y40

2.4

2.8

Y146

3.0

2.6

2.9

D174

Fig.2 morimoto

2.8

H265

2.9

3.0

R268

R181

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(a)

Fig.3 morimoto

(b)

(c)

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H265

H265

2.83

(a)

D174

Fig.4 morimoto

2.85

(b)

D174

...

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