Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

5A,NW12/2004G348

Crystal Structure Analysis of L-Lactate Oxidase
Yasufumi UMENA1,2, Kazuko YORITA3, Akiko KITA1 and Yukio MORIMOTO*1,1
Research Reactor Institute, Kyoto University, Kumatori, Osaka 590-0494, Japan
2
Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan
2
Institute for Enzyme Research, The University of Tokushima, Tokushima 770-8503, Japan
Introduction
The α-hydroxyacid oxidases are a group offlavoproteins which catalyze the flavin mononucleotide(FMN)-dependent oxidation of their respective substrates.
These enzymes have been found to share remarkablesimilarities in catalytic properties with common structural
motifs, making it likely that they constitute a family. Llactate oxidase (LOX) from Aerococcus viridans
catalyzes the oxidation of L-lactate using molecularoxygen and produces pyruvate and H2O2. We had madethe site-directed mutant, LOX-R181M, where Arg181 has
been replaced by Met to determine the effect of removing
the positive charge at this position.
The LOX-R181M was crystallized well to good quality
for structure analysis with a small amount of additivereagent(1). Here, we describe the crystal structure of theLOX wild obtained by the same crystallization method ofthe mutant, and discuss how the substrate recognition
does.
Materials and Methods
Crystals were transferred to a drop of reservoir solution
and soaked for about 10 min. Due to the presence of PEG
as a precipitant in the crystallization protocol, theconcentration of PEG in the drop is gradually increasing
during this time. A crystal was mounted in a cryo-loop
and flash-frozen in a nitrogen-gas stream at 100K. X-ray
diffraction data were collected with a CCD detector usingsynchrotron radiation at beam-line BL5A and NW12 of
PF/AR KEK, Japan. The wavelength was 1 Å, and thecrystal-to- detector distance was maintained at either 200
or 130 mm. A total of 180 flames were collected with 1°oscillation and 5 sec exposures. The data were collected
up to 1.8 Å resolution and were processed with theprogram HKL 2000.
Crystals of the LOX belong to the tetragonal spacegroup I422, with unit-cell parameters a = b = 191.096Å,and c = 194.497Å. With the molecular weight of the LOX(41,000Da), we assume that four LOX monomers exist in
an asymmetric unit, resulting in a Matthews coefficient,VM, of 2.71 Å3Da-1. This value is in the normal range forglobular protein crystals. Solvent content of the crystal is50.4%.
The structure was determined by the molecularreplacement method using the program CNS. Self- andcross-rotation functions showed a tetramer formation of
the enzyme, the initial search model (LOX-R181Mmonomer) was generated to a tetramer with non-crystallographic symmetry. All refinements were carriedout using CNS and CCP4:REFMAC5. Refined model ofthe wild type LOX consisted of four molecules (Wild-A,Wild-B, Wild-C and Wild-D).
Results and Discussion
The overall fold of the LOX monomer was a typical(β/α)8-barrel first observed in a triose-phosphate
isomerase. The r.m.s. deviations of the main chain amongfour monomers whose both terminals and flexible regions
are excluded in the calculation are 0.701 Å (wild-A/B),
0.621 Å (wild-A/C), 0.635 Å (wild-A/D), 0.451 Å (wildB/C), 0.513 Å (wild-B/D) and 0.421 Å (wild-C/D).
Molecular exclusion chromatography of the enzyme bySephadex G-100 provided the basis for the report thatwild-type LOX was a tetramer in 50 mM potassium
phosphate buffer, pH 7.0, in the presence of 0.2 M
potassium chloride (2). Thus our LOX crystals have fourmonomers in the asymmetric unit, it suggests that theenzyme catalyses actively as a tetramer in the solution.
The LOX enzyme has a catalysis activity from L-lactate
to pyruvate with an oxigenation by the FMN group. In thereaction, the proton donation from the Arg181 residue isimportant. We looked into the structure around acitive
site, we've found some additional amino acids.

References[1] Y.Umena et al., Acta. Cryst., F61, 439 (2005)[2] J.D.Duncan et.al., Biochem Biophys Res Commun.
164, 919 (1989)*morimoto@rri.kyoto-u.ac.jp

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