Photon Factory Activity Report 2017 #35 (2018)

AR-NW12A/2011G010

Structure of a cysteine (hydroxyl) lyase from hydrogen sulfide-producing oral
pathogen, Fusobacterium nucleatum
Yuichiro KEZUKA* and Takamasa NONAKA
Department of Structural Biology, School of Pharmacy, Iwate Medical University, Yahaba, Iwate
028-3694
1 Introduction
Hydrogen sulfide (H2S) is one of the predominantvolatile sulfur compounds that are primarily responsible
for oral malodor and is an important contributor toperiodontitis. In the human oral cavity, H2S is generally
produced by enzymatic reactions of oral bacteria. βC-Slyase catalyzing the α,β-elimination reaction of L-cysteine
to produce H2S, pyruvate, and NH4+ is known to be acommon H2S-producing enzyme. In Fusobacterium
nucleatum, a heavy H2S producer in the human oral cavity,
two unique pyridoxal 5’-phosphate (PLP)-dependent H2Sproducing enzymes have been identified and characterized[1], in addition to βC-S lyase. One of the two enzymes isFn1055 protein (cysteine (hydroxyl) lyase), whichcatalyzes the production of L-serine and H2S from Lcysteine and H2O (β-replacement reaction). This enzyme
strictly prefers L-cysteine as a substrate. Interestingly, F.
nucleatum lacks genes encoding the three enzymes
essential to bio-synthesize L-serine from 3phosphoglycerate, which are generally found in bacteriasuch as Escherichia coli. Therefore, Fn1055 seems to beimportant to control L-serine levels in this bacterium. As afirst step to reveal the reaction mechanism of the enzyme,
we performed the structural analysis.
2 ExperimentFn1055 from F. nucleatum subsp. nucleatum ATCC
25586 was overproduced in Escherichia coli and purified
as described earlier [2]. Crystals of Fn1055 were obtainedby the hanging-drop vapor diffusion method. Rod-likecrystals were obtained under condition #42 of PEGRx1[4% (w/v) PEG8000 and 0.1 M Hepes pH 7.5] after initial
screening trials of approximately 1000 conditions. Adataset from the crystal was collected at the beamline
NW12A. The data was indexed, integrated, and scaled
using the programs MOSFLM and SCALA, asimplemented in XIA2. The phase determination wasperformed by the molecular replacement technique using ahomology model on the basis of the structure of Oacetylserine sulfhydrylase from Thermotoga maritima(PDB ID: 1O58). The model was fixed and refined using
COOT and REFMAC5.
3 Results and Discussion
The crystal structure of Fn1055 was refined at 2.9 Åresolution. A monomer of Fn1055 is situated in theasymmetric unit. The gel-filtration experiment suggeststhat the enzyme is present as a dimer (data not shown) andthe two subunits are related by a crystallographic two-foldaxis.
The structure revealed an Fn1055-specific residue,
Asp232, which is likely to be important for the nucleophilic
attack of water molecules to the α-aminoacrylateintermediate. The amino acid sequences of the loop
containing Asp232 differ between Fn1055 and related βreplacement enzymes. Therefore, the loop structure could
contribute the substrate specificity of the enzymes.

Fig. 1 Dimeric overall structure of Fn1055
The figure is shown in a ribbon representation. Whereas
one subunit is colored in blue (N-terminal domain) andgreen (C-terminal domain), the other in gray. The PLPcofactor is positioned between the domains and covalently
bound to the ε-amino group of Lys46.
Acknowledgement
This work was supported by JSPS KAKENHI Grant
Nos. JP23792130 and JP25462897 (to Y. K.). We thank thePF staff for the great support for data collection.
References[1] Y. Yoshida et al., Microbiology, 157, 2164-2171 (2011).
[2] Y. Kezuka et al., Biochem. J. 475, 733-748 (2018).
The atomic coordinates and structure factors of Fn1055
have been deposited to Protein Data Bank as # 5B53.
* ykezuka@iwate-med.ac.jp