Photon Factory Activity Report 2018 #36 (2019)

AR-NE3A, AR-NW12A /2017G675

Structure of a phosphotransacetylase from Porphyromonas gingivalis, a keystone
pathogen of periodontitis
Yuichiro KEZUKA1, *, Yasuo YOSHIDA2 and Takamasa NONAKA1
Division of Structural Biology, Department of Pharmaceutical Sciences,
School of Pharmacy, Iwate Medical University, Yahaba, Iwate, Japan2
Department of Microbiology, School of Dentistry, Aichi Gakuin University, Nagoya, Japan
1

1 Introduction
Porphyromonas gingivalis is known to be a keystone
pathogen of periodontitis and is an asaccharolytic
bacterium that gains its metabolic energy by fermenting
amino acids such as glutamate and aspartate. In themetabolic pathways, acetyl-CoA is produced as anintermediate and then is converted to acetate.
Phosphotransacetylase (Pta, EC 2.3.1.8) and acetate kinase(Ack, EC 2.7.2.1) play a role for production of ATP fromexcess acetyl-CoA. Pta produces acetyl phosphate fromacetyl-CoA and resulting acetyl phosphate is subsequently
converted to acetate by Ack with the concomitant
production of ATP through substrate-level phosphorylation.
Despite the importance of Pta and Ack in energy
metabolism in P. gingivalis, these enzymes have not beencharacterized. We report herein the crystal structure of Pta
from P. gingivalis.
2 Experiment
Pta from P. gingivalis ATCC 33277 (PgPta) wasoverproduced in Escherichia coli BL21(DE3) strain as a
glutathione S-transferase-fusion protein and purified aspreviously reported [1]. Crystals of substrate-free PgPta
were obtained under the conditions, 20% (w/v)
polyethylene glycol 3350 and 0.2 M ammonium tartrate
dibasic using the hanging-drop vapor diffusion method.
Crystals of PgPta in complex with acetyl-CoA wereprepared by cocrystallization in the presence of 5 mM
acetyl-CoA. Crystals were cryoprotected by soaking inmother liquor containing 10% (v/v) 2-methyl-2,4pentanediol, and then data from the crystals were collected
at the beamlines NW12A and NE3A. The data wereindexed, integrated, and scaled using the programs DIALSand SCALA, as implemented in XIA2. The phasedetermination was performed by the molecularreplacement technique using a homology model on thebasis of the structure of Methanosarcina thermophila Pta(MtPta) (PDB ID: 2AF4). The models were fixed andrefined using COOT and REFMAC5.
3 Results and Discussion
The crystal structures of PgPta and its complex withacetyl-CoA were determined at 1.7 Å and 2.0 Å resolution,respectively. The subunit is an α/β protein composed of
two α/β domains (Fig. 1).
Two acetyl-CoA molecules were bound to the dimer,
and the binding site in each subunit is identical. Arg89 andArg135 interact with the 3’-phosphate and β-phosphate ofacetyl-CoA, respectively. Replacement of these residues toalanine resulted in a 58% and 21% decrease in the specificactivity, respectively. In MtPta, two CoA molecules werebound to each subunit [2], and the two binding sites wereshown to have different affinities. One site (site 1) issuggested to be the active site on the basis of the mutational
study. The other site (site 2) is suggested to be a loadingsite to preorient the substrate or a regulatory site to control
the activity. Superposition of the structures of PgPta andMtPta revealed that acetyl-CoA molecules were boundonly to site 2 in both subunits of PgPta, even though crystal
packing of PgPta does not appear to interfere with bindingof acetyl-CoA to site 1. Since interference of the bindingof acetyl-CoA to site 1 of PgPta has not been observed insolution, such contradictory findings are possibly due tocrystallization artifacts. Dynamic aspects, such as domain
motion, can affect substrate binding and are difficult toestimate from the static picture revealed by a crystalstructure. Further analyses to investigate the binding ofacetyl-CoA to PgPta are required.

Arg135Arg89

y let oAcA C

Fig. 1: Dimeric overall structure of PgPta in complex withacetyl-CoA. Each subunit is shown with independentcolors. Bound acetyl-CoA, Arg89, and Arg135 are shownas spheres. The position with a circle is likely tocorrespond to the putative active site.
Acknowledgement
This work was supported by JSPS KAKENHI Grant
Nos. JP16K11485 (to Y. K.) and JP17K11634 (to Y. Y.).
We thank the PF staff for the great support for datacollection.
References[1] Yoshida et al., J. Oral Microbiol. 11, 1588086 (2019).
[2] Lawrence et al., J. Bacteriol. 188, 1143-1154 (2006).
The atomic coordinates and structure factors of PgPtaand its complex with acetyl-CoA have been deposited toProtein Data Bank as # 6IOW and 6IOX, respectively.
* ykezuka@iwate-med.ac.jp