Photon Factory Activity Report 2018 #36 (2019)

AR-NE3A /2017G675

Structure of an acetate kinase 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
11 Introduction
Porphyromonas gingivalis is an asaccharolytic
periodontal pathogen which mainly gains its metabolicenergy from amino acids such as glutamate and aspartate.
In the metabolic pathways, this bacterium has
phosphotransacetylase (CH3COSCoA + HPO42- ↔
CH3CO2PO32- + HS-CoA) and acetate kinase(CH3CO2PO32- + ADP ↔ CH3COO- + ATP) for theconversion of acetyl-CoA to acetate with formation of ATP .
We reported that inactivation of phosphotransacetylase
gene (ack) or acetate kinase gene (ack) in P. gingivalis byhomologous recombination was successful only when theinactivated gene was expressed in trans [1]. These results
strongly suggest that both genes are essential for thispathogen. Despite the importance of these two enzymes inenergy metabolism in P. gingivalis, these enzymes havenot been characterized. We report herein the crystal
structure of acetate kinase from P. gingivalis.

domains relative to the C-terminal domain are different,
which is leading to opening and closing of the putative
catalytic cleft. While the cleft of subunit A is the most open,that of subunit D is the most closed (Fig. 2). Subunits B
and C take the intermediate conformations (data notshown). In the cleft of subunit D, there are two boundsulfate ions probably derived from mother liquor. This may
mimic binding of the phosphate groups of ADP and/orATP, resulting the most closed conformation. These results
indicate a remarkable conformational change in PgAck
during catalysis.

2 Experiment
Acetate kinase from P. gingivalis ATCC 33277 (PgAck)
was overproduced in Escherichia coli BL21(DE3) strain asa hexahistidine tag-fusion protein and purified as describedearlier [1]. Crystals of PgAck were obtained under theconditions, 0.2 M lithium sulfate monohydrate, 0.1 M
Tris/HCl pH 8.5, and 24% (w/v) polyethylene glycol (PEG)
3350 using the hanging-drop vapor diffusion method.
Crystals were soaked in mother liquor containing 30%(v/v) PEG 3350 to cryoprotect, and then a dataset from thecrystals was collected at the beamline NE3A. The data wasindexed, 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 Thermotoga maritima Ack (PDBID: 2IIR). The model was fixed and refined using COOT
and REFMAC5.

Fig. 1: Dimeric overall structure of PgAck (subunits C andD colored in purple and pink, respectively). Bound two
sulfate ions are shown as spheres.

3 Results and Discussion
The crystal structure of PgAck was refined at 1.9 Åresolution (Fig. 1). Two dimers are contained in theasymmetric unit of the P21 cell. The subunit is composedof the N-terminal domain (1-149 and 384-398) and the Cterminal domain (150-383). Comparison of the four
subunits in the asymmetric unit indicated that there are four
distinct conformations, with root mean square deviations
ranging from 0.8 to 5.2 Å using all 398 Cα atoms. As
shown in Fig. 2, the structure of C-terminal domain isalmost identical, and the position of the N-terminal

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.

Pro37

ain
domnalrmi
C -t eN-terminal
domainFig. 2: Superposition of PgAck subunits A (light blue) andD (pink) using Cα atoms of the C-terminal domain. The
bound sulfate ions and Pro37 positioned at the edge of thecleft are shown as spheres. The difference between the two
Pro37 is ~18 Å.

References[1] Yoshida et al., J. Oral Microbiol. 11, 1588086 (2019).
The atomic coordinates and structure factors of PgAck
have been deposited to Protein Data Bank as # 6IOY.
* ykezuka@iwate-med.ac.jp