Photon Factory Activity Report 2017 #35 (2018)

BL-1A, BL-5A/2015G645

Structure analysis of characteristic enzymes regarding phosphate addition or release
Masahiro Fujihashi,* Ryuhei Nagata, and Kunio Miki
Department of Chemistry, Graduate School of Science, Kyoto University,
Sakyo-ku, Kyoto 606-8502, Japan
1 Introduction
A free serine kinase (SerK) was recently identified in
Thermococcus kodakarensis [1]. SerK is involved in thecysteine biosynthesis and phosphorylates L-serine (Ser)using ADP to produces O-phospho-L-serine (Sep), which
is a precursor of L-cysteine.
SerK is expected to possess a novel reaction
mechanism because of its low sequence similarity toknown kinases. This enzyme had been annotated as achromosome-partitioning protein ParB based on theprimary structure. However, no kinase activity of ParB
has been reported. Thus, we determined the crystal
structures of SerK complexed with the substrate or
product and proposed its reaction mechanism.

E30A-product complex possesses Sep in the cavity
between the two domains and is in the closed
conformation (Fig. 1B). This implies that the two-ligand
binding leads to the conformational closure, which may
occur with two substrates, Ser and ADP. Consistent withthis implication, the E30-AMP complex is in an openform.

2 Experiment
We crystallized the wild-type (WT) or its mutants(E30A and E30Q) using poly(ethylene glycol) 3350 as aprecipitant. Diffraction data sets were collected using thebeamlines of BL-1A and BL-5A as well as some other Xray sources. Phases were determined by molecularreplacement with the atomic coordinates of PF0380(Protein Data Bank ID: 1VK1). This protein is 85%
identical to SerK, but it was annotated as a ParB-like
nuclease. Finally, the six complex structures weredetermined at 1.43–2.0 Å resolution: WT-AMP, E30Aproducts, E30A-AMP, E30A-ADP, E30Q-products, andE30Q-AMP.
3 Results and Discussion
The WT enzyme was crystallized with Ser and AMP,
but the determined structure possesses only AMP. No
electron density map corresponding to Ser was observed.
The overall structure is divided into two domains: domain
I and II (Fig. 1A). AMP is bound to domain I.
We chose four acidic residues (E4, E30, E36, and D69)
near to the α-phosphate of AMP as candidates for thecatalytic residue (Fig. 2A), because many kinases
deprotonate a hydroxyl group of the phosphate acceptors
using the acidic residue. The four residues were eachreplaced with alanine, and the activities of the resultant
mutants were measured. No activity of E30A and D69A
mutants were detected, while E4A and E36A mutants
exhibited significant activity. This suggests that E30 andD69 may be important for the catalysis.
The E30A and D69A mutants were crystallized withSer and ADP in order to determine the structure justbefore the reaction. The E30A structure was determined,
whereas that of D69A was not due to its poor diffraction.
Unexpectedly, the products (Sep and AMP) are found in
the E30A structure (Fig. 1B), suggesting that E30A cancatalyze the reaction in a long crystallization period. TheFig. 1: Overall structures of the WT-AMP complex (A)and the E30A-product complex (B).
The structure indicates that three of the four catalytic
residue candidates (E4, E36, and D69) are involved in themagnesium binding. The other (E30) is the probablecatalytic residue because structural comparison between
the WT-AMP and E30A-product complex suggests thatE30 is located near to the Oγ atom of Sep (Fig. 2B).
Supporting this suggestion, Q30 in the E30Q-productcomplex is nearer to the Oγ atom than that in the E30QAMP complex (Fig. 2C).
We also determined the structure of the E30A-ADP
complex. The electron density corresponding to the βphosphate of ADP (phosphate donor of the reaction) wasambiguous. Thus, we collected the data sets using X-ray

Photon Factory Activity Report 2017 #35 (2018)of the wavelength of 2.70 Å. The anomalous Fourier map
corresponding to the β-phosphate was clearly observed.
Comparison between the E30A-ADP and E30A-productcomplex led us to propose that, in the closed form, the βphosphate of ADP may be recognized in the same manneras that of the phosphate group of Sep (Fig. 2D).

Fig. 2: Structures of the ligand-binding site. (A) AMP
binding in the WT-AMP complex. (B, C) Comparison of
E30 and the corresponding residues. The structures ofWT-AMP, E30A-product, E30Q-product, and E30QAMP complexes are shown in yellow, pink, cyan, andlight orange, respectively. (D) Structural comparison ofthe ligand-binding site between the E30A-productcomplex (pink) and the E30A-ADP complex (black).
Our structural analyses on SerK lead us to propose thereaction mechanism with the conformational shift. SerK
is in the open form without its substrates, Ser and ADP, insolution. The substrate binding to the ligand-binding site
results in the conformational closure. Then, the side chainof E30 approaches the hydroxyl group of Ser anddeprotonates it. The deprotonated hydroxyl group attacks
the phosphorus atom of the β-phosphate of ADP toproduce Sep and AMP. Finally, Sep and AMP are released
with opening of the cavity [2].

References[1] Y. Makino et al., Nat. Commun. 7, 13446 (2016)[2] R. Nagata et al., ACS Chem. Biol. 12, 1514–23 (2017)* mfuji@kuchem.kyoto-u.ac.jp