Photon Factory Activity Report 2016 #34 (2017)

BL-1A, AR-NE3A/NW12A 2014G661

Crystallographic study of novel enzymes necessary for CoA biosynthesis in archaea
Yoshiki Aikawa1, Akiko Kita2, Yuichi Nishitani1, and Kunio Miki1, *Graduate School of Science, Kyoto University, Sakyo-Ku, Kyoto, 606-8502, Japan
2
Research Reactor Institute, Kyoto University, Kumatori, Sennan, Osaka 590-0494, Japan
1

1 Introduction
Coenzyme A (CoA) plays pivotal roles in a variety of
metabolic pathways in all organisms. The biosynthesis
pathway of CoA has been studied well in bacteria andeukaryotes. In this pathway, two enzymes which convert
pantoate to 4’-phosphopantothenate are involved. Thesetwo enzymes are pantothenate synthetase (PS), whichcatalyzes the ATP-dependent condensation of pantoateand β-alanine to form pantothenate, and pantothenate
kinase (PanK), which catalyzes the phosphorylation ofpantothenate. The CoA biosynthesis pathway is regulated
by feedback inhibition by CoA to PanK.
These two enzymes, PS and PanK are absent in almost
all archaea. Instead, it was reported that two peculiar
enzymes of archaea, pantoate kinase (PoK) andphosphopantothenate synthetase (PPS), are responsible
for these conversion [1]. PoK catalyzes the ATP mediatedphosphorylation of pantoate to form 4’-phosphopantoate.
On the other hand, PPS catalyzes the ATP-dependent
condensation of 4’-phosphopantoate and β-alanine. In
the archaea PoK/PPS pathway, the order of thecondensation reaction with β-alanine and thephosphorylation reaction are reversed compared to thePS/PanK reactions in bacteria/eukaryotes. In archaea,
CoA biosynthesis feedback inhibition is not regulated by
CoA binding to PoK or PPS, but to ketopantoatereductase (KPR) [2]. We have previously reported thecrystal structure of PPS and its reaction mechanisms [3].
We report here the crystallographic studies of PoK andKPR from Thermococcus kodakarensis.
2 Experimental
Crystals of PoK belonging to the space group of P321
with the cell dimensions of a=94.8Å, c=60.0Å wereobtained from PEG8,000 solutions. X-ray diffraction data
were collected at the AR-NW12A beamline of the Photon
Factory.
Crystals of the ternary complex of KPR from T.
kodakarensis (Tk-KPR), CoA, and 2-oxopantoate wereobtained from 2-methyl-2,4-pentanediol solution. The
crystals belong to the space group P212121 with the celldimensions of a=53.5Å, b=70.8Å, and c=163.9Å. The
crystallization was also performed in the presence ofNADH to investigate the effect of crystal packing.
Crystals belonging to the different space group, P1, withthe cell dimensions of a=37.6Å, b=45.0Å, c=183.1Å,α=84.9˚, β=87.8˚, γ=65.2˚, were obtained (P1-KPR) from
PEG3,350 solutions. X-ray diffraction data were collectedat the BL-1A, ARNE3A, and ARNW12A beamlines ofthe Photon Factory, and were processed with theHKL2000 package.
3 Results and Discussion
The crystals of PoK using glycerol as a cryoprotectant
diffracted up to 2.7Å resolution. The asymmetric unit
contained one PoK monomer. The crystal structure
analysis is in progress.
The crystal structure of Tk-KPR was solved [4] by themolecular replacement method using the coordinates of E.
coli KPR (2OFP.pdb) [5], and that of P1-KPR was solved
using Tk-KPR coordinates [6]. The R/Rfree values for TkKPR are 0.169/0.185 at 1.65Å resolution, and those ofP1-KPR are 0.222/0.260 at 2.3Å resolution. In each TkKPR and P1-KPR crystal, protein molecules exist as adimer (Fig. 1) in the same dimerization manner. This
result suggests that the dimer structure is not an artifact ofcrystallization. The dimerization is also consistent withthe result of size-exclusion chromatography. In Tk-KPR,+NADP , which presumably derives from E. coli cells used
as expression host, is bound to one of the two monomers(referred to as monomer 1 in Fig. 1), whereas CoA and 2oxopantoate are bound to the other (referred to as
monomer 2 in Fig. 1).

Fig. 1: Tk-KPR dimer structure.
When we superimposed two monomers in Tk-KPR
crystal, the binding site of CoA is overlapped with that of+NADP . This result may indicate that the activity
inhibition by CoA is competitive with NADH. Notably,monomer 2 adopts a relatively closed conformationcompared to monomer 1. These results suggest that CoAand 2-oxopantoate cooperatively trigger a conformationalchange from an open form to a closed form.
Comparison of two Tk-KPR monomers with E. coli
KPR indicates that monomer 1 of Tk-KPR represents aPhoton Factory Activity Report 2016 #34 (2017)+resting state as previously reported for NADP -bound E.
coli KPR, and monomer 2 in its inhibited state is similar
to products-bound E. coli KPR that represents an activestate [5,7]. In E. coli KPR, the binding of pantoate is a
key to adopt a closed form. Accordingly, comparisons ofTk-KPR and E. coli KPR also support the idea that 2oxopantoate, located at the same position, is essential toadopt a closed form for Tk-KPR. Comparison between
Tk-KPR and E. coli KPR also shows that the residues
involved in CoA recognition in Tk-KPR are also involvedin NAD(P)H recognition and highly conserved in archaea,
whereas some are not conserved in E. coli KPR. Thus, theaffinity of CoA for E. coli KPR would be lower than thatfor Tk-KPR, implying that E. coli KPR may not be
inhibited by CoA. Significantly, Cys84, which isconserved among several Thermococcus strains but notamong bacteria or eukaryotes, forms a disulfide bondwith CoA (Fig. 2). This disulfide bond prevents
dissociation of CoA from the active site, and enables
strict inhibition of Tk-KPR activity upon binding of CoA.

Fig. 2: Disulfide bond with CoA.

References[1] Y. Yokooji et al., J. Biol. Chem., 284, 28137 (2009).
[2] H. Tomita et al., Mol. Microbiol., 90, 307 (2013).
[3] A. Kishimoto et al., PROTEINS, 82, 1924 (2014).
[4] Y. Aikawa et al., PROTEINS, 84, 374 (2016).
[5] A. Ciulli et al., J. Biol. Chem., 282, 8487 (2007).
[6] Y. Aikawa et al., Acta Cryst., F72, 369 (2016).
[7] CM. Lobley et al., Biochemistry, 44, 8930 (2005).
* miki@kuchem.kyoto-u.ac.jp