Photon Factory Activity Report 2014 #32 (2015) B
AR-NE3A/2013G003

Unique Cofactor Binding Mode of Homoserine Dehydrogenase from
Hyperthermophilic Archaeon Pyrococcus horikoshii
Junji Hayashi1, Shota Inoue1, Kazunari Yoneda2, Toshihisa Ohshima3 and Haruhiko Sakuraba1
1
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University,
Ikenobe 2393, Miki-cho, Kagawa 761-0795, Japan2

Department of Bioscience, School of Agriculture, Tokai University,Aso, Kumamoto, 869-1404, Japan3
Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology,
5-16-1, Ohmiya, Asahi-ku, Osaka, 535-8585, Japan
1 Introduction
Homoserine dehydrogenase (HseDH, EC 1.1.1.3) is akey enzyme in the biosynthetic pathway from aspartate tohomoserine (Hse), which is a common precursor for thesynthesis of three amino acids, methionine, threonine andisoleucine in plants and microorganisms. In this pathway,
aspartate is first phosphorylated to -aspartyl phosphate(-Ap) by aspartate kinase, after which L-aspartate-semialdehyde (Asa) dehydrogenase catalyzes theconversion of -Ap to Asa. The third enzyme in thepathway, HseDH, catalyzes the NAD(P)H-dependent
reduction of Asa to Hse. Subsequent metabolism of Hse
yields methionine, threonine or isoleucine, which are all
essential in humans. HseDH is thought to be a potential
target for the structure-based design of antibiotics or
herbicides, as the enzyme is not present in mammals.
Moreover, stable HseDH is a potentially useful target forthe development of new method for homocysteine
biosensing [1].
Up to now, structure of HseDH from Archaea or fromhyperthermophiles has not been reported. The structuresof putative HseDHs from Archaeoglobus fulgidus,
Thermoplasma acidophilum and T. volcanium weredeposited in the Protein Data Bank (PDB), but the
enzymological properties and structural details of theseenzymes have not been reported. Within the genomic
sequence of the hyperthermophilic archaeon Pyrococcus
horikoshii, we found a gene (ORF PH1075) whose
predicted amino acid sequence exhibits 31.2% identity tothat of the A. fulgidus HseDH homolog. In the present
study, we expressed that gene, characterized the enzyme
produced and revealed the enzyme to be a highly
thermostable HseDH. We then determined the crystal
structure of this enzyme [2]. Refinement of the structure
showed the presence of a bound cofactor, NADP(H),although it was not added during crystallization, whichindicates earlier high-affinity binding of the cofactor tothe protein. Surprisingly, however, we found that NADP
does not act as a cofactor for this enzyme, but as a strong
inhibitor of NAD-dependent oxidation of Hse. Structural
analysis and site-directed mutagenesis showed that thelarge number of interactions between the cofactor and theenzyme are responsible for the lack of reactivity of theenzyme towards NADP. This observation suggests thisenzyme exhibits a new variation on cofactor binding to a

dehydrogenase: very strong NADP binding that acts as anobstacle to NAD(P)-dependent dehydrogenase catalytic
activity.
2 Experiment
Diffraction data were collected (=1.0 Å) on thebeamline AR-NE3A at the Photon Factory. The initial
phases for the structure were determined by molecularreplacement; the structure of A. fulgidus HseDH
homologue (PDB entry 3DO5) served as the searchmodel.
3 Results and Discussion
The gene (PH1075) was overexpressed in Escherichia
coli, and its product was purified and characterized. The
expressed enzyme is the most thermostable HseDH yet
described, retaining 70% of its activity even afterincubation for 10 min at 95ºC. The crystal structure of theenzyme was determined at a resolution of 2.30 Å.
Refinement of the structure and mass analysis showed thepresence of the bound cofactor NADPH, although it has
not been added during crystallization.

Fig. 1: NADPH-binding site in P. horikoshii HseDH.

Photon Factory Activity Report 2014 #32 (2015) B

The C2 phosphate group of the cofactor adenine ribose istightly held at the nucleotide-binding site through three
direct hydrogen bonding interactions with the side chainsof Arg40 and Lys57.
Unexpectedly, however, NADP did not act as acofactor with this enzyme, but as a strong inhibitor ofNAD-dependent Hse oxidation. Most strikingly, K57A
mutant acquired high reactivity against NADP. The
crystal structure of the K57A mutant with bound NADPH
was then determined to a resolution of 2.43 Å [2].

O3X

Fig. 2: Comparison of the NADPH-binding site structuresin P. horikoshii HseDH wild-type (green and blacklabels) and the K57A mutant (cyan and red labels).
Superposition of the NADPH-bound K57A structure
onto the NADPH-bound wild-type structure showed thatthe NADPH molecule in the mutant structure waspositioned/configured nearly identically to the NADPH
molecule in the wild-type structure, except for thepositioning of the C2 phosphate group of the adenine
ribose (Fig. 2). In the wild-type enzyme, the C2
phosphate is tightly held in position through fivesurrounding hydrogen bonds. In K57A mutant, however,
the C2 phosphate group is rotated in a clockwise direction
around C2B of NADPH by about 30 relative to the wildtype structure. In addition, the guanidino group of Arg40
in the mutant is also rotated clockwise by about 90
around the NE atom of Arg40 relative to the wild-typestructure. As a result, only one interaction between O3Xof the C2 phosphate and the NE of Arg40 was observed at
the corresponding position in K57A (Fig. 2). Nearly all
the interactions between the cofactor NADH moiety andthe enzyme are conserved between the two enzymes,except for a few water-mediated interactions. Given thatno substantive difference in the domain organization of
the two enzymes was found, these observations suggestthat the larger number of interactions around the C2
phosphate of the cofactor strengthen the affinity of wild
type HseDH for NADP, making the binding too tight toutilize NADP as the cofactor.

Because NAD differs from NADP only in the C2
phosphate group of the adenine ribose, the amino acidresidues interacting with this region are thought to beresponsible for the cofactor specificity of the enzymes. In
the medium-chain dehydrogenase/reductase familyenzymes, the coenzyme binding domains possess similar
-- motifs centered around a highly conserved
GXGXXG/A sequence. Among the amino acid residuesof this -- motif, the primary determinant of NADspecificity is the presence of an Asp or Glu residue, whichforms hydrogen bonds with both the C2 and C3 hydroxyl
groups of the NAD adenine ribose and occupies the spacethat would be occupied by the C2 phosphate group of theNADP adenine ribose. In NADP dependent enzymes, on
the other hand, this residue is usually replaced by asmaller residue such as Gly, Ala or Ser, accompanied byone or more positively charged residues, Arg and/or Lys,
which form a binding pocket for the C2 phosphate group.
Similar features are also observed in short-chain
dehydrogenase/reductase family enzymes. In someenzymes with dual cofactor specificity, replacement of thehydrogen bonds associated with each cofactor binding has
been observed. HseDH belongs to an expansive anddiverse class of oxidoreductases, and although the overall
fold of the catalytic region is unique, the nucleotidebinding domain conforms to the Rossmann fold, likeconventional NAD(P)-dependent dehydrogenases. The
structure of the nucleotide-binding site in our model
indicates that P. horikoshii HseDH would have a strong
preference for NADP. However, NADP does not act as
the cofactor for this enzyme, but as a strong inhibitor for
NAD-dependent Hse oxidation. To the best of ourknowledge, this is the first reported example of an
enzyme in which the very strong binding of NADP is an
obstacle to its catalytic activity, in this case NAD(P)dependent dehydrogenase activity. Although thephysiological significance of the inhibitory effect ofNADP remains unclear, the present study indicates thatthe molecular details underlying cofactor preference inNAD(P)-dependent dehydrogenases are more complex
than expected, and the cofactor specificity cannot bepredicted even from the structural information in theabsence of biochemical data.
Acknowledgement
We are grateful to the staff of the Photon Factory for
their assistance with data collection, which was approvedby the Photon Factory Program Advisory Committee.
References[1] Y. Tomonaga, T. Komoriya, H, Kohno, T. Ohshima,K, Yoshimune, (2013-12-7) ISSN 2186-5647, P-59
( 第 46 回日本大学生産工学部学術講演概要)[2] J . H a y a s h i , S . I n o u e , K . K i m , K , Yo n e d a ,Y, Kawarabayasi, T. Ohshima and H. Sakuraba,Sci. Rep. 5, 11674 (2015).
* sakuraba@ag.kagawa-u.ac.jp