Photon Factory Activity Report 2022 #40 (2023)

AR-NW12A/2021G143

Crystal structure of a novel type of ornithine -aminotransferase

Ryushi KAWAKAMI1, Tatsuya OHSHIDA2, Junji HAYASHI1, Kazunari YONEDA3, Toshihisa
OHSHIMA4, and Haruhiko SAKURABA2, *1
Division of Bioscience and Bioindustry, Graduate school of Technology, Industrial and Social
Sciences, Tokushima University, 2-1, Minamijosanjima-cho, Tokushima, Tokushima 770-8513,Tokushima, Japan2
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393
Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0795, Japan3
Department of Food and Life Sciences, School of Agriculture, Tokai University, 871-12 Sugido,
Mashiki-machi, Kamimashiki-gun, Kumamoto 861-2205, Japan
4
Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology, 516-1 Ohmiya, Asahi-ku, Osaka, 535-8585, Japan.
1 Introduction
Ornithine -aminotransferase (Orn-AT, EC 2.6.1.13) is
one of the -aminotransferases and a fold type I pyridoxal
5’-phosphate (PLP)-dependent enzyme. This enzyme
catalyzes the transfer of the -amino group from Lornithine (L-Orn) to 2-oxoglutarate, yielding L-glutamate(L-Glu) and L-glutamate 5-semialdehyde, which
spontaneously cyclizes to form L-1-pyrroline-5carboxylate (P5C). In the first half reaction of Orn-AT(PLP-form), L-Orn is converted to L-glutamate 5semialdehyde and the PMP-form intermediate is produced.
In the second half-reaction, 2-oxoglutarate binds to theintermediate and after transamination, L-Glu and PLPform enzyme are produced as the final product (Fig. 1).
This means that the enzyme can selectively recognize the-amino group of L-Orn and the -amino group of Lglutamate for the two half reactions. However, themechanism in the dual substrate specificity of Orn-AT
remains to be fully elucidated.

Fig. 1. The two half-transamination reactions catalyzed
by Orn-AT. E-PLP: pyridoxal form of the enzyme, E-PMP:
pyridoxamine form of the enzyme.
Recently, an Orn-AT has been detected in thehyperthermophilic archaeon Thermococcus kodakarensis
and reported to be required for its growth in the absence ofexogenous proline [1]. That enzyme is the only archaeal
Orn-AT so far reported, and genetic analysis confirmed its
physiological function as the enzyme involved in thebiosynthesis of L-proline. Interestingly, the amino acidsequence of the T. kodakarensis Orn-AT (TK2101) is notsimilar to the previously reported sequences of bacterial
and eukaryotic Orn-ATs. The enzyme was originallyannotated as -aminobutyrate aminotransferase (GABAAT) in the genome database, and phylogenetic analysis
revealed that the clade containing T. kodakarensis Orn-AT
is clearly distinct from that of previously characterized
Orn-ATs [1].
We have been focusing on GABA-AT homologs fromPyrococcus and Thermococcus strains and found that thePH1423 gene product from Pyrococcus horikoshii sharesnearly 60% sequence identity with T. kodakarensis Orn-ATand exhibits a high level of Orn-AT activity. A structural
analysis of the PH1423 gene product may shed light on thesubstrate-recognition mechanism of a novel group ofarchaeal Orn-ATs, which are phylogenetically distant fromconventional Orn-ATs. In the present study, crystal
structures were determined for the enzyme in complex
with PLP and L-Orn, and in complex with N-(5’phosphopyridoxyl)- L-glutamate (PLP- L-Glu).
2 Experiment
Data were collected under cryo conditions at theBeamline AR-NW12A at Photon Factory in Japan. Theprogram Phaser-MR in the PHENIX was used formolecular replacement phase determination. The crystalstructure of putative GABA-AT from Sulfolobus tokodaii(PDB ID: 2eo5) was served as the search model. Modelbuilding was performed using the program Coot, andrefinement was carried out using REFMAC5.
3 Results and Discussion
The structure of the L-Orn/PLP-bound enzyme wasrefined to a resolution of 1.92 Å.
The crystal structure of human Orn-AT (PDB ID: 2oat)in complex with the inhibitor 5-fluoromethylornitine(FMO) has been reported previously. Based on thestructure of inhibitor-bound human Orn-AT, the natural
substrate L-Orn has been modeled into the active site as anexternal aldimine intermediate. In that model, the mainPhoton Factory Activity Report 2022 #40 (2023)

residues responsible for the specific binding of L-Orn were
thought to be Tyr55 and Arg180; the former is likely
involved in a hydrogen bond with the -amino group andthe latter forms a salt bridge with the -carboxylate (Fig.
2A, the human Orn-AT active site with inhibitor bound isindicated). In addition, the two aromatic residues, Tyr85and Phe177, were observed to form a hydrophobic
sandwich that accommodates the side chain of L-Orn. On
the other hand, mutation analysis of human Orn-AT
suggests that Tyr85 is a major determinant of specificity
toward L-Orn; Y85I mutation greatly decreased thereaction rate of the enzyme with L-Orn (1/1000). Becausethe -amino group of L-Orn is held between the OH groupsof Tyr55 and Tyr85, both of those residues are likely
responsible for the suitable binding of L-Orn.
Within P. horikoshii Orn-AT, Arg180 and Phe177 in
human Orn-AT were respectively conserved as Arg154 andPhe151, whereas the residues corresponding to Tyr55 andTyr85 were replaced by Thr30 and Ile63, respectively (Fig.
2B). Although the side chain of Ile63 forms hydrophobic
interactions with the side chain of L-Orn, no interaction
was observed between Thr30 and the substrate. Instead, theside chains of Thr92* and Asp93*, which arise from a loop
(residues 90-94) in the neighboring subunit (subunit B),
form hydrogen bonds with the -amino group of L-Orn(Fig. 2B). The residues involved in the corresponding loop
in human Orn-AT have no interactions with the substrate.
These observations mean that the manner by which the amino group of L-Orn is recognized totally differs between
the two enzymes.

human Orn-AT are conserved as Glu236 and Arg419,respectively. In the L-Orn-bound P. horikoshii Orn-AT, theside-chain of Glu236 forms a salt bridge with that ofArg419, closing the “Glu switch” and thus preventing
unsuitable L-Orn binding (Fig. 3B).
To test the “Glu switch” hypothesis, we performed acrystal structure analysis of the PLP- L-Glu-bound enzyme.
The structure was refined to a resolution of 2.99 Å.
Although the electron density for the covalent bondconnecting the C atom of L-Glu and the pyridine ring of
PLP was discontinuous, the significant electron density at
the L-Glu and PLP moieties enabled us to deduce aplausible structure for PLP-L-Glu (Fig. 3C). In this model,
we found that the side-chain of Glu236 rotates clockwise
by about 56 around the C atom of Glu236 relative to theL-Orn-bound P. horikoshii Orn-AT structure. As a result,the side-chain carboxylate of Glu236 faces in the direction
opposite to the side-chain of Arg419. Instead, the carboxylate of the L-Glu moiety in PLP-L-Glu interacts
with the side-chain of Arg419. These observations clearly
indicate that the “Glu switch” functions in this archaeal
Orn-AT. Moreover, the P. horikoshii Orn-AT structure incomplex with the external aldimine PLP-L-Glu adductprovides the first structural evidence for a “Glu switch”
mechanism [2].

Fig. 2. Close-up of FMO bound to human Orn-AT (A) andL-Orn bound to P. horikoshii Orn-AT (B).
Within the active site of the L-Orn-bound model ofhuman Orn-AT, the side chain of Arg413 is covered by theside chain of Glu235, and the -carboxylate of L-Orn binds
to the side chain of Arg180 (Fig. 3A; the active site ofinhibitor-bound human Orn-AT is indicated). Therefore, a“Glu235 switch” mechanism that prevents L-Orn frombinding in an orientation that would lead to transaminationof the -amino group has been proposed. In thatmechanism, the function of Glu235 is thought to be toneutralize the positive charge of Arg413 when L-Orn binds
to the active site. In the second half-reaction, the side chainof Glu235 is supposed to switch its position, uncovering
Arg413, thereby enabling the Arg413 side-chain to interact
with the -carboxylate of 2-oxoglutarate or L-Glu. Within
the P. horikoshii Orn-AT structure, Glu235 and Arg413 inFig. 3. Close-up of FMO bound to human Orn-AT (A), LOrn bound to P. horikoshii Orn-AT (B), and PLP-L-Glubound to P. horikoshii Orn-AT (C).
The present study describes the first structure of anovel type of Orn-AT, and our results may provide criticalinformation that will facilitate better understanding of thePhoton Factory Activity Report 2022 #40 (2023)

structure-function relationships within archaeal GABA-AT
homologues.
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] R.C. Zheng et al., J. Biol. Chem. 293, 3625 (2018).
[2] R. Kawakami et al., Int. J. Biol. Macromol. 208, 731(2022).
* sakuraba.haruhiko@kagawa-u.ac.jp