Photon Factory Activity Report 2017 #35 (2018)

BL-5A, AR-NE3A/2017G010

Crystal structure of the novel amino-acid racemase isoleucine 2-epimerase fromLactobacillus buchneri
Junji Hayashi1, Yuta Mutaguchi2, Kazunari Yoneda3, Taketo Ohmori4, and Toshihisa Ohshima4, andHaruhiko Sakuraba5
1Department of Biotechnology, College of Life Sciences, Ritsumeikan University Biwako-Kusatsu
Campus, 1-1-1 Noji-higashi, Kusatsu, Shiga, 525-8577, Japan
234

Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University,
241-438 Kaidobata-Nishi, Nakano, Shimoshinjo, Akita 010-0195, Japan

Department of Bioscience, School of Agriculture, Tokai University, 9-1-1 Toroku, Higashi-ku,
Kumamoto-shi, Kumamoto, 862-8652, Japan.

Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology, 516-1 Omiya, Asahi-ku, Osaka, 535-8585, Japan
5

Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393
Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0795, Japan

1 Introduction
Free D-branched-chain amino acids (D-BCAAs) suchas D-isoleucine (D-Ile), D-leucine (D-Leu) and D-valine(D-Val) are thought to act as specific molecular signals insome bacterial species. For example, D-Ile, D-Leu and DVal are reportedly involved in regulating cell-wall
remodelling in Vibrio cholera. It has been suggested thatD-BCAAs are produced by a few pyridoxal 5’-phosphate(PLP)-dependent amino-acid racemases with broad
substrate specificity, which include arginine racemasefrom Pseudomonas graveolens and amino-acid racemasefrom P. putida. However, these two racemases showmuch lower activity towards Leu than their main
substrate, lysine, and no activity towards Ile or Val.
Alanine racemase from P. putida is known to catalyze LIle racemization, but this enzyme exhibits much lower
activity towards L-Ile than L-Ala. Thus, the available
biochemical and structural information on the mechanismof bacterial D-BCAA production remains limited. In a
recent study, a marked accumulation of D-Leu, D-allo-Ileand D-Val was observed in the growth medium of thelactic acid bacterium Lactobacillus otakiensis JCM 15040,
and the enzyme responsible was identified as a racemase
catalyzing the production of D-amino acids from thecorresponding L-amino acids [1]. On the basis of the Nterminal amino-acid sequence of the purified enzyme, agene encoding a homologue of this racemase wasidentified in the genome of L. buchneri JCM 1115 [1],which is closely related to L. otakiensis JCM 15040. The
product is a PLP-dependent racemase that preferentiallycatalyzes epimerization between L-Ile and D-allo-Ile, butalso catalyzes the racemization of nonpolar amino acids,including Leu and Val [1]. This enzyme was therefore
named isoleucine 2-epimerase (ILEP). ILEP is a novel
enzyme that catalyzes the racemization of BCAAs as its
main substrate and is thought to be responsible for theproduction of D-BCAAs in lactic acid bacteria. L.
buchneri ILEP has been classified as a fold-type I enzyme
on the basis of its amino-acid sequence [1]. Before thedetection of L. buchneri ILEP, α-amino-ε-caprolactam
racemase (ACLR) from Achromobactor obae was the
only known bacterial racemase that was classified into
fold type I; ILEP is the second. Crystal structures of A.
obae ACLR have previously been reported, but thereaction mechanism of this enzyme remains unclear. A
structural analysis of L. buchneri ILEP may shed light on
the substrate-recognition and reaction mechanisms offold-type I racemases.
In the present study, we solved structures of L.
buchneri ILEP bound by the reaction-intermediate
analogues N-(5’-phosphopyridoxyl)-L-isoleucine (PLP-LIle)andN-(5’-phosphopyridoxyl)-D-allo-isoleucine(PLP-D-allo-Ile), in addition to apo and PLP-boundstructures. We also evaluate the candidate acid/basecatalytic residues for L-Ile epimerization by means ofsite-directed mutagenesis [2].
2 Experiment
Diffraction data for crystals of PLP-bound, apo form,
PLP-L-Ile-bound, and PLP-D-allo-Ile-bound ILEP werecollected using an ADSC Quantum CCD detector systemon the BL-5A and AR-NE3A beamlines at the Photon
Factory, Tsukuba, Japan. The structure of the PLP-bound
ILEP was solved to a resolution of 1.94 Å by molecularPhoton Factory Activity Report 2017 #35 (2018)

replacement using the MOLREP program in the CCP4
program suite with the tetramatic structure of GABA-AT
from Sulfolobus tokodaii (PDB code: 2eo5) serving as thesearch model. The structures of apo form, PLP-L-Ilebound, and PLP-D-allo-Ile-bound ILEP were solved toresolutions of 2.77, 2.65 and 2.12 Å, respectively, bymolecular replacement using MOLREP. As a searchmodel, the structure of chain C from the PLP-bound ILEP
was used for the apo and PLP-L-Ile-bound ILEP and thestructure of chain A from the PLP-L-Ile-bound enzyme
was used for the PLP-D-allo-Ile-bound enzyme.
3 Results and Discussion
The L. buchneri ILEP assembled as a tetramer, whichis consistent with the subunit assembly of the enzyme as
estimated by gel filtration and SDS–PAGE (nativeenzyme 200 kDa, subunit 49 kDa). A very clear electron
density corresponding to the PLP molecule was observed
within the cofactor-binding site of PLP-bound enzyme,
which enabled us to place the ligand with reasonableaccuracy (Fig. 1A). The 5’-phosphate group of PLP
interacts with the side-chain O and backbone N atoms of
Ser116 and Thr309* (the asterisk indicates a residue inthe neighbouring subunit), and the backbone N atom of
Gly115. The phosphate group also interacts with thebackbone N atoms of Lys280 and Thr310* and the sidechains of Thr310* and Asn113* via water molecules (W1and W2). The pyridine ring of PLP is sandwiched
between Tyr142 and Val252. Via another water molecule(W3), the 3-hydroxyl group of PLP interacts with the sidechains of Glu217 and Asp222 and the backbone N atomof Asp222. The C4 atom of PLP connects covalently tothe ε-amino group of Lys280 to form an internal aldimine(Schiff-base) linkage. The pyridine N atom of PLP makes
salt bridge with the side chain of Asp250. This interaction
is conserved in all known fold-type I enzymes.
In the apo ILEP, amino-acid residues –11 to 28, 52 to
54 and 302* to 309* were disordered and were not visible
in the electron-density map. When we superimposed thestructure of apo ILEP on that of the PLP-bound enzyme,
the configuration of the loops around the active site
differed greatly between the two enzymes (Fig 1B). Loop
302*–309*, including Thr309*, which forms hydrogenbonds to the 5’-phosphate group of PLP in the PLP-boundstructure, is disordered in the apo structure. In addition,
loop 81*–87* in the apo structure moves towards theopposite side of the bound PLP molecule. These twoloops are derived from the neighbouring subunit, and theelements surrounding the two loops (amino-acid residues–11 to 28 and 52 to 54) are also disordered in the apostructure. It is noteworthy that all of the residues thought
to form hydrophobic interactions with the substrate L-Ile(Ile24, Ala54, Ala83*, Tyr84* and Leu307*; see below)
are included in the abovementioned loops and elements.
As a result, the active-site cavity in the apo structure ismuch more solvent-accessible than that in the PLP-boundstructure. These observations suggest that the binding of
PLP to the apo enzyme gives rise to a large structuralchange around the active site, which in turn provides asolvent-inaccessible environment for the enzyme reaction.

Fig. 1: (A) PLP bound to L. buchneri ILEP. The PLP
molecule is shown in yellow. The water molecules areshown as red spheres. Residues that interact with PLP are
shown in magenta, and those belonging to theneighbouring subunit are in light gray. (B) Close-up
views of the loops and elements surrounding the activesite. The apo and PLP-bound structure are shown in lightgray and magenta, respectively. PLP molecule, Lys280
and Thr309* in the PLP-bound structure is shown as astick model.
In the initial electron-density map of PLP-L-Ile andPLP-D-allo-Ile bound enzyme, extra density wasobserved within the active-site cavity, and afterrefinement of the peptide chain, PLP-L-Ile or PLP-Dallo-Ile could be modelled into this density (Figs. 2B andC). Comparison of the two structures revealed that the 5’phosphopyridoxyl moiety of PLP-D-allo-Ile occupies
almost the same position as that of PLP-L-Ile (Fig. 2A).
In addition, the carboxyl group of the substrate isoleucine
forms hydrogen bonds to the side chain of Arg408 in bothstructures. However, the positioning of the covalent bonds
connecting the Cα atom of isoleucine and the pyridine
ring of PLP is totally different between the two enzymes.
The Cβ atom of L-Ile in PLP-L-Ile is rotated in aclockwise direction around the Cα atom by about 49°
relative to that of the D-allo-Ile in PLP-D-allo-Ile.
Moreover, the Cδ atom in L-Ile is positioned on theopposite side to that in D-allo-Ile. In the PLP-L-Ile-boundenzyme, as a result, the side chain of L-Ile forms ahydrophobic core mainly with Ile24, Tyr142 and Leu307*,
while that of D-allo-Ile forms hydrophobic interactions
mainly with Ala54, Ala83* and Tyr84*.
Alanine racemase from Bacillus stearothermophilus, afold-type III PLP-dependent enzyme, utilizes a two-basemechanism in which Lys39 and Tyr265* act as generalacids/bases. In this mechanism, the Lys residue isresponsible for the deprotonation of the substrate Damino acid, which creates a quinonoid intermediate that is
reprotonated by the Tyr residue on the opposite face of
the intermediate to produce the L-amino acid. In thereverse reaction (L→D), proton abstraction is performed
by Tyr, while reprotonation is accomplished by Lys.
Although the overall structure of A. obae ACLR is quite
different from that of alanine racemase, the position of thePhoton Factory Activity Report 2017 #35 (2018)

side chain of Tyr137 in A. obae ACLR, which is situatedon the re-face of the PLP ring, symmetrically corresponds
to that of Tyr265* in alanine racemase, which is situatedon the si-face of the PLP ring. Lys39, which forms aninternal aldimine with PLP in the alanine racemase, isconserved as Lys267 in A. obae ACLR. Therefore, Tyr137
and Lys267 are thought to be candidate acid/basecatalytic residues. In addition, another possibility thatAsp210, the side chain of which is located near to the side
chain of Tyr137, acts as the acid/base catalytic residue
instead of Tyr137 has also been described. However, thedetails of the reaction mechanism of A. obae ACLR
remain unclear.
The L. buchneri ILEP monomer showed high structuralsimilarity to ACLR from A. obae (PDB codes: 3dxv and2zuk). Tyr137, Asp210 and Lys267 in A. obae ACLR are
strictly conserved in L. buchneri ILEP as Tyr142, Asp222
and Lys280, respectively. In the PLP-L-Ile-bound enzyme,
the side chain of Lys280 points towards the α-hydrogenof the substrate L-Ile. The interatomic distance from theCα atom of PLP-L-Ile to the side chain NZ atom ofLys280 is 3.6 Å, which is close enough to cause areaction, suggesting that Lys280 is a catalytic base thatabstracts the α-hydrogen of L-Ile (Fig. 2B). In the D-alloIle-bound enzyme, on the other hand, the residue whoseside chain is most proximate to the Cα atom of D-allo-Ileis Asp222: the interatomic distance from the Cα atom ofD-allo-Ile to the side chain OD2 atom of Asp222 is 3.2 Å(Fig. 2C). The distance between the Cα atom of D-allo-Ileand OH of Tyr142 (5.1 Å) is too long to form aninteraction. To assess the role of Tyr142 and Asp222, weconstructed Y142F, D222A, and D222N mutants andobserved that the Y142F mutant retained Vmax values of
85 and 132 μmol min–1 mg–1 for the epimerization of LIle and D-allo-Ile, respectively, which are about 46–56%
of the wild-type Vmax values. In contrast, the activities of
the D222A and D222N mutants were almost abolished(about 0.33–0.38% and 0.0047–0.0049%, respectively, ofthe wild-type Vmax values). This suggests that, in the caseof L-Ile epimerization, Lys280 is responsible forremoving the α-hydrogen from L-Ile, while Asp222, but
not Tyr142, serves as the catalytic residue adding αhydrogen to the quinonoid intermediate to form D-allo-Ile.
In the reverse reaction, proton abstraction andreprotonation would be performed by Asp and Lys,respectively. Although having a low reaction rate, theD222A and D222N mutants still retained catalytic activity.
Therefore, the hydroxyl anion in the solvent water may
function as the catalytic acid/base, as in the case ofalanine racemase from B. stearothermophilus. This is thefirst description of the structure of a BCAA racemase, andour results may provide critical information that will
facilitate a better understanding of the reaction
mechanism of fold-type I racemases.

Fig. 2: (A) Comparison of the structures around the PLPIle binding site. The structure with bound PLP-L-Ile(gray) is superimposed on that with bound PLP-D-allo-Ile(magenta). The hydrogen bonds are shown as dotted lines.
(B) PLP-L-Ile binding site. (C) PLP-D-allo-Ile bindingsite. The final σA-weighted Fo – Fc OMIT electrondensity maps for PLP-L-Ile and PLP-D-allo-Ile are shownat the 2.5σ level, respectively. The hydrogen bonds areshown as dotted lines.
Acknowledgement
We are grateful to the staff of the Photon Factory for
their assistance with data collection
References[1] Y. Mutaguchi et al., J. Bacteriol. (2013). 195, 5207–5215.
[2] J. Hayashi et al., Acta Crystallogr. D73. (2017). 428–437.
* sakuraba@ag.kagawa-u.ac.jp