Photon Factory Activity Report 2016 #34 (2017)

BL-5A, AR-NW12A/2015G001

Structure based engineering of an NADP+-dependent D-amino acid dehydrogenase

Haruhiko Sakuraba1, , Junji Hayashi1, Tomonari Seto1, Kazunari Yoneda2, and Toshihisa Ohshima3
123

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

1 Introduction
meso-Diaminopimelate dehydrogenase (DAPDH; EC1.4.1.16) catalyzes the reversible NADP+-dependentoxidative deamination of meso-diaminopimelate (mesoDAP) to produce L-2-amino-6-oxopimelate . The enzyme
acts stereoselectively on the D-center of meso-DAP,
indicating it recognizes the difference between D- and Lconfiguration carbons. It was therefore expected that thisenzyme would be useful for one-step production of Damino acids, which are often utilized as source materials
for industrial production of medicines, seasonings andagrochemicals. However, its high substrate specificity formeso-DAP and instability have proved to be the major
disadvantage for the practical application of DAPDH. We
recently identified and characterized a highly stable
DAPDH from the thermophilic bacterium Ureibacillus
thermosphaericus. By introducing five point mutations,
we succeeded in creating a stable NADP+-dependent Damino acid dehydrogenase (DAADH) from U.
thermosphaericus DAPDH. This DAADH does not act on
meso-DAP but catalyzes the reversible deamination of Damino acids such as D-cyclohexylalanine (relative activity,100%), D-isoleucine (73%), D-2-aminooctanoate (61%)
and D-lysine (53%). As the next step, creation of anenzyme exhibiting different substrate specificity andhigher catalytic efficiency is a key to the further
development of D-amino acid production. We havedetermined the structures of U. thermosphaericus
DAPDH in the apo form and in complex with NADP+ andN-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid[1]. However, the information needed for production ofnovel DAADH mutants with different substratespecificities remains limited.
In the present study, we determined the crystal
structure of apo-DAADH. We compared the active-sitearchitecture of the apo-DAADH (open-form) with that ofSymbiobacterium thermophilum DAPDH in complex withsubstrate (closed-form) and sought the amino acidresidues responsible for substrate binding. We used sitedirected mutagenesis to identify the residues responsiblefor determining the enzyme’s substrate specificity. As aresult, we found that a single mutation (D94A), which has
not been tested previously, caused a striking change in thesubstrate specificity of DAADH. We then succeeded indetermining the structures of a D94A/Y224F double
mutant in complex with NADP+ and in complex withboth NADPH and 2-keto-6-aminocapronic acid (KACA),
the keto acid analogue of lysine. Based on this structural
information, the factors responsible for the difference insubstrate specificity between U. thermosphaericus
DAADH and its mutant were evaluated [2].
2 Experiment
Diffraction data for crystals of apo-DAADH and theD94A/Y224F mutant enzyme were collected using anADSC Quantum CCD detector system on the BL-5a andAR-NW12 beamlines at the Photon Factory, Tsukuba,Japan. The structure of the apo-DAADH was solved to aresolution of 1.78 Å by molecular replacement using theMOLREP program in the CCP4 program suite; thestructure of chain A from U. thermosphaericus DAPDH(PDB code: 3wyb) served as the search model. The
structures of NADP+-bound and NADPH/KACA-bound
D94A/Y224F mutant enzymes were solved to resolutionsof 1.59 Å and 1.74 Å, respectively, by molecularreplacement using MOLREP with the apo-enzymestructure (chain A) as a search model.
3 Results and Discussion
The crystal structure of the S. thermophilum
DAPDH/NADPH/meso-DAP ternary complex has beenreported. Superposition of this structure (PDB code: 3wbf,subunit B) onto that of U. thermosphaericus apoDAADH (subunit B) enabled estimation of the aminoacid residues involved in substrate binding, as well as theinduced-fit movement of these residues. In S.
thermophilum DAPDH, the side chain of Tyr205 forms ahydrogen bond with the side chain of Asp92 and throughthis interaction Tyr205 is held at the active-site entrance,
where it acts as part of the substrate-binding site. Theseresidues were respectively conserved as Tyr224 andAsp94 in U. thermosphaericus DAADH. Thus,interactions among these residues and the substrate may
also occur upon substrate binding to DAADH, though theside chain of Tyr224 is situated about 13 Å farther awayfrom that of Asp94 in the apo-structure. Although both ofPhoton Factory Activity Report 2016 #34 (2017)

the residues are strictly conserved among all known
DAPDHs and DAADHs, and may play a key role insubstrate binding, there have been no reported mutationalanalyses of these residues. We therefore constructed
D94A and Y224F mutants and examined their substratespecificities.
When we compared the substrate specificities foroxidative deamination catalyzed by the parent DAADHand the D94A and Y224F mutants (substrateconcentration: 30 mM), we found that the substratespecificity of D94A markedly differed from that of theparent enzyme. The D94A mutant utilized Dphenylalanine as the most preferable substrate and had aspecific activity of 5.33 mol/min/mg at 50C, which is
54 times higher than the parent enzyme. Although therelative activities with D-leucine, D-norleucine, Dmethionine, D-isoleucine, D-tryptophan and D-histidine
were lower than the activity with D-phenylalanine, thespecific activities with all these substrates were 6-25times higher than was observed with the parent enzyme.
For the reductive amination, D94A also showedsignificantly
higherspecificactivity
towardphenylpyruvate (16.1 μmol/min/mg, 8.3 times), 2-oxooctanoate (65.5 μmol/min/mg, 5.0 times), 2-oxohexanoate (3.8 times), 2-oxo-4-methylthiobutanoate (3.9times), 2-oxo-4-methylpentanoate (3.3 times), 2-oxopentanoate (1.6 times) and 2-oxo-3-methylpentanoate (1.7times) than the parent enzyme [5 mM of 2-oxo-acidanalogues were used except for phenylpyruvate (2 mM)].
On the other hand, Y224F exhibited less activity than theparent enzyme with all substrates, especially for oxidative
deamination, though the substrate spectrum wascomparable. This suggests Tyr224 is essential for theproper catalytic activity, but is not very important for thesubstrate recognition.
Reportedly, the H227V mutant created from S.
thermophilum DAPDH has the specific activity of 2.4
mol/min/mg at 30C with phenylpyruvate in thereductive amination. The Clostridium tetani DAPDH
mutant also shows catalytic activity toward the synthesisof D-phenylalanine, but the specific activity with phenylpyruvate is only 0.11 mol/min/mg at 30C. The
extremely high activity for reductive amination ofphenylpyruvate (Vmax of 92.5 mol/min/mg at 50C) is
likely a major advantage of using the D94A mutant for
direct synthesis of D-phenylalanine.
To examine the factors responsible for the difference insubstrate recognition, we modeled the phenylpyruvate
molecule into the active site of NADPH/KACA-bound
D94A/Y224F based on the orientation of KACA and thenminimized the energy of the complex using CNS (Fig. 1).
Within this model, an interaction was formed between thecarboxyl group of the substrate and the nicotinamide
ribose phosphate, as well as between the C2 carbonylgroup of the substrate and the side chain of Lys150. On
the other hand, the phenyl group of the substrate wasobserved to rotate 53 around C3 in a clockwise direction
relative to the C3C4 bond of KACA. The phenyl group
was held at that position via stacking interactions with

Trp148 and the nicotinamide ring, in addition tohydrophobic interactions with the side chains of Lys150(C and C; 8 interactions), Trp123 (CD2, CE3, and CZ3;
5 interactions) and Ala94 (C, 2 interactions). Thisbinding mode of the phenylpyruvate would not beallowed in the parent DAADH, because the phenyl groupis supposed to make unusual short contacts with the side
chain of Asp94 (the Asp94 side chain is shown in yellow
in Fig. 1). The large substrate-binding pocket formed by
Trp148, Lys150, Trp123, and Ala94 may also be related
to a high activity of the D94A mutant towards 2oxooctanoate with bulky side chain. Taken together, ourobservations suggest that the D94A substitution enlarged
the substrate-binding pocket and enhanced thehydrophobicity of the pocket around the side chain of thesubstrate. Consequently, the D94A mutant gained strong
reactivity toward hydrophobic D-amino acids such as Dphenylalanine, D-leucine, D-norleucine, D-methionine andD-isoleucine.

Fig. 1: Proposed model for phenylpyruvate binding.
NADPH/KACA-bound D94A/Y224F and phenylpyruvate-bound model are shown in white and cyan,respectively. Phenylpyruvate and NADPH molecules in
the phenylpyruvate-bound model are in green andmagenta, respectively. The hydrogen bonds aroundphenylpyruvate are shown as dotted lines.
Through the use of U. thermosphaericus DAADH, wehave already succeeded in developing novel methods forproducing D-branched-chain amino acids, for subsequent
generation of compounds labeled with stable isotopes,
and for assaying D-isoleucine without interference fromthe three other isomers. As the next step, creation of an
enzyme exhibiting different specificity and higher
catalytic efficiency became a key for the further
development of D-amino acid production by the method
at the industrial level. In this study, we succeeded in thecreation of a novel mutant with an extremely highcatalytic activity for phenylpyruvate amination. Moreover,the much higher catalytic activity of the mutant observed

Photon Factory Activity Report 2016 #34 (2017)with several 2-oxo acid analogues of D-amino acids
would be useful for further development of practicalapplications.
Acknowledgement
We are grateful to the staff of the Photon Factory for
their assistance with data collection
References[1] H. Akita et al., Acta Crystallogr. D71, 1136 (2015).
[2] J. Hayashi et al., Appl. Env. Microbiol. in press (2017).
doi: 10.1128/AEM.00491-17* sakuraba@ag.kagawa-u.ac.jp