Product Release Mechanism Associated with Structural Changes in

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Product release mechanism associated with structural changes in monomeric L-threonine 3-dehydrogenase Tomoharu Motoyama, Shogo Nakano, Yuta Yamamoto, Hiroaki Tokiwa, Yasuhisa Asano, and Sohei Ito Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00832 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 11, 2017

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Biochemistry

Product release mechanism associated with structural changes in monomeric L-threonine 3-dehydrogenase

Tomoharu Motoyama‡, ¶, 1, Shogo Nakano‡, ¶, 1*, Yuta Yamamoto#, Hiroaki Tokiwa#, †, Yasuhisa Asano§, ¶, and Sohei Ito‡, ¶

‡

Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan §

Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan

#

Department of Chemistry, Rikkyo University, Nishi-ikebukuro, Toshimaku, Tokyo 171-8501, Japan

†

Research Center of Smart Molecules, Rikkyo University, Nishi-ikebukuro, Toshimaku, Tokyo 171-8501, Japan

¶

Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu, Toyama 9390398, Japan

1

These authors contributed equally to this work.

*To whom correspondence may be addressed: School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan, Tel.: +81-54-264-5578 (Ext.); Fax: +81-54-264-5578, E-mail: [email protected]

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Abstract A short-chain dehydrogenase like L-threonine 3-dehydrogenase (SDR-TDH) from metagenome data (mtTDH) was identified by database mining. Its enzymatic properties suggested that mtTDH has unique characteristics relative to other SDR-TDHs, including two mesophilic and thermophilic SDR-TDHs identified in this study. The activation energy of mtTDH was the lowest (29.6 kJ/mol) among SDR-TDHs, indicating that it is a psychrophilic enzyme. Sizeexclusion chromatography analysis revealed mtTDH is monomer. Crystal structures of mtTDH in apo, binary, and two ternary complexes (L-Ser and L-Thr soaked forms) were determined at resolutions of 1.25–1.9 Å. Structural and computational analysis revealed the molecular mechanism of switching between the open/closed state induced by substrate binding and product release. Furthermore, six residues and two water molecules at the active site contributing to product release were assigned. The residues could be categorized into two groups based on the enzymatic properties of their variants: S111, Y136, and T177; and S74, T178, and D179. The former group appeared to affect L-Thr dehydrogenation directly, because the kcat value of their variants was > 80-fold lower than that of mtTDH wild type. On the other hand, the latter group contributes to switching between the open and closed states, which is important for the high substrate specificity of SDR-TDH for L-Thr: the kcat and Km toward L-Thr value of variants in these residues could not be determined because the initial velocity was unsaturated at high concentrations of L-Thr. Based on these findings, we proposed a product release mechanism for SDR-TDH associated with specific structural changes.


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Biochemistry

Abbreviation:

TDH,

L-threonine

3-dehydrogenase;

SDR-TDH,

short

chain

dehydrogenase/reductases type TDH; MDR-TDH, medium chain dehydrogenase/reductases type TDH; FMO, fragment molecular orbital; mtTDH, SDR-TDH from the uncultured archaeon MedDCM-OCT-S05-C57; MD, molecular dynamics; AKB, 2-amino 3-ketobutyrate; QM/MM, quantum mechanics/molecular mechanics; WT, wild type

Keywords: L-threonine 3-dehydrogenase; product release mechanism; crystal structures; enzyme kinetics; metagenome library; psychrophilic and monomer enzyme


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Introduction All species metabolize 20 L-amino acids using various enzymes. In general, L-threonine (LThr) is initially catabolized by L-threonine 3-dehydrogenase (TDH, EC 1.1.1.103), an NAD+dependent enzyme that catalyzes oxidation of L-Thr to 2-amino 3-ketobutyrate (AKB). AKB, which is unstable, is promptly converted to aminoacetone + CO2 by a non-enzymatic reaction or glycine + acetyl-CoA by 2-amino 3-ketobutyrate CoA ligase (4). There are two distinct types of TDHs:

medium

chain

dehydrogenase/reductases

(MDR-TDHs)

and

short

chain

dehydrogenase/reductases (SDR-TDHs). Many MDR-TDHs have been characterized in bacteria (5-7), and their 3D structures have been determined (8, 9). MDR-TDHs are metal-dependent enzymes (11) with broad substrate specificity, and can recognize not only L-Thr but also L-Ser (12) and certain alcohols (6). Although the names of SDR-TDHs and MDR-TDHs are similar, these enzymes share no sequence and structural similarity. In this study, we focused on SDRTDHs. The first SDR-TDH was described by Kazuoka et al., who reported that this enzyme requires no metal ion and exhibits high specificity for L-Thr (13). SDR-TDHs are also expressed in various species, and their physiological functions have been characterized by several groups. For example, Millerioux et al. reported that Trypanosoma brucei metabolize L-Thr using SDR-TDHs in order to use L-Thr as a carbon source for biosynthesis of lipids and sterols (14). Wang et al. reported that SDR-TDHs play important roles in growth of mouse embryonic stem cells (15). Regarding the applications of the enzymes, Ueatrongchit et al. proposed that SDR-TDHs could be used to quantify L-Thr concentration in human serum and plasma (16). As shown previously, there is no doubt that SDR-TDHs have biological significance and potential applications. Many groups have tried to elucidate the enzymatic functions of SDR-TDHs at the molecular level. To date, crystal structures have been determined for five SDR-TDHs from 4 ACS Paragon Plus Environment

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distinct species, and the structural information is available from the PDB database. Yoneda et al. determined the first crystal structure of an SDR-TDH using the enzyme from Flavobacterium frigidimaris KUC-1 (FfTDH) (17). Subsequently, by structural and biochemical analysis of SDRTDH from Thermoplasma volcanium, the same authors also succeeded in predicting the molecular mechanism underlying proton and hydride transfer by SDR-TDHs during dehydrogenation of L-Thr (1). Furthermore, using SDR-TDH from Cupriavidus necator (CnTDH), based a combined approach consisting of structural, computational, and biochemical analyses, Nakano et al. suggested that stepwise structural changes induced by binding NAD+ and L-Thr are important for the high substrate specificity of SDR-TDH (18). Structural changes were

also observed in SDR-TDH from mouse (mTDH), the first enzyme of this superfamily to be structurally characterized in a mammal (19). Structures of SDR-TDH from Trypanosoma brucei are available from PDB (5L9A and 5LC1). Despite progress in the structural and functional analysis of SDR-TDHs, the detailed reaction mechanisms, such as the role of water molecules in catalysis and product release, remain unknown. In this study, using SDR-TDH from a metagenome library (mtTDH, PMID: ADD93128.1) identified by database mining methods based on INTMSAlign (20), we attempted to answer these questions. The mtTDH is one of the sequences in the metagenome of mature mediterranean deep chlorophyll maximum (DCM) which was pyrosequenced (21): here, DCM is a phenomenon that the concentration of chlorophyll becomes maximal at certain depth in the ocean or a lake (22). First, we investigated the enzymatic functions of mtTDH, including its enzyme properties and oligomeric state under solution condition, by comparing two different SDR-TDHs from Cyclobacterium marinum DSM 745 (CmaTDH, GI:343085198) and Thermoanaerovibrio acidaminovorans DSM 6589 (TaTDH, GI: 269793245), also newly obtained using the database mining method. Both of these proteins were originally annotated as “NAD-dependent 5 ACS Paragon Plus Environment

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epimerase/dehydratase family” in their locus tags, making it difficult to predict whether the enzymes are SDR-TDHs. We selected CmaTDH and TaTDH are selected for comparison of enzymatic properties with mtTDH because the enzymes share moderate sequence identity (40 60%) and are expected to have distinct properties relative to each other. For example, CmaTDH and TaTDH are expressed in mesophilic and thermophilic bacteria: the optimal cultivation temperature of C. marinum DSM745 and T. acidaminovorans DSM6589 are 22 °C and 55 °C, respectively (DSMZ web site [https://www.dsmz.de/]), implying that the enzymes themselves may have the corresponding temperature–activity profiles. In addition, we proposed a product release mechanism for SDR-TDH based on combined analysis of high–resolution structures (1.25–1.9 Å resolution), interaction energies calculated by the fragment molecular orbital (FMO) method, and biochemical analysis of mtTDH variants.


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EXPERIMENTAL PROCEDURES Site-directed Mutagenesis of mtTDH variants Plasmids cloned mtTDH into pET15b were utilized as templates. Site-directed mutagenesis was performed using the QuikChange Lightning Multi-site mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). Primers used to design the variants are indicated in Table S1. Confirmation of mtTDH variants was performed by DNA sequencing.

Overexpression and Purification of mtTDH(WT), mtTDH variants, TaTDH, and CmaTDH A plasmid encoding mtTDH(WT) was transformed into Escherichia coli strain BL21 (DE3). Overexpression of mtTDH(WT) was achieved as previously described (18). The overexpressing cells were suspended in buffer A (10 mM potassium phosphate (pH 7.0), 50 mM NaCl), and sonicated. The insoluble fraction was removed by centrifugation (11,000 g, 30 min), and the resultant supernatant was applied to a Ni2+-Sepharose column. The column was washed with 50 mL of buffer A, and then with 30 mL of buffer A containing 50 mM imidazole. The samples were eluted and collected in buffer A containing 100 mM imidazole. Eluted samples were concentrated and applied to a gel filtration column (Superdex 200 pg) equilibrated with buffer A. Fractions containing the samples were collected and concentrated for crystallization. Purity was confirmed by SDS-PAGE. The same procedure was applied to purification of two other mtTDH variants, TaTDH and CmaTDH.

Analysis of oligomeric state of mtTDH, CmaTDH and TvoTDH by size-exclusion chromatography (SEC) Purified samples were applied to a gel filtration column equilibrated with buffer B (30 mM potassium phosphate [pH 7.0], 150 mM NaCl). Elution was monitored by plotting UV absorbance 7 ACS Paragon Plus Environment

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at 280 nm. Molecular weights of the samples in solution, reflecting the oligomeric state, was estimated by checking elution volume at the peak of purpose fraction with calibration curve. The calibration curve for determining molecular weight was depicted by plotting the elution volume at the peaks corresponding to cytochrome c (12.5 kDa), chymotrypsin (25 kDa), ovalbumin (45 kDa), albumin (68 kDa), and aldolase (158 kDa) (Fig. 2b).

Crystallization of apo, binary, and tertiary forms of mtTDH Screening for crystallization of mtTDH was carried out using the PEG/ION screen kit (Hampton Research, Aliso Viejo, CA, USA) at 4°C using the sitting-drop vapor diffusion method. The mtTDH sample was concentrated to 22 mg/mL using an Amicon Ultra-15 Centrifugal filter (Millipore). A 2.0 µL sample was mixed with 1.0 µL of each reservoir solution from the screening kit. mtTDH (apo form) crystals appeared in PEG/ION screen condition 22 (20% [w/v] PEG3350 and 0.2 M potassium formate). mtTDH (binary form) crystals appeared in PEG/ION screen condition 1 (20% [w/v] PEG3350 and 0.2 M sodium fluoride) containing 2.5 mM NAD+. Ternary complex crystals of mtTDH, i.e., the L-Ser–soaked and L-Thr–soaked forms, were obtained by soaking mtTDH (binary form) crystals quickly in cryo-reservoirs containing 100 mM L-Ser and 100 mM L-Thr, respectively. All crystals were quickly soaked in cryo-reservoirs prior to X-ray data collection. The cryo-reservoir used in crystallization of mtTDH was prepared by addition of 20% (w/v) trehalose.

X-ray data collection and MD simulation The soaked crystals were mounted and flash-cooled under a nitrogen stream (-173 °C). Diffraction data were collected using PILATUS 2M-F at BL1A and the Quantum 270 detector at NE3A of the Photon Factory (Tsukuba, Japan). The collected data were integrated and scaled 8 ACS Paragon Plus Environment

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using HKL2000 and SCALEPACK (23). Initial phases were determined by the molecular replacement method using MOLREP (24), using the crystal structure of CnTDH(holo) as a template (PDB ID: 3WMX). Model building and structure refinement, including unrestrained structural refinement, were performed using COOT (25) and REFMAC (26), respectively. All figures were prepared using PyMOL (27). Crystallographic parameters are listed in Table 2. Molecular dynamics (MD) simulation was performed utilizing the crystal structure of mtTDH L-Ser soaked form. Preparation of parameter files, such as protonation of proteins and solvation of water molecule (TIP3P) into a cuboid box (box size, 90 × 80 × 90 Å), were performed as described previously (18). The simulation was performed using the NAMD 2.9 software (28) with periodic boundary conditions. Electrostatic interactions were estimated by the particle mesh Ewald method (29), and the RATTLE algorithm (30) was adopted by constraining all hydrogen bonds. The simulation steps were 2 fsec, and an isothermal–isobaric (NPT) ensemble was applied to the system. The temperature was set to 300 K with a Langevin thermostat, and pressure was set to 1.0 bar. Collection of trajectories from the simulation was started after completion of energy minimization and simulated annealing, as indicated in the reference. A total of 50 nsec of productive MD simulation was performed. Analysis of the trajectories was performed using the Wordom software (31).

Fragment molecular orbital calculation First-principles calculation based on the fragment molecular orbital method (FMO) calculations was applied to the L-Thr–soaked structure of mtTDH, in which AKB and NADH are bound to the active site. The structure was protonated by Protonate3D implemented in MOE (32). Energy minimization was performed for hydrogen atoms of the mtTDH structure utilizing the Amber10EHT force field. The mtTDH structure was divided into one-residue fragments; the cut9 ACS Paragon Plus Environment

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off points were at the Cα atom of each residue, as described in a previous study (33). Fragmentation of NAD molecules was performed as shown in Fig. S3. The FMO calculation was executed by PAICS (34) at the resolution of the identity approximation of the second-order Møller−Plesset perturbation theory (RI-MP2), with the double zeta correlation-consistent polarized valence sets (cc-pVDZ) level. HF, vDW, and MP2 indicate Hartree-Fock, van der Waals, and the second-order Moellar-Presset perturbation theory, respectively. Interaction energy between AKB and amino acid residues of mtTDH were quantitatively evaluated using IFIEs calculated using FMO method (34). IFIEs between AKB and amino acid residues in mtTDH were colored utilizing previously described tools (35).

Assay of Enzyme activity toward L-Thr and NAD+ Enzyme activities of mtTDH(WT), TaTDH, and CmaTDH were measured by quantitating the amount of NADH produced by the TDH reaction at various L-Thr concentrations and temperatures (5–50 °C). Enzyme samples were kept in icy water until just before measurements of enzyme kinetics. Enzyme activity toward L-Thr was measured using assay buffer A (0.10 M Gly-KOH (pH 10.0) 2.5 mM NAD+, and 5–300 mM L-Thr). To start the reaction, 195 µL of assay buffer was added to a cuvette, and then 5 µL of enzyme solution was added. The cuvette was placed in a UV-Vis spectrometer (DU800, Beckman), and the time-dependent spectrum change of NADH at 340 nm was measured for 3 min. NADH concentration was calculated using the molar extinction coefficient (6300 M-1 cm-1) of NADH at 340 nm. Initial velocity at different concentrations of L-Thr was determined and plotted using the ORIGIN software. The enzyme kinetics parameters kcat and Km toward L-Thr (Km,L-Thr) were determined using the Michaelis– Menten equation and applying the nonlinear least-squares method. For mtTDH variants, the Michaelis constant toward NAD+ (Km,NAD+) was determined utilizing 10 ACS Paragon Plus Environment

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assay buffer B (0.10 M Gly-KOH [pH 10.0], 0.010–1.0 mM NAD+, and 50 mM L-Thr). The procedure for measuring enzyme activity and calculating enzyme kinetics parameters was identical to the method used to measure activity toward L-Thr.


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RESULTS & DISCUSSION Characterization and enzymatic functional analysis of mtTDH, CmaTDH, and TaTDH mtTDH was originally registered as a member

of

the

epimerase/dehydratase

NAD-dependent family from

the *

uncultured archaeon MedDCM-OCT-S05C57 (GenBank ID: ADD93128.1, 308 amino acid residues). The mtTDH gene was

*

*

identified from the metagenome of the Mediterranean deep chlorophyll maximum (21).

Like

exhibits

other

TDH

SDR-TDHs,

activity

and

***

mtTDH

has

high

specificity for L-Thr (13, 16). Amino-acid sequence alignment of mtTDH with five bacterial SDR-TDHs is shown in Fig. 1. The alignment

reveals that mtTDH shares

moderate sequence identity with the other enzymes: 49% (FfTDH), 46% (CnTDH), 45% (CmaTDH), 43% (TaTDH), and 42% (TvoTDH) (Fig. 1). Based on the sequence length of mtTDH, the protein contains no characteristic insertion/deletion of sequence in comparison with other bacterial SDRTDHs (Fig. 1).

FIGURE 1, Multiple sequence alignment of bacterial SDR-TDHs from various species. Abbreviations are as follows: mtTDH, SDR-TDH from uncultured archaeon MedDCM-OCT-S05-C57 (GI: ADD93128.1); CmaTDH, SDR-TDH from Cyclobacterium marinum DSM 745 (GI: WP_014020555.1); TaTDH, SDR-TDH from Thermoanaerovibrio acidaminovorans DSM 6589 (GI: YP_003318149.1); FfTDH, SDR-TDH from Flavobacterium frigidimaris KUC-1 (GI: BAC05433.1); CnTDH, SDR-TDH from Cupriavidus necator (GI: BAJ61594.1); TvoTDH, SDR-TDH from Thermoplasma volcanium (WP_010916712.1). The helix-forming fourhelix bundle in dimeric SDR-TDHs (α4 and α5 region) are indicated by blue mesh. The residues represented in Fig. 3a are indicated by straight arrows. Residues interacting with the substrate, L-Thr, are indicated by asterisks (*). Alignment was performed using MAFFT (3), and the figure was prepared using ESPript (10).


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mtTDH has unique residues at the dimer interface recognized in other TDHs, including C84, H85, Y89, E93, T98, and T149, whose hydrophobicities and side chain-volumes are inconsistent with the corresponding residues of other SDR-TDHs (blue meshed region, Fig. 1). This observation implies that the oligomeric state of mtTDH differs from that of other SDR-TDHs. As expected, mtTDH adopted a monomeric form in solution. To prove this, we examined the oligomeric states of three different SDR-TDHs, mtTDH, CmaTDH, and TaTDH, by sizeexclusion chromatography (SEC) (Fig. 2a and b). The results demonstrated that TaTDH and CmaTDH existed in dimeric forms, like previously reported SDR-TDHs (black and gray lines in Fig. 2a and b), whereas mtTDH existed as a monomer (red line in Fig. 2a and b). Next, we characterized the enzymatic properties of mtTDH by comparing its kinetic parameters with those of TaTDH and CmaTDH. Here, the cloned SDR-TDHs are expected to bear different thermophilicity to each other. To confirm this, we measured the enzyme kinetics toward L-Thr for mtTDH (Fig. 2c), TaTDH (Fig. 2d), and CmaTDH (Fig. 2e) by plotting initial velocity as a function of L-Thr concentration and temperature from 5 to 50 °C. The parameters at each temperature were indicated in Table 1. At lower temperatures (< 20 °C), the kcat/Km value for mtTDH was higher than those of TaTDH and CmaTDH (Table 1). By contrast, at 50 °C the enzymatic activity of mtTDH was completely lost, whereas TaTDH and CmaTDH exhibited the highest kcat/Km values in this temperature range (Table 1). Activation energies (Ea) for the three SDR-TDHs were calculated from analysis of Arrhenius plots (Fig. 2f), revealing that mtTDH is a psychrophilic SDR-TDH. In fact, the Ea value of mtTDH was 29.6 kJ/mol, lower than those of TaTDH (48.1 kJ/mol, Table 1), CmaTDH (40.8 kJ/mol, Table 1), and SDR-TDH from Cytophaga sp. Strain KUC-1 (34.4 kJ/mol, Table 1), also reported to be psychrophilic SDR-TDH (13). 13 ACS Paragon Plus Environment

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Table 1 Enzymatic properties and activation energy of mtTDH, TaTDH, and CmaTDH at various temperaturesa mtTDH kcat

°C

Km

-1

s

mM

kcat/Km -1

s mM

-

kcat s

Km

-1

mM

1

5 10 20 30 40 50

18.4 ± 0.5 19.5 ± 0.4

22.9 ± 1.7 13.0 ± 1.0

CmaTDHb

TaTDH kcat/Km -1

s mM

-

kcat

Km

kcat/Km

-1

s

mM

s-1 mM-1

1

0.80

7.73 ± 1.5

42.4 ± 12

0.18

37.5 ± 7.0

90.3 ± 20

0.42

1.5

12.2 ± 2.3

36.1 ± 11

0.33

81.6 ± 4.5

153 ± 11

0.53

35.4 ± 2.0

20.7 ± 3.5

1.71

19.4 ± 1.6

14.9 ± 2.5

1.30

118 ± 7.2

119 ± 9.6

1.0

48.8 ± 3.7

25.3 ± 5.4

1.93

32.6 ± 2.7

12.6 ± 2.3

2.6

185 ± 5.7

142 ± 5.4

1.3

75.4 ± 6.4

46.9 ± 8.8

1.61

73.6 ± 5.5

12.9 ± 2.1

5.7

n.d.d

n.d.

2.2

n.d.

n.d.

161 ± 3.4

27.6 ± 1.0

5.8

n.d.

n.d.

3.8

n.d.

c

Activation Energy (kJ/mol) 29.6

48.1

40.8

a

The Km value toward L-Thr was determined in this table. The numbers of biological and technical replicates were two and six, respectively. b Enzyme kinetics parameters for CmaTDH were determined using Hill plots. The Hill coefficient ranged from 1.39 to 2.04. c The parameters could not be determined due to inactivation of the mtTDH sample. d For CmaTDH, the Km was too high to determine the parameters.

Thus, relative to other SDR-TDHs, mtTDH has unique features: namely, mtTDH is psychrophilic and monomeric in solution (Fig. 2a and b). This is the first case of a monomeric SDR without insertion/deletion of a sequence segment. Indeed, the majority of SDRs, including all of the structure-determined SDR-TDHs (1, 17-19) are dimers or tetramers (36). The porcine carbonyl reductase, a monomeric SDR, contains an insertion of 41 residues before the catalytic Tyr, and this sequence appears to prohibit dimer formation (37). One hypothesis that might explain why monomeric SDR-TDHs are comparatively rare is that, like as other monomeric enzymes, monomeric SDR-TDHs may evolve into dimeric forms to improve thermal stability (38-40).


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Biochemistry

a,

b,

c,

d,

e,

f,

FIGURE 2, Characterization of oligomeric state of mtTDH, TaTDH, and CmaTDH (a and b). Size-exclusion chromatography spectra of mtTDH (red, a), TaTDH (black, a), and CmaTDH (gray, a), plotted on a calibration curve (b). About 2.5 mg of each sample was applied to SEC. Temperature-dependent enzyme kinetics plots of mtTDH (c), TaTDH (d), and CmaTDH (e). The initial velocities at 5, 10, 20, 30, 40 and 50 °C are shown as squares, circles, upward-pointing triangles, downward-pointing triangles, and diamond, respectively. Kinetics parameters are shown in Table 1. Arrhenius plots for kcat values of mtTDH, CmaTDH, and TaTDH (f). From the plots, activation energies (Ea) were calculated as shown in Table 1. Data are means ± s.d. 15 ACS Paragon Plus Environment

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Overall structural analysis of mtTDH Structural analysis of mtTDH would help to elucidate its characteristic properties in comparison with those of the reported SDR-TDHs. Accordingly, in this study, we determined the high-resolution structures of four forms of mtTDH: apo (1.9 Å), binary (NAD+ binding form, 1.25 Å), L-Ser–soaked (1.9 Å), and L-Thr–soaked forms (1.35 Å), respectively. For the binary and L-Thr–soaked forms, we determined structures at near-atomic resolution, which should be helpful in elucidating the roles of water molecules in the reaction mechanism of SDRTDHs. The crystallographic parameters are presented in Table 2. Table 2 Statistics of X-ray diffraction data collection for mtTDHs mtTDH mtTDH (apo) (NAD+) Space group P21 C2 Unit cell parameters a (Å) 56.39 113.5 b (Å) 49.32 48.93 c (Å) 113.6 56.36 α (degree) 90.0 90.0 β (degree) 89.9 104.7 γ (degree) 90.0 90.0 X-ray source PF PF-AR BL-1A NE3A Wavelength (Å) 1.10 1.00 Resolution (Å) 113.6–1.90 54.9–1.25 (1.93–1.90) (1.28–1.25) No. of reflectionsa 236910 475655 No. of unique reflections 46791 75876 Completeness (%) 95.7 (96.5) 91.5 (99.5) I/sig(I) 16.2 (3.2) 28.9 (3.9) Rmergeb 0.106 (0.423) 0.073 (0.360) CC1/2 0.959 (0.857) 0.985 (0.986) 2 B of Wilson plot (Å) 8.6 7.7 Rc 0.190 0.157 d Rfree 0.233 0.181 RMSD of geometry Bond length (Å) 0.010 0.014 Bond angle (degree) 1.335 1.652 Geometry Ramachandran outlier (%) 0 0 Ramachandran favored (%) 100 100 Average B factor (Å)2 Protein atoms 18.1 13.1 e Ligand atoms n.d. 9.1

mtTDH (L-Ser–soaked) C2

mtTDH (L-Thr–soaked) C2

112.6 50.86 55.3 90.0 105.6 90.0 PF BL-1A 1.10 54.2–1.90 (1.95–1.90) 154598 23337 98.7 (97.1) 17.2 (3.1) 0.080 (0.445) 0.977 (0.911) 12.6 0.160 0.199

112.6 49.27 55.64 90.0 105.3 90.0 PF BL-1A 1.10 54.3–1.35 (1.38–1.35) 425984 64955 99.7 (99.5) 30.2 (4.1) 0.056 (0.345) 0.986 (0.947) 6.7 0.141 0.164

0.013 1.489

0.015 1.715

0 100

0 100

19.2 17.1

11.5 10.1


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Biochemistry

Solvent atoms 22.0 25.6 22.8 22.8 PDB code 5Y1D 5Y1F 5Y1E 5Y1G a Sigma cutoff was set to none (F > 0σF). b Rmerge = ΣhklΣj|Ihkl,j|/ ΣhklΣjIhkl,j, where Ihkl,j is the jth measurement of reflection [h,k,l], and is the mean value of the symmetry-related reflection intensities. Values in brackets are for the shell of the highest resolution. c R = Σ||Fo||Fc ||/ Σ|Fo |, where Fo and Fc are the observed and calculated structure factors used in the refinement, respectively. d Rfree is the R-factor calculated using 5% of the reflections chosen at random and omitted from the refinement. e n.d. means “not determined”.

The crystal structures of mtTDH also supported the idea that this enzyme is monomeric in solution. The number of molecules in the asymmetric unit (ASU) was one for three forms (binary, L-Ser and L-Thr–soaked forms), consistent with the results of SEC of mtTDH. There were two

molecules in the ASU of the apo form, but the contact area (379 Å2) was > 3-fold smaller than those of other bacterial SDR-TDHs (1166 Å2 on average). This observation implied that the contact observed in the apo form is derived from crystal packing. Structural comparison at dimer interface between mtTDH and other SDR-TDHs suggested that the paucity of hydrophobic interactions at the interface keeps mtTDH in the monomers. Currently, three crystal structures of bacterial SDR-TDHs (FfTDH (17), CnTDH (18), and TvoTDH (1)) are available. All of these structures share high structural similarity each other: the root-mean-square deviation value for Cα atoms are 0.656 (FfTDH [PDB ID: 2YY7] vs mtTDH(apo)), 0.726 (CnTDH [PDB ID: 3WMX] vs mtTDH(apo)), and 0.820 Å (TvoTDH [PDB ID: 3A4V] vs mtTDH(apo)). With the exception of mTDH, the structures are dimeric, and contain a four-helix bundle formed by an interaction between the α4 and α5 helices (blue meshed region, Fig. 1). The structures at the interface for bacterial SDR-TDHs are shown in Fig. 4a; aromatic (W92 and W154 in CnTDH, Y85 and Y93 in TvoTDH) and hydrophobic residues (L100 in CnTDH and L147 in TvoTDH) formed hydrophobic interactions, such as π-π stacking interaction, at the interface (Fig. 3a). Based on the results of the sequence alignment (Fig. 1), other bacterial SDR-TDHs appear to


Biochemistry

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form similar interactions; in fact, homologous residues were occupied by aromatic and hydrophobic residues (indicated by arrows in Fig. 1). However, in mtTDH these residues are occupied by hydrophilic (H85 and E93) and hydrophobic (L146) residues (Fig. 3a). Taken together, these observations suggest that mtTDH exists as a monomer because the comparative lack of hydrophobic interactions at the interface of the helix bundle in other bacterial SDR-TDHs inhibits dimer formation. Steric interaction may be another factor, because several amino-acid mutations were detected between mtTDH and other SDR-TDHs (region indicated by blue mesh in Fig. 1). The number of hydrogen bonds in enzymes would become a factor to determine their thermophilicity. However, for the case of psychlophilic (mtTDH), mesophilic (CnTDH) and thermophilic SDR-TDHs (TvoTDH) of which structures are available, the number of hydrogen bonds are almost identical: the numbers for mtTDH, CnTDH and TvoTDH in chain A were 241, 239 and 245, respectively, which calculated by VADAR software (41). For other parameters, such as parameters for accessible surface area and dihedral angles, we could not find clear differences; these inferred that other factors, like protein dynamics, affect to thermophilicity of SDR-TDHs. In a previous study, based on a computational analysis, we predicted that the enzymatic functions of SDR-TDH, such as its high specificity for L-Thr, is due to a dynamic structural change induced by binding NAD+ and L-Thr (18); however, this change could not be directly captured in crystal structures. In this study, by determining four mtTDH structures, we succeeded in capturing conformation change in the loop region containing amino acids 171–178; this loop corresponds to one of the two regions (e.g., the 180–186 loop region in CnTDH) that are important for the high specificity of SDR-TDH for L-Thr (18). The 171–178 region is clearly altered between the open (apo [green] and binary form [magenta], Fig. 3b) and closed state (L-Ser [orange] and L-Thr–soaked form [cyan], Fig. 3b), indicating that induced fit occurs when SDR18 ACS Paragon Plus Environment

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Biochemistry

TDH changes from the binary to ternary complex structure. Structures around the 171–178 loop are shown in Fig. 3c; conformational change at D179 is associated with the state change (Fig. 3c). Furthermore, D179 is highly conserved among SDR-TDHs (Fig. 1), suggesting that this residue may work as a switch to convert the states of SDR-TDHs. On the other hand, for the 72–80 region, which is corresponding to another region (77–87 in CnTDH) that affects the substrate specificity of SDR-TDH, no structural changes were observed in mtTDH. We speculated that the conformation change in the 72–80 region is suppressed by a, W154’ L147’ Y85

W92

H85

L100’ W92’

W154

Y93’ L146

L147 Y85’

E93

Y93

L100

CnTDH

mtTDH

TvoTDH c,

b,

Closed

178 171-178 loop

178 Flip 171

NAD

D179

Open

171

FIGURE 3, Structure comparison of the dimer interfaces of three bacterial SDR-TDHs (a). The structures of CnTDH, TvoTDH and mtTDH are colored cyan, yellow, and green, respectively. Here, W92, L100, and W154 in CnTDH are consistent with Y85, Y93, and L147 in TvoTDH and H85, E93, and L146 in mtTDH, respectively. Bacterial SDR-TDHs for which crystal structures have been determined appear to form π–π stacking interaction at the interface; however, there is no such interaction for mtTDH. Overall structure of apo (green), binary (magenta), L-Ser–soaked (orange), and L-Thr–soaked (cyan) forms of mtTDH (b). Structure comparison at and near the 171–178 loop region between the apo and L-Ser–soaked forms (c). Apo and binary forms are in the “open” state (green), whereas the L-Ser–soaked and L-Thr–soaked form are in the “closed” state (orange). 19 ACS Paragon Plus Environment

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crystal packing effects. In fact, the 72–80 region forms interaction with other mtTDH molecules in the crystal: N72 and Y263 form hydrogen bonds with N38’ and H40’, respectively (Fig. S1). Furthermore, the results of MD simulation suggested that the 72–80 region is flexible (Fig. S2a and b, red colored region, orange colored in Fig. S2c and d): the RMSF values at the region (average value was 5.47 Å) is larger than other regions (average value was 2.42 Å) and the open state formed in the MD simulation, as confirmed in other SDR-TDHs (18, 19).

Active-site structures of L-Ser– and L-Thr–soaked forms of mtTDH Ternary complex structures of mtTDH could be obtained by soaking crystals of mtTDH binary form in reservoirs containing L-Ser or L-Thr. In the structure of the L-Ser–soaked form, there is electron density at the active site corresponding to L-Ser (Fig. 4a). The interaction model (Fig. 4b) indicated that the residues interacting with L-Ser are almost identical with those in the L-Thr– bound form of TvoTDH (1). The side chains of S74 and T178 and the main chains of T177 and T178 form hydrogen bonds with the amide group of L-Ser, and the side chain of S111 and Y136 interact with the hydroxyl group of L-Ser (Fig. 4b), suggesting that proton transfer during the reaction occurs through Y136, as suggested by Yoneda et al. (1). We discovered that D179 interacts with L-Ser via a water molecule (WatA, Fig. 4b); the interaction could only be observed in the closed state, suggesting that the side chain of D179 contributes to the reaction mechanism of SDR-TDHs. The structure of the L-Thr–soaked form contained electron density similar to that of the soaked compound (Fig. 4c). The interaction mode between mtTDH and the amide and carboxyl groups of the soaked compound are identical to that of the L-Thr–bound form of TvoTDH; however, the interaction mode between mtTDH and the side-chain atoms of the compound are different (Fig. 4d). First, the hydroxyl group of the compound does not interact with the side chains of S111 and 20 ACS Paragon Plus Environment

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Biochemistry

Y136 (Fig. 4d); instead, WatB formed the interaction (Fig. 4c). Secondary, the Cα–Cβ bond of the compound rotated about 60°, and the C4 atom shown in Fig. 4D was oriented toward a hydrophobic pocket formed by P164, G165, and W271 (Fig. 4D). a,

b, NAD S111

T178 L-Ser WatA

Y136 T177

S74

c,

d, NADH S111

T178

AKB

WatA

WatB

Y136

T177

S74

FIGURE 4, Active-site structures of L-Ser–soaked (a) and L-Thr–soaked forms (c) of mtTDH. The 2Fo-Fc electron density map (blue color) is contoured at 1.0 σ. We assigned the electron density observed in the L-Thr–soaked form (c) as AKB. 2D schematic interaction model between mtTDH and L-Ser (b) or AKB (d). C, N, and O atoms are colored black, blue, and red, respectively. Hydrogen bonds, including bond length, are shown as dashed lines (green). Hydrophobic residues located near ligands are represented as radiating spokes. The figures were prepared using LigPlot+ (2).

We assigned this electron density as AKB, a reaction product of mtTDH, for the following reasons. First, L-Thr should be dehydrogenated immediately by binding to the active site of mtTDH, as indicated by structural comparison between the L-Ser– and L-Thr–soaked form. LThr should interact with mtTDH as well as L-Ser shown in Fig 4a because their chemical structures are almost identical to each other. However, their interaction modes are different, 21 ACS Paragon Plus Environment

Biochemistry

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suggesting that L-Thr is promptly dehydrogenated and that the product, AKB, is trapped at the active site. Second, NAD+ appeared to be reduced, judging from the geometry of nicotine ring; in other words, dehydrogenation of L-Thr had already occurred. The geometry of the NAD+ binary form and L-Thr–soaked form was calculated by applying unrestrained structural refinement; application of the refinement is necessary not to reflect library information of NAD+ implemented in REFMAC (42). The refinement could be applied to these two structures because they were determined at high resolution (< 1.35Å, Table 2). The C4-C5 bond distance, αC bond angle, and αN bond angle were increased by 0.05 Å, 2.0 and 3.7°, respectively in the L-Thr–soaked form in comparison with the binary form (Table 3), suggesting that the nicotine ring is puckering and the NAD+ in L-Thr–soaked form is reduced. Here, the geometry change was moderate relative to those in structures of other proteins that bind NADH (42); the change may be repressed by the interaction between NAD+ and other surrounding residues, including water molecules, in mtTDH.

Table 3 Bond distances (Å) and distortion angle (°) of nicotinamide rings calculated by unrestrained structural refinement Structure N1–C2 (Å) C2–C3 C3–C4 C4–C5 C5–C6 C6–N1 αCa (°) αNa NAD+ 1.41 1.34 1.43 1.36 1.38 1.36 0.7 2.0 NADH-AKB 1.38 1.37 1.45 1.41 1.39 1.37 2.7 5.7 a Distortion angles (αC and αN) were calculated as defined in reference (43).

Taken together, these findings indicate that the structure of L-Thr–soaked form could be regarded as the AKB–NADH-bound form. Several factors could explain why we obtained this form; for example, the crystallization condition may work to trap the form.

Analysis of interactions between AKB and amino–acid residues in mtTDH using the FMO method The crystal structure of the AKB–NADH-bound form revealed that six residues (S74, S111, Y136, T177, T178, and D179) and two waters (WatA and WatB), in other word eight groups (Fig.


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Biochemistry

5a), participate in product release. To

a,

consider the release mechanism from

NADH

S111

WatA

the viewpoint of interaction between

T178

WatB

mtTDH and AKB, we calculated the

Y136 AKB

IFIEs using the FMO method (44); D179

recently,

several

T177

protein–ligand

S74

interactions have been quantitatively evaluated using IFIEs (35, 45-48).

b,

Accurate atomic coordinates of proteins are required to correctly calculate IFIEs; and therefore, we calculated IFIEs utilizing the crystal structure of the L-Thr–soaked form of mtTDH, which was determined at near-atomic resolution (1.35 Å). IFIEs were calculated for the eight fragments around AKB (Fig. 5a) by applying the Hartree-Fock (HFIFIEs)

and

Møller−Plesset

Total IFIEs for MP2: -4.4 kcal/mol FIGURE 5, Representation of IFIEs for MP2 of AKB, depending on the magnitude of the energy value (A). Negative and positive IFIEs are colored red and blue, respectively. Residues strongly interacting with AKB are colored deep red, and residues forming repulsive interactions are colored deep blue. IFIEs of the interacting six residues and two water fragments (B). HF-IFIEs and dMP2-IFIEs are represented as blue and red bars, respectively. MP2-IFIEs are the sums of the values depicted by the blue and red bars.

perturbation theories (MP2-IFIEs), respectively: the HF-IFIEs and MP2-IFIEs were represented as electrostatic interactions and the sum of electrostatic and dispersion interactions, respectively. To estimate only dispersion interactions, we used dMP2-IFIEs calculated by subtracting HF-IFIEs from MP2-IFIEs. Plots of IFIEs are shown in Fig. 5b. Here, the fragments bearing negative and positive MP223 ACS Paragon Plus Environment

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IFIEs generate attractive and repulsive interactions with AKB, respectively. Five of the eight fragments (S74, T177, T178, Y136, and WatA) formed attractive interactions, and in particular S74, T177, and T178 interacted strongly with AKB (Fig. 5b): the MP2-IFIEs for S74, T177 and T178 were -43.9, -34.4, and -20.6 kcal/mol, respectively. The contribution of HF-IFIEs (blue bar in Fig. 5a) was larger than that of dMP2-IFIEs (red bar in Fig. 5a), suggesting that the three residues formed electrostatic interactions with AKB. The remaining three fragments (S111, D179, and WatB) generate repulsive interaction, and there is an especially strong repulsive interaction between AKB and D179 (Fig. 5b) even when the stabilization provided by the interaction between D179 and WatA (-16.7 kcal/mol) is taken into account: the MP2-IFIE for D179 was 45.7 kcal/mol. WatB also formed a repulsive interaction with AKB: the MP2-IFIE was 2.3 kcal/mol.

Thus, we can identify the fragments forming attractive interactions or generating repulsive interactions with AKB. In this study, the interaction of the L-Thr–bound form could not be estimated by the FMO method due to the lack of a high-resolution structure; however, we speculate that the interaction of L-Thr with mtTDH should be stronger than that of AKB. The reason for this is that, in addition to the interaction between amide and carboxyl groups of L-Thr and the three residues observed in the AKB–NADH-bound form (S74, T177, and T178, Fig. 5a and b), the hydroxyl group of L-Thr should form a hydrogen bond with S74 and Y136, as observed in a previous study (1).

Enzymatic properties of mtTDH variants The residues interacting with L-Thr in SDR-TDH were revealed by TvoTDH (1) and structural and FMO analyses of mtTDH. In order to determine the reaction mechanism of SDR-TDH, it is necessary to estimate how the residues affect enzymatic function. In this study, we estimated their 24 ACS Paragon Plus Environment

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Biochemistry

function of a total six of variants (S74A, S111A, Y136F, T177A, D179A, and D179N) by determining enzymatic properties (Table 4).

Table 4 Enzymatic properties of mtTDH wild type (WT) and variants Variantsa kcatb Km, L-Thrc Km, NAD+ c kcat/Km, L-Thr Functiong s -1 mM μM s -1 mM -1 WT 48.8 ± 3.7 25.3 ± 5.4 50.7 ± 3.6 1.9 --Y136F 0.60 ± 0.03 40.6 ± 5.7 428 ± 8.2 0.015 1 S111A 0.52 ± 0.04 246 ± 26 19.6 ± 4.2 0.002 1 T177A 0.07 ± 0.00 23.7 ± 2.2 25.7 ± 3.2 0.003 1 D179A n.d.d n.d. 24.3 ± 1.9 0.053 2 D179N n.d. n.d. 15.5 ± 2.0 0.010 2 S74A n.d. n.d. 14.0 ± 1.6 0.006 2 [Example of CnTDH]e WT 50.0 ± 3.7 15.2 ± 2.6 120 ± 20 3.3 --T186Nf n.d. n.d. 97.0 ± 4.0 0.01 2 a Locations of mutated sites are shown in Figure 4b and d. Recombinant enzymes were purified as described in “Experimental Procedures.” The enzymatic properties were determined at 30 °C condition. b kcat values of the variants and WT were derived from the data shown in Figure 5. c Km, L-Thr and Km, NAD+ values represent the Michaelis constant values of the enzyme toward L-Thr and NAD+, respectively. d n.d., not determined. e The enzyme properties for CnTDH were cited from the reference. f T186 in CnTDH is corresponding to T178 in mtTDH from their structural comparison. g The mutation affecting dehydrogenation of L-Thr directly is indicated by “1”, and the mutation affecting switching between the open and closed forms is indicated by “2”.

First, we will summarize the properties of SDR-TDH variants, based on previous studies. Variants of SDR-TDHs can be classified into two patterns. In one group, the kcat value is significantly decreased (> 50-fold lower than that of WT), but the Km, L-Thr value is hardly changed. This pattern was observed in variants in which the conformational change induced by binding of NAD+ and L-Thr to SDR-TDH is not affected by the mutation, whereas dehydrogenation is inhibited. A representative example is the Y137F variant of TvoTDH (1). In the second group, Km, L-Thr

value is too high to determine both kcat and Km, L-Thr values accurately. This implies that the

conformational change is inhibited by the mutation; in other words, SDR-TDH remained in the open state when L-Thr bound to the active site. L80G, G184A, and T186N in CnTDH (18) and


Biochemistry

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R180K in mTDH (19) are consistent with this pattern. Taking this into consideration, we tried to classify the residues interacting with the substrate (Fig. 4b and 4d) into two groups, based on the enzyme kinetics of the variants. The residues corresponding to the former pattern (pattern “1” in Table 4) in mtTDH are S111, Y136, and T177; the plot of enzyme kinetics indicated that the initial velocity was saturated at high concentration of L-Thr, but significantly lower than that of WT (Fig. 6a). The kcat values of Y136F and S111A, which directly interacted with hydroxyl groups of substrates (Fig. 4b and d), were > 100-fold lower than that of WT (Table 4), suggesting that not only Y136, which shown by Yoneda et al. to dehydrogenate at the OH group of L-Thr, but also S111 contributes to enzyme catalysis of SDR-TDH. For the S111 variant, the Km,L-Thr value was > 5-fold higher than that of WT (Table 4). S111 is not located on the loop that affected switching between the open and closed states, suggesting that the increase in the Km,L-Thr value of S111A variant may be due to the loss of the interaction with the OH group of L-Thr. In the three variants, T177A had the lowest kcat value despite the fact that the side chain of T177 does not interact with substrate (Fig. 4b and d); the kcat value of T177A is > 800-fold lower than that of WT, and > 8-fold lower than those of Y136F and S111A (Table 4). Thus, the side chain of T177 may be important to desolvate the active site and/or place substrate in the optimal position to promote the reaction. On the other hand, the three other residues (S74, T178, and D179) appeared to be classified into the second pattern (pattern “2” in Table 4); the plot of enzyme kinetics indicated that initial velocity did reach a maximum value at a high concentration (300 mM) of L-Thr (Fig. 6b), thus, only the kcat/Km,L-Thr values of the corresponding variants (S74, D179A, and D179N) could be determined (Table 4). Like T178 in mtTDH, which corresponds to T186 in CnTDH (Table 4), S74 also contributes to switching between open and closed states in SDR-TDHs. In fact, the kcat/Km,LThr

value of S74A is ~300-fold lower than that of WT mtTDH, and this reduction was similar in 26 ACS Paragon Plus Environment

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Biochemistry

magnitude to that of T186N in CnTDH. D179 is the newly identified residue in this study whose structure is correlatively changed between the binary and ternary complex forms (Fig. 3c). The kcat/Km,L-Thr values of D179A and D179N variants were ~35- and ~190-fold lower than that of WT (Table 4), suggesting that, as expected from the structural analysis (Fig. 3c), the side chain carboxyl group of D179 is important for switching between the open and closed states. Mutations in SDR-TDH often affect the Km value for NAD+. Hence, we determined Km,NAD+ for the six designed variants of mtTDH (Table 4). In the variants, only Y136F exhibited change of Km,NAD+ value: the value was about 9-fold higher than that of WT (Table 4). On the other hand, for the other five variants (S111A, T177A, D179A, D179N, and S74A), the Km,NAD+ value was hardly altered in comparison with the WT; in fact, their values were ~30 μM (Table 4). This observation suggested that the change in the enzymatic properties of these five variants is mainly due to differences in L-Thr binding and formation of ternary complex.

a,

b,

FIGURE 6, Enzyme kinetics of mtTDH(WT) and six variants toward L-Thr. Variants exhibiting the former (S111A, Y136F and T177A) and latter pattern (S74A, D179A, and D179N) of enzymatic properties were shown in Fig. 6a and b, respectively. The data are shown as means ± S.D.

Product release mechanism of SDR-TDH associated with structural change Based on these results, we propose a product release mechanism of SDR-TDH associated with the structural change described above. A schematic model of the mechanism is shown in Fig. 7. 27 ACS Paragon Plus Environment

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As demonstrated in a previous study, the binary form of SDR-TDH switches from the open to closed state by binding L-Thr (18). Structural and biochemical data for mtTDH suggested that four residues (S74, T178, D179, and the main chain of T177) contribute to this switching, and the enzyme kinetics of the corresponding variants also supported this prediction. FMO analysis revealed that the four residues appeared to play different roles in switching. As expected, S74, T178, and the main chain of T177 keep mtTDH in the closed state by forming attractive interactions with L-Thr. However, there is a repulsive interaction between D179 and the L-Thr, and this would be cancelled by formation of another attractive interaction, such as the interaction between the L-Thr and the other four residues (S74, S111, Y136 and T178). Thus, D179 might work as a sensor to judge whether L-Thr has been converted to AKB. The predicted mechanism can be described as follows: in the closed state, the dehydrogenation of L-Thr progresses as indicated by Yoneda et al. (1): the hydroxyl group is dehydrogenated (step a in Fig. 7), and the hydride at the Cβ atom of L-Thr is concomitantly transferred to NAD+ (step b in Fig. 7). Although the mechanism following hydride transfer remains unknown, we predicted a mechanism based on our structural analysis of mtTDH: The Cα–Cβ bond rotates about 60° (step c, Fig. 7), and S111 and Y136 form a hydrogen-bond interaction among, causing the oxygen atom on the product to be cleaved (step d, Fig. 7). A solvent water molecule (WatB) comes into the active site and forms an interaction with S111 and Y136 (step e, Fig. 7). A repulsive interaction is generated between the WatB and AKB; this step is supported by FMO analysis (step f, Fig. 7). Due to the deficiency in the hydrogen-bond network between the hydroxyl group of L-Thr and two residues (S111 and Y136) (step d, Fig. 7) and generation of a repulsive interaction (step f, Fig. 7), the repulsive interaction between D179 and AKB cannot be cancelled; this may be the signal that induces the conformation change of D179, as observed in open-state structure of mtTDH (step g, Fig. 7). Simultaneously, the structures are converted from closed to open state, 28 ACS Paragon Plus Environment

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and the hydrogen-bond interactions between AKB, S74, and T178 are cleaved (step h, Fig. 7). Finally, AKB is released into the solvent.


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Open L-Thr

AKB ・・・

How to release products?

NAD+ binding form [Open state]

NADH binding form [Open state]

L-Thr

AKB

b

c

h

NAD+

NADH f

h

a g

d

e AKB

L-Thr

NAD+-L-Thr binding form [Closed state]

NADH-AKB binding form [Closed state]

FIGURE 7, Proposed reaction mechanism for SDR-TDH associated with structural change. After binding of L-Thr to the active site, dehydrogenation of the hydroxyl group (a) and Cβ atom (b) occurs by the following scheme previously proposed by Yoneda et al. (1). Then, the Cα–Cβ bond rotates about 60 ° (c), a hydrogen bond forms between the oxygen atom of L-Thr and the hydroxyl group of S111, and Y136 is cleaved (double line, d). Solvent water molecule forms an interaction with S111 and Y136 (e). The water molecule and AKB generate a repulsive interaction due to close contact with each other (f). D179 may sense generation of AKB and flip into position, as observed in the open-state structure of mtTDH (g). Simultaneously, mtTDH adopts the open state, and hydrogen bonds between AKB and other residues are cleaved (double line, h). Finally, AKB is released into the solvent.


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In this study, we predicted the product-release mechanism of SDR-TDH based on highresolution structures, FMO analysis, and analysis of the enzymatic properties of mtTDH and its variants. These results also reinforced the validity of our proposed mechanism, in which structural changes of SDR-TDHs are induced by binding of NAD+ and substrates (18). However, we must acknowledge that there is a weakness in the predicted mechanism. Specifically, we cannot collect experimental evidence to prove that the electron density observed in L-Thr–soaked form is really AKB, e.g., by preparing AKB–soaked crystals of mtTDH. This is due to the fact that handling of AKB under experimental conditions (such as those for crystallization and enzyme kinetics analysis) is difficult because the compound is too unstable and rapidly catabolized to other compounds. In this situation, computational analysis was the only choice for confirming the validity of the mechanism at the molecular level. Indeed, the total IFIE energies between AKB and other residues in mtTDH supported the validity of this structure (total MP2-IFIEs were -4.4 kcal/mol, Fig. 5b). Currently, high-resolution structures of mtTDH are available, so we are now trying to prove the mechanism by a computational approach, e.g., QM/MM calculation.


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Acknowledgements: X-ray data were collected at the synchrotron facilities of the Photon Factory (PF) using beamlines BL-1A and NE3A (proposal No. 2016G102). This work was supported by JSPS KAKENHI Grant Number 16K18688, 17KT0010 and JST by ERATO grant number JPMJER1102.

Supporting Information. Results of MD simulation for mtTDH binary form, fragmentation of NADH.


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Biochemistry

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D179 senses product formation.

TOC Figure


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Product Release Mechanism Associated with Structural Changes in

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