2023/2/22
CCS-KISTI WORKSHOP

Unique reaction mechanism of copper amine oxidase revealed bytheoretical QM/MM and experimental approaches

Mitsuo Shoji1,2
1Biological Function and Information Group, Division of Life Science, CCS, U.Tsukuba、
2JST-PRESTO

mshoji@ccs.tsukuba.ac.jp
1

Acknowledgement
Kenji Mishima (U.Tsukuba)
Yasuteru Shigeta (U.Tsukuba)
Shigenori Nagatomo (U.Tsukuba)
Mauro Boero (Strasbourg U.)

Hideyuki Hayashi (OMPU)
Toshihide Okajima (Osaka U.)
Takashi Ueno (Tokyo Tech)
Eriko Nango (Tohoku U.)

Funds
KAKENHI:
20H05453(representative：Osamu Miyashiya)
16KT0055(representative：Hidetoshi Okajima)
JST-PRESTO:JPMJPR19G6
MEXT Q-LEAP: JPMXS0120330644
20H05088(I4LEC)
Computational resources
MCRP in CCS, Univ. Tsukuba
HPCI system research project (project ID: hp210115)
2

Background１

Copper amine oxidases (CAO)• CAOs catalyze the oxidative deamination of primary amines into their aldehydes.
RCH2NH3 + O2 + H2O –> RCHO + H2O2 + NH3• CAOs exert fundamental functions in aerobic organisms from bacteria to yeast, plant andmammals.
• CAOs contain Cu ion and topaquinone (TPQ) cofactor

Oxidative form of TPQ(TPQox)

Catalytic cycle of CAO

Background２• Catalytic cycle of CAO is composed of reductive half-reaction and oxidative half-reaction
3WA22CWU

2CWV

1.85Å

3X3X

3X3Z

PDBIDsFeatures
••
•
•
•

X-ray structures of all the intermediate states are determined.
Positions of proton in TPQox were determined.
Spectroscopy can trace the intermediate states.
Isotope effect of proton transfer (H/D) is observed. (Target for quantum biology)
Large conformational change during the TPQamr -> TPQsq transition (Target for SFX study)Large conformational change of TPQ
Off-Cu(TPQamr)→On-Cu (TPQsq)

Asn381

TPQsq

TPQamrCu2+

Asp298

His592

His433Tyr284

TPQamr: 3X3Z
TPQsq: 3X3X

His431• Asn381 is a conserved residue• Reaction mechanism is unsolved（impossible ?）5

Quite different from a classical picture of enzyme reaction
Structural changes are quite small → substrate specificity： classical enzyme• Key and keyhole model: E.Fischer(1890) substrate-binding site is rigid• Induced fit model: Koshland(1995) substrate-binding site is more flexible（active site is rigid）
enzyme酵素

1. 基質結合substrate binding

補酵素
cofactor酵素

補酵素

２. 触媒反応

catalytic reaction

酵素

補酵素

CAO
enzyme酵素

cofactor

結合残基
触媒残基

• Really happen？
• Why and is meaningful？
6

Purpose of this study• Elucidate the most difficult reaction step in TPQamr → TPQsq• Solve the role of Asn381
Theoretical approach：
Reaction mechanism of WT by using QM/MM*
Reaction mechanism of N381A mutant
Structural change of N381A mutant in TPQox
Experimental approach：
X-ray crystal structures of N381A and kinetic spectroscopy*QM/MM: hybrid quantum mechanics and molecular mechanics(This study took 2-3 years to find appropriate reaction pathways,
because it is a significantly difficult problem)
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Off-Cu → On-Cu transition
25-H+

5244

HOff-Cu (TPQamr)

On-Cu (TPQsq)• steps of the TPQ movement

Slide

Turnover8

2 x 2 = 4 candidate pathways
1. Turnover + Slide

A. clockwise-turnover

444Slide

Turnover
Off-Cu (TPQamr)

4On-Cu (TPQsq)

2. Slide + TurnoverB. counterclockwise-turnover

444SlideOff-Cu (TPQamr)

Turnover
On-Cu (TPQsq)

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Rotation of TPQ is quit difficult for steric repulsion fromsurrounding residues

1. Turnover + Slide
TPQ382

Tyr384
Asp298Asn381

Val282

1A. clockwise turnover: ⊢Asn381, Tyr384
1B. counterclockwise turnover: ⊢ main
chain of TPQ382, Val282

2. Slide + TurnoverAsn381

Phe407

Tyr384
His4332A. clockwise turnover: ⊢ main chain ofPhe407, Tyr384
2B. counterclockwise turnover: ⊢Asn381,His433

Inevitable reaction step: counterclockwise turnover of TPQ (1B,2B)
1B. Counterclockwise rotation @ Off-Cu

2B. Counterclockwise rotation @ On-Cu

TPQ382
TPQ382

His433⊢ main chain of TPQ382⊢His433

Two candidate pathways: clockwise-turnover of TPQ (1A, 2A)
1A. Turnover (clockwise) + Slide

Turnover
Off-Cu (TPQamr)

Slide
On-Cu (TPQsq)

2A. Slide + Turnover (clockwise)

Slide
Off-Cu (TPQamr)

Turnover
On-Cu (TPQsq)
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Computational details
Initial settings• Crystal structure; PDBID: 3X3Z (copper amine oxidase from Arthrobacter globiformis (AGAO) inthe TPQamr state)• Homodimer model• Protonation state is referred to the neutron structure (PDBID: 6L9C)• Added 36 Na+ to charge neutral• A spherical water droplet of 60Åradius• Proton positions are relaxed by 10ps MD (amber99fprce field, 0->250K, 250K->0K).
• Heavy atoms are fixed to the X-ray structure

3X3Z+ 22,000 Waters

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Computational details
QM/MM• Program Package: NWChem version 6.8• Method: UB3LYP/(LANL-2DZ, 6-31G*)| amber 99• Optimization for 10Å around the QM center• #QM=84, #Basis=789

Asn381
Asn381

TPQ382

TPQ382

Tyr384

Asp298

Cu2+

Asp298His433Tyr284Thick Tube: QM region

Tyr284

His431

His592QM atoms (side view)
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-H+,
conformational change

Calculated resultskcal mol-1 is simplified as kcal

Intermediate states (IMS) determined by QM/MMMap of IMS

1A: 1h → 2h → 2 → 4 → 5

Map of IMS

2A: 1h → 1 → 6 → 5

Energy profile of 1A (1h → 2h → 2 → 4 → 5)
O(4) deprotonatedO(4) protonated
WT
N381A

turnover DE‡=23.3 kcal (TS(1h,2h))

WTN381A

silde DE‡=10.0 kcal (TS(4,5)))

Energy profile of 2A (1h → 1 → 6 → 5)
O(4) deprotonatedsilde DE‡=19.8 kcal (TS(1,6))

turnover DE‡=49.3 kcal (TS(6,5))

Energy profile of N381A mutant (NA)O(4) protonatedO(4) deprotonated
WT
N381A

WT
N381A

turnover DE‡=14.9 kcal

-slide DE‡=16.3 kcal

slide DE‡=19.0 kcal

slide DE‡=11.1 kcal

Map of IMS Energy profile of N381A (1hNA→2hNA(⇄3hNA)→2NA→ 4NA→ 5NA)

Possibilities of turnover of TPQox in WT and N381A

WTN381A• In WT, turnover of TPQox becomes unstable (DE =1.7 kcal)• In N381A, turnover of TPQox becomes stabilized (DE =-2.6 kcal).
• In N381A, DE‡ also becomes low

Kinetics measurements by UV-vis spectroscopy• TPQ formation decreased in N381A (1/180)• kcat is reduced in N381A (1/160)

UV-vis spectra changes by Stopped-flow spectrometer (A) the N381A
mutant and (B) the WT AGAO with 2-PEA.
• Reaction stoped at TPQamr in N381A• TPQsq formed in WT

X-ray crystal structures in N381A

1.50 Å resolution

1.90 Å resolution

TPQ flipped form can be observed (52%)=> OxrotNA

N381A TPQamr take a TPQ flipped form=> 3hNA

Conclusion
Theoretical study• In WT, 1A:turnover(clockwise)& slide pathway is possible for TPQamr→ TPQsq• In N381A, reaction barriers of turnover and slide steps become low, and TPQ
flipped form, TPQamr(3hNA), is most stabilized even to TPQsq.
• In N381A, a flipped form of TPQox is stabilized. This conformation is inactive
for the reductive half reactions.
• N381 controls the conformation of TPQ in TPQox and TPQamr . In theformation of TPQsq, the conformational change of TPQ contributes to stabilize
TPQsq.
Experimental study• Detection of intermediate states by Spectroscopy• X-ray crystal structures of N381A in TPQox and TPQamr
Proposed reaction mechanism for TPQamr→ TPQsq

M. Shoji* et al., Molecular mechanism of a large conformational change of the quinone cofactor in thesemiquinone intermediate of bacterial copper amine oxidase, Chem. Sci. 13, 10923 (2022).

CAO is a nonclassical enzyme（nonstatic、dynamical、quantum？）• Reaction really possible？
There is a pathway• Why the conformation takes
place？
stabilization of TPQsq• Multistep reaction (reaction 1 and 2)• State specific reaction• important for highly efficient catalysis

Collaborative researches in a research area, Grant-in-Aid for Scientific Research on Innovative Area,of “Molecular Movies”
1. Light-harvesting protein, C-Phycocyanin

A01: Y. Umena
3. Hemoglobin M Iwate (Hb M Iwate)

2. Copper Amine Oxidase (CAO)

A01: T. Murakawa
4. Lysozyme・Mn(CO3) complex
CO ax

Wateq1

CO eq1

S.Nagatomo, A01: S.-Y. Park, C01: M.Kubo

S.Nagatomo*, M.Shoji, et al., Heme-bound tyrosine vibrations inhemoglobin M: Resonance Raman, crystallography, and DFT calculation,
Biophysical Journal, 121(14), 2767-2780, 2022

Wateq2Mn(I) CO eq2Imiax

B01: Basudev Maity29Collaborative researches in the research area of“Molecular Movies”
5. Heliorhodopsin (HeR)

6. 2-oxoglutarate-dependent dioxygenase(2OGD)

M.Hashimoto et al, JPCL 2020

A01: K. Katayama, H. Kandori
M.Hashimoto, K.Miyagawa, M.Singh,
K.Katayama, M.Shoji, Y.Furutani, Y. Shigeta,
H.Kandori, “Specific zinc binding toheliorhodospin”, PCCP, 25, 3535 (2023).

A01: S. Nagano

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