Computational Evidence for the Enzymatic Transformation of 2

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Computational Evidence for the Enzymatic Transformation of 2-hydroxypropylphosphonate to Methylphosphonate Yanwei Li, Xiaodan Wang, Ruiming Zhang, Junjie Wang, Zhongyue Yang, Likai Du, Xiaowen Tang, Qingzhu Zhang, and Wenxing Wang ACS Earth Space Chem., Just Accepted Manuscript • DOI: 10.1021/ acsearthspacechem.8b00070 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 16, 2018

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ACS Earth and Space Chemistry

1 2

Computational Evidence for the Enzymatic

3

Transformation of 2-hydroxypropylphosphonate to

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Methylphosphonate

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Yanwei Li†, Xiaodan Wang†, Ruiming Zhang†, Junjie Wang†, Zhongyue

8

Yang‡, Likai Du§*, Xiaowen Tangǁ, Qingzhu Zhang†*, Wenxing Wang†

9 †

10

Environment Research Institute, Shandong University, Jinan, 250100, P. R. China ‡

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Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States

12 §

13

Hubei Key Laboratory of Agricultural Bioinformatics, College of Informatics, Huazhong Agricultural University, Wuhan, 430070, P.R. China.

14 15

ǁ

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, P. R. China

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Keywords

18 19

Quantum

mechanics/molecular

20

Greenhouse

21

efficiency

22

___________________________________________________________

23

*

24

Fax: 86-531-8836 1990

gas

methane,

mechanics,

Enzymatic

Methylphosphonate,

transformation,

Enzymatic

Corresponding authors. E-mail: [email protected]；[email protected]

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ACS Paragon Plus Environment

ACS Earth and Space Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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Understanding the origins of the greenhouse gas methane in the ocean is

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of great environmental importance, especially for global climate change and the flow

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of

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2-hydroxyethylphosphonate dioxygenase (HEPD) has been reported to catalyze the

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transformation of 2-hydroxypropylphosphonate (2-HEP) to methylphosphonate

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(MPn), a compound that can be easily transformed to methane by C–P lyase in marine

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microbe. Here, HEPD E176H-catalyzed transformation of 2-HEP to MPn was

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investigated at the molecular level by using QM/MM method. The results evidenced

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the feasibility of the transformation of 2-HEP to MPn and highlighted that the

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transformation contains five elementary steps: H-abstraction, O-O bond cleavage,

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H-transfer, C-C bond cleavage, and MPn formation. H-abstraction was found to be the

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rate-determining step with an energy barrier of 17.8 kcal/mol, which is in reasonable

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accordance with the experimentally determined rate constant (0.38 s-1, correspond to

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18.0 kcal/mol). Three intersystem crossing events were involved in H-abstraction,

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H-transfer, and MPn formation steps. Residue electrostatic analysis on the rate

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determining step suggests that proper mutation of Tyr174 may improve the enzymatic

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efficiency.

carbon

within

the

earth

surface

system.

45 46 47 48 49 50 51 52

2


A

mutant

(E176H)

of

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1. Introduction

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Methane, a greenhouse gas with more than 20-fold more warming

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potential than carbon dioxide, is supersaturated in the ocean relative to the atmosphere

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1, 2

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Understanding the origin of the excess methane in the ocean is of great environmental

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importance, especially for global climate change and the flow of carbon within the

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earth surface system 4. It has been hypothesized that methane comes from the

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transformation of methylphosphonate (MPn) by carbon-phosphorus lyase

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hypothesis was once challenged by the fact that no MPn was detected in the marine

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ecosystem. Recently a breakthrough finding has supported that MPn can be vastly

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produced

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exopolysaccharides in ubiquitous marine archaeon Nitrosopumilus maritime, adding a

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strong evidence to support the hypothesis that the excess methane in the ocean comes

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from MPn 9, 10.

. The ocean contributes up to 4 % of the annual global methane emission 3.

by

methylphosphonate

synthase

(MPnS)

to

build

3-8

the

. This

polar

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MPnS is a non-heme iron-dependent oxygenase that converts

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2-hydroxyethylphosphonate (2-HEP) to MPn at a moderate rate constant of 0.18 s-1 11.

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Lowering its catalytic efficiency may help control the methane emission from the

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ocean and regulate the global climate while improving its efficiency may be helpful in

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rapidly harnessing methane for industrial and daily use. It is thus of great significance

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to understand the enzymatic processes both kinetically and structurally, however,

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efforts to crystalize MPnS have not been successful

3


12

. Lacking crystal structure


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hinders further molecular-level investigation of the catalytic mechanism of MPnS. It

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has been reported that the active site of MPnS is highly conserved with an enzyme

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known as 2-hydroxyethylphosphonate dioxygenase (HEPD), whose crystal structure

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has been successfully deposited

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2-HEP to MPn, just like MPnS does

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alternative of MPnS to investigate the enzymatic transformation of 2-HEP to MPn.

12-16

. Actually, HEPD E176H mutant can convert 12

. HEPD E176H mutant can thus serve as an

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The transformation of 2-HEP to hydroxymethylphosphonate (HMP) by

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wild type HEPD (rather than HEPD E176H mutant) has been extensively investigated

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through many state-of-the-art experimental techniques

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theoretical investigations which used DFT and QM/MM methods to elucidate the

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detailed mechanism at molecular level

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such as a ferric superoxide was found to active 2-HEP and start the reaction, the

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H-abstraction from 2-HEP was demonstrated to be the rate-determining step, and

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valence state change [Fe(III)→Fe(IV)→Fe(III)] was spotted during the transformation

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from 2-HEP to HMP. Based on these results, possible reaction mechanism of HEPD

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E176H-catalyzed transformation of 2-HEP to MPn has been proposed

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However, an in-depth understanding at molecular level is still not available.

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Understanding the detailed mechanism is essential for regulating the transformation

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efficiency of 2-HEP to MPn. For instance, with a deep understanding of the

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mechanism, people can perform site-directed mutation studies to accelerate or

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decelerate the reaction rate. This can be particularly useful for regulating the methane

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emission from the ocean to the atmosphere or harnessing methane for industrial or

22-25

12-21

. There are also several

. Many valuable results were obtained,

4


11, 12, 20

.

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daily use.

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Here the detailed mechanism of HEPD E176H-catalyzed transformation

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of 2-HEP to MPn was investigated by using QM/MM method. QM/MM is a

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multiscale computational method that balances the accuracy and efficiency in

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modelling enzymatic reactions. It has become an increasingly powerful tool to

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complement experimental enzyme chemistry

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feasibility of the transformation of 2-HEP to MPn and highlighted that the

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transformation contains five elementary steps: H-abstraction, O-O bond cleavage,

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H-transfer, C-C bond cleavage, and MPn formation. H-abstraction was found to be the

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rate-determining step. Interestingly, three intersystem crossing (ISC) events were

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observed along the transformation processes.

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2. Methods

26-30

. The results evidenced the

The selected model of HEPD E176H was obtained based on the QM/MM

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24, 25

109

calculations previously as described

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work was similar to our previous studies and only important details of the methods are

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briefly summarized here. The QM/MM calculations were performed by using

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ChemShell 31 software, which interfaces QM program Turbomole 32 to MM program

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DL-POLY 33. The charge shift model

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used during the QM/MM calculations. The geometries of the intermediates and

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transition states were optimized by using hybrid delocalized internal coordinates

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optimizer and microiterative TS optimizer under the B3LYP/def-SV(P)//CHARMM22

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level 36. Frequency calculations were performed to verify the one imaginary frequency

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character of transition state structures, and the suitability of the transition vector was

34

. The QM/MM protocol used in the present

and electrostatic embedding method

5


35

were


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also confirmed. Additional single point energy calculations were carried out at

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B3LYP/def2-TZVPP//CHARMM22 level for a better description of the energy profiles.

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Similar choice of method has been successfully applied in different systems by many

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individual groups 37, 38. For HEPD E176H catalyzed reaction, the QM region contains

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Fe, His129, His176, His182, 2-HEP, O2, and two water molecules (Scheme 1). This

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resulted in 57 QM atoms. Atoms within 20 Å of Fe were allowed to move (~3700

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MM atoms) while the rest of the system (~29000 MM atoms) was fixed during

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QM/MM calculations. The total charge of the whole system is 0. Five different spin

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multiplicities (singlet, triplet, quintet, septet, and nonet) were considered during

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QM/MM calculations to determine the most feasible reaction pathway. Additional

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computational details were provided in the Supporting Information.

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3. Results and Discussion

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By using QM/MM method, we have previously identified the detailed

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reaction mechanism of wild type 2-hydroxyethylphosphonate dioxygenase (HEPD)

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catalyzed transformation of 2-HEP to HMP

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mechanism of HEPD E176H-catalyzed transformation of 2-HEP to MPn was fully

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investigated. Five elementary steps were found: H-abstraction, O-O bond cleavage,

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H-transfer, C-C bond cleavage, and MPn formation, as indicated in Scheme 1. Each

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individual elementary step will be discussed in detail in the following paragraphs.

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3.1 Reaction mechanism and energy profiles

24, 25

. Here the detailed reaction

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It is critical to determine the most stable spin state in the reactant (R-1).

140

Literally, triplet, quintet or septet state was found to be more stable for non-heme 6


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22-25, 37, 38

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iron-dependent systems

. Here the singlet, triplet, quintet, septet, and nonet

142

state have been systematically analyzed, and the order of relative energies is

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determined as: septet

Computational Evidence for the Enzymatic Transformation of 2

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