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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
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Computational Evidence for the Enzymatic
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Transformation of 2-hydroxypropylphosphonate to
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Methylphosphonate
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Yanwei Li†, Xiaodan Wang†, Ruiming Zhang†, Junjie Wang†, Zhongyue
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Yang‡, Likai Du§*, Xiaowen Tangǁ, Qingzhu Zhang†*, Wenxing Wang†
9 †
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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 §
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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
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Quantum
mechanics/molecular
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Greenhouse
21
efficiency
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___________________________________________________________
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*
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Fax: 86-531-8836 1990
gas
methane,
mechanics,
Enzymatic
Methylphosphonate,
transformation,
Enzymatic
Corresponding authors. E-mail:
[email protected]；
[email protected] 25 1
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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.
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A
mutant
(E176H)
of
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ACS Earth and Space Chemistry
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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
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. 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,
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.
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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
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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
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. The QM/MM protocol used in the present
and electrostatic embedding method
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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).
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Literally, triplet, quintet or septet state was found to be more stable for non-heme 6
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iron-dependent systems
. Here the singlet, triplet, quintet, septet, and nonet
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state have been systematically analyzed, and the order of relative energies is
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determined as: septet
