Simulations of Cellulose Synthesis Initiation and Termination in

Subscriber access provided by IDAHO STATE UNIV

B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Simulations of Cellulose Synthesis Initiation and Termination in Bacteria Hui Yang, John B. McManus, Daniel Oehme, Abhishek Singh, Yaroslava G Yingling, Ming Tien, and James David Kubicki J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b02433 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 22, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 20 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

The Journal of Physical Chemistry

Simulations of Cellulose Synthesis Initiation and Termination in Bacteria Hui Yang1, John McManus2, Daniel Oehme3, Abhishek Singh4, Yaroslava G. Yingling4, Ming Tien2, James D. Kubicki3* 1 Department of Biology, The Pennsylvania State University, University Park, PA 16802, United States 2 Department of Biochemistry and Molecular Biology, The Pennsylvania State University, PA 16802, United States 3 Department of Geological Sciences, University of Texas at El Paso, El Paso, TX, United States 4 Department of Materials Science and Engineering, North Carolina State University, Raleigh, North Carolina 27695, United States

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Hui Yang: 0000-0001-6331-6801

ABSTRACT The processivity of cellulose synthesis in bacterial cellulose synthase (CESA) was investigated using molecular dynamics (MD) simulations and the hybrid quantum mechanics and molecular mechanics (QM/MM) approach. Our results suggested that cellulose synthesis in bacteria CESA can be initiated with H2O molecules. The chain length or degree of polymerization (DOP) of the product cellulose is related to the affinity of the cellulose chain to the transmembrane (TM) tunnel of the enzyme. This opens up the possibility of generating mutants that would produce cellulose chains with desired chain lengths that could have applications in the biofuel and textile fields that depend on the DOP of cellulose chains.

1. INTRODUCTION Cellulose is a major component in plant cell walls and the most abundant renewable hydrocarbon biofuel source on Earth

1-3.

Composed of aggregates of chains with β-1,4-linked glucose units

cellulose can be synthesized by a range of organisms including plants and microbes

4

by the 1

ACS Paragon Plus Environment

The Journal of Physical 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

Page 2 of 20

enzyme cellulose synthase (CESA). Studies with bacterial systems have provided a model for plant cellulose synthesis. Genes encoding cellulose synthase were first cloned from bacteria and then used to identify plant genes. The crystal structure of a cellulose synthase was determined with the BcsA/BcsB complex from R. sphaeroides

5-8.

This enzyme complex utilizes UDP-α-D-

glucose, as substrate 9-10. The BcsA subunit is the catalytically-active subunit responsible for the formation of bacterial cellulose and translocation across the inner membrane 10. Within BcsA is the transmembrane (TM) tunnel from which the cellulose chain is extruded during the course of synthesis

5-6, 8.

The chain length or degree of polymerization (DOP) of cellulose is a major

determinant of chemical and physical properties with different species of bacteria able to generate cellulose with different length 11-13. A high DOP can be desirable for the manufacture of paper and cotton cloth, whereas a low DOP may be desirable for digestibility for biofuels

14.

However, the structural components of cellulose synthases and the molecular mechanism that control the length of cellulose synthesized are not known. Recently, our group, McManus et al. 13 performed kinetic analysis of BcsA/BcsB and proposed a mechanism accounting for processivity and mechanism of initiation. The study involved a combination of kinetic and chemical analysis, and kinetic simulations

13.

McManus et al. (13)

determined that initiation of bacterial cellulose synthesis is primer-independent. After initiation, the enzyme proceeds to an elongation phase that involves cyclic rounds of glucose addition from UDP-glucose to the non-reducing end of the chain

15

followed by translocation of the chain by

one glucose unit such that the active site is empty and preparing the enzyme for the next elongation cycle

16.

After multiple cycles of elongation, translocation can occur without the

addition of a glucose residue, which results in termination of the cellulose chain synthesis and chain release. In this paper, MD simulations were used to estimate the free energy cost of cellulose synthesis termination and to investigate the molecular mechanism governing the length of cellulose chain produced by the enzyme. The molecular mechanism of cellulose synthesis initiation was also modeled using the hybrid quantum mechanics/molecular mechanics (QM/MM) approach. We observed that cellulose synthesis initiation is primer-independent.

2 ACS Paragon Plus Environment

Page 3 of 20 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


Figure 1. A proposed mechanism of cellulose synthesis catalyzed by bacterial CESA. Panel A: After release of the cellulose chain, the chain-harboring tunnel is devoid of cellulose. Water, located in the acceptor site of the tunnel then undergoes nucleophilic attack of the UDP-αglucose located in the active site. Panel B: the resultant glucose molecule can then enter the tunnel where the carbon 4 hydroxyl can then participate in chain elongation (Panels C and D). Elongation entails adding one glucose residue to the non-reducing end of the chain via a glycosyl transfer (GT) process and translocation (Trans) by one glucose unit to the acceptor site ready for the next elongation cycle. Panels C, E: After multiple cycles of elongation, a translocation greater than one glucose unit will result in the cellulose synthesis termination. After the termination, H2O molecules take up the receptor position ready to initiate a new cellulose chain.

2. Methods 2.1 MD simulations



Page 4 of 20

All MD simulations were conducted using AMBER14 17. The enzyme system contained a cyclic di-GMP bound BcsA, the TM anchor of BcsB 7 and a cellulose chain with eleven glucose units, as shown in Figure S1. It is assumed that the UDP has been released and thus there is no UDP or UDP-Glc molecules bound at the active site. The configuration of the enzyme and bound cyclic di-GMP were constructed based on the X-ray crystal structure of BcsAB (PDB entry: 4P02). The conserved gating loop (residue 499 to 517) is in the “open” state, interacting with an amphipathic interface helices (IF2). The positioning of the cellulose chain in the TM tunnel was also taken from PDB entry: 4P02, with the final glucose unit at the non-reducing end (closest to the active site) positioned on top of W383, representing the cellulose chain at the pre-termination conformation. The cellulose chain for the post-termination conformation was extracted from the PDB entry: 4HG6, superimposed onto the 4P02 BcsA structure and then had the final two nonreducing end glucose units (donor and acceptor glucose) removed. A further system was generated for both conformations of the cellulose chains by mutation of Phe404 to Ala using the Mutator plugin in VMD 18. The enzyme systems were imbedded in a POPC lipid membrane using the Membrane plugin in VMD

18.

Phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylethanolamine

(PE) are the major components of R. sphaeroides membrane

19-20.

An equimolar mixture of

POPC and POPE has been applied to model the lipid membrane for BcsA by Knott and coworkers

21

to study the cellulose translocation process right after the glycosyl transfer in the

elongation phase, as shown in Figure 1. It has been deemed that the composition of the lipid membrane would have inconsequential effects on the simulation results 21. For simplicity, in all simulations, the enzyme systems were imbedded in a POPC lipid membrane. Counter ions (0.15 M NaCl) were added to neutralize the system which was then solvated with TIP3P water molecules 22. The size of the initial systems was 101x108x186 Å3 and contained ~200,000 atoms. Minimization and equilibration stages/phases were conducted by following the standard protocols for membrane embedded protein systems

23

and Lipid 14 force field

24

which place

gradually reducing constrains on different parts of the system. Firstly, a 1000 step minimization consisting of 400 steps of steepest decent and 600 steps of conjugated gradient minimization was performed with the positions of the lipids and the whole enzyme system (except for water molecules and counter ions) were constrained using a force constant of 250 kcal/mol/Å2. After 4 ACS Paragon Plus Environment

Page 5 of 20 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


minimization the system temperature was increased to 300K through two sequential runs, with a relative weak 10 kcal/mol/Å2 constraint placed on the positions of the lipid and the enzyme system. First the system was heated to 100 K in 20 ps in the NVT ensemble, then it was slowly heated to 300 K in 100 ps at 1 atm in the NPT ensemble, with 2 fs time step, 10 Å non-bonded interaction cutoff, and SHAKE-constrained hydrogen bonds. Short 500 ps NPT simulations were then performed with no constraints on lipids and enzyme prior to production simulations. Conventional MD simulations were conducted on the systems (both wild-type and Phe404Ala mutant) for 200 ns with periodic boundary conditions, temperature of 300K, pressure of 1 atm, 2 fs time step, 10 Å non-bonded interaction cutoff, SHAKE-constrained hydrogen bonds. In all simulations, the protein was described by the FF14SB force field, the cellulose chain by the Glycam06 carbohydrate force field 25, POPC lipid molecules by the Lipid14 force field 26, cyclic Di-GMP by the AMBER General Force Field for organic molecules (GAFF) Antechamber package

28

with the AM1-BCC charge method

29-30,

27

and the

and H2O molecules and

counter ions were described by the TIP3P model 31. 2.2 Free energy calculations The cellulose chain at the pre-termination conformation properly positioned (after 200 MD simulations) in the TM tunnel was then pulled up by one more register using the Targeted MD utility in AMBER 14

17.

The pulling was done in 65 increments of 0.1 Å in magnitude. Each

increment has run for 10 ps. The final configuration of each increment provided the starting configuration for a 5 ns umbrella sampling simulation on that window which was conducted by using the targeted MD utility in AMBER 14 along RMSD-based coordinates with reference to the post-termination conformation. The C1 and C4 atoms of the middle five glucose rings within the TM tunnel were utilized as the RMSD coordinates with a tMD force constant of 30 kcal/mol/Å2 applied. A potential of mean force (PMF) was constructed by the weighted histogram analysis method (WHAM) for the last 3 ns of the 65 windows (0.1 Å separations) of the umbrella sampling simulation 32. 2.3 QM/MM model



Page 6 of 20

After the targeted MD simulation by which the cellulose chain was pulled out of the receptor position in the tunnel by one extra register, H2O molecules have taken the position that belonged to the non-reducing end acceptor glucose, the glycosyl acceptor stabilized by van der Waals interactions with the tryptophan residue (W383). One of the H2O molecules is ~ 2.6 Å away from the general base, D343, forming a strong hydrogen bond (nearly a low-barrier hydrogen bond) 33. This indicated that this H2O molecule could be a potential glycolsyl acceptor to initiate a new chain of cellulose. In order to investigate whether UDP-Glc can transfer its glucose residue to a H2O molecule with a reasonable energy cost, and the molecular mechanism of the reaction, a QM/MM model was generated following the same protocol established in our previous QM/MM study on the cellulose elongation catalyzed by BcsA 15. The cellotriose acceptor in our previous study was replaced by five H2O molecules. One of them formed a strong hydrogen bond with the general base D343. The hybrid QM/MM calculation was conducted using the multilayered (Our own N-layered Integrated molecular Orbital and molecular Mechanics) ONIOM scheme 34 in Gaussian 09 35. In this study, the MM system, treated by the AMBER force field 36,was composed of the UDP-α-DGlc donor, Mg2+, one H2O molecule (to fulfill the hexa-coordination of Mg2+) 37-38, residues 13– 759 of the cytosolic domain in the crystallographic structure (PDB entry: 4HG6) and five H2O molecules taking up the acceptor site. The QM system, as shown in Figure S2, was composed of the UDP-α-D-Glc donor, Mg2+, one H2O molecule coordinating with Mg2+, and the side chains of seven residues involved in the catalytic reaction, or binding with the substrates, Y149, K226, D246, D248, E342, D343, and W383, and five H2O molecules taking up the acceptor site. The density functional M05-2X

39

was used to treat the model system. The geometries of the model

system were optimized with the 6-31G(d) basis set 40-41. Hydrogen link atoms were added to the MM-bounded QM atoms to fulfill the valence of the QM system. All atoms in both layers were free to move in the geometry optimization calculations. Following the same protocol

15,

the reaction mechanism was monitored by the reaction

coordinate rOw–C1, which is defined as the distance between O of the H2O molecule that formed a strong hydrogen bonding with the general base D343 and the anomeric carbon C1 of the incoming glucose, representing the nucleophilic attack of Ow on the anomeric carbon C1. The energy profile of the reaction path is determined by the potential energy surface (PES) scan. In 6 ACS Paragon Plus Environment

Page 7 of 20 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


order to avoid a possible “overshooting” of the potential energies

15,

both forward and reverse

scans were conducted. In the forward scan, the reaction coordinate was changed by −0.1 Å increments between 3.4 and 1.5 Å. In the reverse scan, the reaction coordinate was varied by 0.1 Å increments, between 1.5 and 3.4 Å. All atoms in both layers were free to move in the geometry optimization calculations. Defining the C1 and Ow distance as the reaction coordinate could lead to overestimation of the calculated ΔGa compared to the true reaction path,

21

so this transition

state and ΔGa should be further refined. The results presented here represent a reasonable firstorder approximation of the reaction mechanism. On the basis of the obtained energy profile, the structure with the maximum energy was used to start the transition state search using Gaussian 09. The transition state was characterized by the vibrational frequency calculation on the whole QM/MM system. All the figures are generated using Maestro 42 and PyMOL 43.

3. Results 3.1 The Termination Step



Page 8 of 20

Figure 2. The energy profile for cellulose synthesis termination. Cellulose synthesis termination is associated with an extra step of translocation of the cellulose chain. Panel A: the cellulose chain prior to translocation. Panel B: the cellulose chain post terminating translocation. Panel C: The free energy barrier for cellulose synthesis termination as calculated via umbrella sampling simulations with RMSD measured to the cellulose chain position in ‘B’ and the two minima resulting from the structures in (A) and (B).

Elongation of cellulose synthesis involves cyclic translocation of the cellulose chain. Part of this process requires translocation of the newly-added glucose moves from the active site into the TM tunnel. The length of this movement has to equal one glucose length such that it becomes properly positioned to become the new “attacking” glucose of non-reducing end (Figure 1D). As 8 ACS Paragon Plus Environment

Page 9 of 20 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


shown in Figure 1, the cycle starts with UDP-glucose binding to the active site. Chemistry occurs when the C4 hydroxyl of the acceptor glucose (the non-reducing end of the cellulose chain) undergoes SN2 attack of C1 of the UDP-glucose to form a new glycosidic bond 13, 15. The newly-added glucose then needs to translocate so that the newly-added (donor) glucose is now positioned at the acceptor glucose site 16. However, by definition, for termination to occur (strand release), translocation has to occur at a length greater than one glucose unit (Figure 1C to 1E) 13. Using umbrella sampling simulations, we have been able to estimate the free energy profile of a second translocation that resulted in the cellulose synthesis termination. The energy barrier of this translocation is approximately 80 kJ/mol, as shown in Figure 2C.

3.2 The initiation Step Termination of elongation and release of the cellulose chain from the TM tunnel requires initiation for continued rounds of synthesis. Our previous work, McManus et al. no primer is required for initiation. We

13

13

showed that

also showed that cellulose synthase has UDP-glucose

hydrolase activity. In the current simulations performed with the cellulose chain in a posttermination conformation, several H2O molecules took up positions in the acceptor glucose binding site (previously occupied by the non-reducing end of the cellulose chain in the elongation phase). The conformation of the conserved gating loop (residue 499 to 517) remained in the “open” state during the simulation of the termination process, which may facilitate H2O molecules entering the active site. Those H2O molecules formed H-bonding networks in the acceptor glucose binding site adjacent to Trp383, as shown in Figure S2. This indicated that the H2O molecule can potentially react with the incoming UDP-glucose, resulting in hydrolysis. The glucose molecule would then be free to initiate a new cellulose chain. In order to investigate the molecular mechanism and the free energy cost of such a reaction, QM/MM calculations following the same protocol established in our previous study on the cellulose chain elongation were conducted 15. The energy profile obtained by QM/MM PES scans is shown in Figure 3D with both forward and reverse scans conducted, in order to avoid a possible “overshooting”. The structure of the transition state was found during the reverse scan, labeled TS in Figure 3D, with the structure of 9 ACS Paragon Plus Environment


Page 10 of 20

the SN2-type transition state very similar to the one obtained for the cellulose elongation process 15.

Given that key structural parameters in Table S1 for the initiation process are also similar to

the elongation process, this indicates that cellulose initiation and elongation have followed the same catalytic mechanism. The O atom of H2O molecule (Ow) occupies the same location as the C4 hydroxyl O of the non-reducing end (acceptor glucose). This is followed by a similar nucleophilic attack of the Ow on the anomeric carbon C1, leading to simultaneous breaking of the glycosidic bond C1–O1 and transfer of a proton from the H2O molecule to the general base Asp343. The transition state, as shown in Figure 3B, has been confirmed by a vibrational frequency calculation on the whole QM/MM system. The only imaginary frequency, at −188 cm–1, is the vibration corresponding to forming the new glycosidic bond, the breaking of the glycosidic bond C1–O1, and the transfer of a proton to the general base Asp343, as shown in Figure 3B. The calculated activation electronic energy (ΔEa) for this SN2-type transition state is ∼100 kJ/mol. In consideration of the thermal correction to the Gibbs free energies obtained via vibrational frequency calculations on the whole QM/MM system of the transition state and enzyme– substrate complex, the calculated activation Gibbs free energy (ΔGa) for this SN2-type transition state is ∼73 kJ/mol, close to the one for the elongation process, ~68 kJ/mol.


Page 11 of 20 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


Figure 3. (A) Enzyme-substrate complex, (B) transition state (TS), (C) enzyme-product complex models obtained by QM/MM calculations. (D) Energy profiles obtained by QM/MM PES scan calculations. Arrows in panel B represent the normal mode of the calculated imaginary frequency. Only H2O H atoms are shown.

3.3 The Termination reaction for F416A Mutant



Page 12 of 20

Figure 4. The energy profile of cellulose synthesis termination of F416A mutant. Panel A: before the translocation. Panel B: after the translocation. Panel C: The free energy barrier for cellulose synthesis termination of F416A mutant is ~50 kJ/mol estimated via umbrella sampling simulations.

As shown in Figure 1, the cycle of elongation involves the enzyme adding one glucose residue to the non-reducing end of the chain and translocating by one glucose unit in the TM tunnel. A translocation distance greater than one glucose unit in the cellulose exit tunnel will result in the synthesis termination. As such, we rationalized that the binding affinity of the BcsA enzyme to the cellulose chain in the exit tunnel would affect the processivity of the enzyme. Located in this tunnel are three aromatic residues (Trp383, Phe301, and Phe416) stacked at the entrance of the tunnel that interact with the first three glucose residues (at the non-reducing end of the cellulose chain) via carbohydrate-aromatic interactions 44. Trp383 and Phe301 are highly conserved across 12 ACS Paragon Plus Environment

Page 13 of 20 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


all CESA enzymes and are positioned too close to the active site. Mutagenesis was performed at Trp383 and the Phe301 yielding the Ala mutants. We found that none of the mutants were active 13, 45.

The third aromatic residue, Phe416, is not as well conserved and is positioned a bit further

from the active site so was mutated to an Ala and was subjected to umbrella sampling simulations to determine the potential of mean force (PMF) of cellulose synthesis termination in this mutant system. Classical MD simulations were also performed to get the starting structure for pulling then umbrella sampling simulations. The energy barrier for this process was 50 kJ/mol, as shown in panel C of Figure 4, a decrease of 30 kJ/mol compared to the WT. The lowered energy barrier in F416A mutant is most likely due to the decreased strength of the interaction between Ala416 and the cellulose chain. This suggests that the interaction strength between residues in the TM tunnel and the cellulose chain can have a strong influence on the processivity of the enzyme.

4. Discussion 4.1 Cellulose synthesis initiation catalyzed by bacteria CESA is primer-independent. An extra round of translocation of the cellulose chain within the TM tunnel of BcsA before further glucose transfer will force the glucose residue at the non-reducing end (acceptor glucose) to leave the acceptor site, causing termination of cellulose synthesis. Once this occurs, H2O molecules can then fill the acceptor site. Using a hybrid QM/MM approach, it was found that a single H2O molecule can form a strong H-bond with the general base, Asp343, and can function as an acceptor for the glucose residue from an incoming UDP-Glc bound in the BcsA active site. This suggested that in addition to its synthesis activity, BcsA can also catalyze UDP-glucose hydrolysis, which we previously demonstrated

13.

This also suggests that the glucose molecule

generated in the BcsA active site via its hydrolysis activity could diffuse out of the enzyme, instead of being trapped in the TM tunnel to initiate a new cellulose chain. As BcsA has demonstrated both synthesis and hydrolysis activities, it could be expected that both activities followed a similar molecular mechanism, since the same substrate was bound in the active site of both processes. Using the hybrid QM/MM approach, we found that BcsA 13 ACS Paragon Plus Environment


Page 14 of 20

catalyzed cellulose synthesis initiation and elongation processes do have identical catalytic mechanisms. Most interestingly, it was found that the cellulose synthesis initiation process had a free energy barrier of +73 kJ/mol, which is similar to the +68 kJ/mol for the elongation process. 4.2 Cellulose chain length (DP) is associated with cellulose synthesis termination During a normal cycle in the elongation phase (Figure 1C and 1D), BcsA will add one glucose unit to the non-reducing end of the growing cellulose chain that is located at the entrance of the TM tunnel. This is followed by a translocation process, in which the growing cellulose chain will be translocated by one glucose unit into the tunnel so that the new non-reducing end will be positioned at the acceptor site for the next cycle (Figure 1D to 1C). Translocation by more than one glucose unit into the tunnel (Figure 1C to 1E) will cause the termination of cellulose synthesis, since there will no longer be a glucose residue at the acceptor site. Combining the kinetic and chemical analyses of cellulose synthesized by AcsA-AcsB (from G. hansenii) and BcsA-BcsB (from R.sphaeroides), and the kinetic simulations, McManus et al. found that the rate of translocation is much faster (~16000 times) than the rate of the termination 13.

We found that this rate difference could be attributed to the different energy barriers.

Umbrella sampling simulations performed on the WT gave an energy barrier for this second translocation (termination process) of ~80 kJ/mol. It is much higher (~6 times) than the barrier for translocation (~13 kJ/mol) that Knott and co-workers have found for the translocation process 16. McManus et al. also found that the rate of the translocation, relative to the rate of termination determines the processivity of the enzyme13. An increased ratio (translocation/termination) increased the DOP

13.

By mutating one of the aromatic residues that interact with the cellulose

chain within the tunnel, Phe416, the free energy barrier of the cellulose synthesis termination was lowered to ~50 kJ/mol. This decrease in the free energy barrier could be attributed to the loss of van der Waals interactions between Phe416 and the glucose residue. Thus the affinity between the BcsA enzyme and the cellulose chain in the TM tunnel can have a strong influence on the processivity of the enzyme.


Page 15 of 20 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


Interestingly, to determine whether tunnel resides can impact chain length, McManus et al. mutated the four amino acids residues of the transmembrane tunnel shown to interact with the first three glucose units of the cellulose chain (Figure S3): Trp383, Phe301, Trp417 and Phe416 to Ala residue 45. Aromatic amino acids have long been shown to be an essential component of cellulose binding domains (26). Of the four mutants, one Phe416Ala was active. The DOP of the cellulose chain produced by this mutant is lower than that of wild type, with a peak DOP of 3,300 and a maximum DOP of 22,000 at the 30 minute time point and longer

45.

The GPC

elution profile of cellulose from a F416A mutant of BcsA has demonstrated that F416A synthesized cellulose that was on average much shorter than that of wild type. 13 This suggests a strong association between the DOP of a cellulose chain and the free energy barrier of the cellulose synthesis termination process. As the free energy decreases so does the DOP and this is consistent with the findings of McManus et al. 13 who found that increasing the rate of cellulose termination resulted in a decreased DOP of the cellulose chain. This suggests that it is possible to produce cellulose chains with desired chain lengths by modifying residues in the TM tunnel (Figure S3) of bacterial CESA. If longer cellulose chains are desired, one could mutate residues in the TM tunnel such that there is an increased affinity to the cellulose chain. For shorter cellulose chains, residues in the tunnel could be mutated to decrease the affinity to the chain. We caution the reader that the model barriers will generally overestimate the real energy barriers because the model can miss something the real enzyme does to lower the barrier. The relative changes in the model energy barriers are significant though and consistent with the DOP observations. In short, we may have ± 10 or 20 kJ/mol absolute error in the calculated energy barrier, but the changes modeled are significant and explain the experimental data. 5. Conclusion Cellulose synthesis and termination within BcsAB has been investigated using molecular dynamics simulations and a hybrid QM/MM approach. Our results suggested that initiation of cellulose synthesis in bacteria CESA is primer-independent with a H2O molecule able to accept a glucose residue, transferred to it from UDP-Glc to initiate the synthesis a new cellulose chain.



Page 16 of 20

The catalytic mechanism and activation free energies were found to be similar for both BcsA catalyzed cellulose synthesis initiation and elongation. Umbrella sampling simulations performed on both a BcsAB wild type and a F416A mutant system where an aromatic residue at the bottom of the TM tunnel was mutated demonstrated that decreasing the affinity of the BcsA enzyme to the cellulose chain in the TM tunnel resulted in a decreased free energy barrier for the termination of cellulose synthesis. Alongside an experimental GLC elution profile of the mutant that showed a decrease in the DOP of the cellulose chain, this suggests that DOP is related to the affinity of the cellulose chain to the TM tunnel of the enzyme. This indicates that it would be possible to design mutated bacterial CESA proteins that would produce cellulose chains with desired chain lengths which could have significant impact on applications in the biofuel and textile fields that are heavily dependent on the DOP of cellulose chains.

Supporting Information The enzyme system imbedded in the POPC lipid membrane (Figure S1), the QM layer of the QM/MM model of cellulose synthesis initiation (Figure S2), geometric parameters for Enzyme– Substrate, Enzyme–Product, and Transition State models (Table S1), residues within 4Å of the glucan chain in the exit tunnel of BcsA (Figure S3). Acknowledgement This work is supported by the Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Basic Energy Sciences under Award # DE-SC0001090. Portions of this research were conducted with Advanced Cyber Infrastructure computational resources provided by the Institute for Cyber Science at The Pennsylvania State University (http://ics.psu.edu). This research also used resources of NERSC, supported by the Office of Science of DOE under Contract No. DE-AC02-05CH11231.


Page 17 of 20 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


References 1. Youngs, H.; Somerville, C., Best practices for biofuels. Science 2014, 344, 1095-1096. 2. Youngs, H.; Somerville, C., Development of feedstocks for cellulosic biofuels. F1000 Biology Reports 2012, 4. 3. Somerville, C., Cellulose synthesis in higher plants. Annu. Rev. Cell Dev. Biol. 2006, 22, 53-78. 4. Delmer, D. P., Cellulose biosynthesis: exciting times for a difficult field of study. Annu. Rev. Plant Biol. 1999, 50, 245-276. 5. Morgan, J. L.; McNamara, J. T.; Fischer, M.; Rich, J.; Chen, H.; Withers, S. G.; Zimmer, J., Observing cellulose biosynthesis and membrane translocation in crystallo. Nature 2016, 531, 329. 6. Morgan, J. L.; McNamara, J. T.; Zimmer, J., Mechanism of activation of bacterial cellulose synthase by cyclic di-GMP. Nat. Struct. Mol. Biol. 2014, 21, 489. 7. Omadjela, O.; Narahari, A.; Strumillo, J.; Mélida, H.; Mazur, O.; Bulone, V.; Zimmer, J., BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 17856-17861. 8. Morgan, J. L.; Strumillo, J.; Zimmer, J., Crystallographic snapshot of cellulose synthesis and membrane translocation. Nature 2013, 493, 181. 9. Römling, U., Molecular biology of cellulose production in bacteria. Res. Microbiol. 2002, 153, 205-212. 10. Lin, F. C.; Brown, R.; Drake, R.; Haley, B., Identification of the uridine 5'diphosphoglucose (UDP-Glc) binding subunit of cellulose synthase in Acetobacter xylinum using the photoaffinity probe 5-azido-UDP-Glc. J. Biol. Chem. 1990, 265, 4782-4784. 11. Evans, R.; Wallis, A. F., Cellulose molecular weights determined by viscometry. J. Appl. Polym. Sci. 1989, 37, 2331-2340. 12. Zhang, Y.-H. P.; Lynd, L. R., Determination of the number-average degree of polymerization of cellodextrins and cellulose with application to enzymatic hydrolysis. Biomacromolecules 2005, 6, 1510-1515. 13. McManus, J. B.; Yang, H.; Wilson, L.; Kubicki, J. D.; Tien, M., Initiation, elongation, and termination of bacterial cellulose synthesis. ACS Omega 2018, 3, 2690-2698. 14. Hallac, B. B.; Ragauskas, A. J., Analyzing cellulose degree of polymerization and its relevancy to cellulosic ethanol. Biofuel. Bioprod. Biorefin. 2011, 5, 215-225. 15. Yang, H.; Zimmer, J.; Yingling, Y. G.; Kubicki, J. D., How cellulose elongates - A QM/MM study of the molecular mechanism of cellulose polymerization in bacterial CESA. J. Phys. Chem. B. 2015, 119, 6525-6535. 16. Knott, B. C.; Crowley, M. F.; Himmel, M. E.; Zimmer, J.; Beckham, G. T., Simulations of cellulose translocation in the bacterial cellulose synthase suggest a regulatory mechanism for the dimeric structure of cellulose. Chemical science 2016, 7, 3108-3116. 17. Case, D. A.; Babin, V.; Berryman, J.; Betz, R.; Cai, Q.; Cerutti, D.; Cheatham III, T.; Darden, T.; Duke, R.; Gohlke, H., Amber 14. 2014. 18. Humphrey, W.; Dalke, A.; Schulten, K., VMD: visual molecular dynamics. J. Mol. Graphics 1996, 14, 33-38. 19. Gorchein, A., Distribution and metabolism of ornithine in Rhodopseudomonas spheroides. Proceedings of the Royal Society B: Biological Sciences 1968, 170, 265-278.



Page 18 of 20

20. Benning, C.; Huang, Z.; Gage, D. A., Accumulation of a novel glycolipid and a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation. Arch. Biochem. Biophys. 1995, 317, 103-111. 21. Knott, B. C.; Haddad Momeni, M.; Crowley, M. F.; Mackenzie, L. F.; Götz, A. W.; Sandgren, M.; Withers, S. G.; Ståhlberg, J.; Beckham, G. T., The mechanism of cellulose hydrolysis by a two-step, retaining cellobiohydrolase elucidated by structural and transition path sampling studies. J. Am. Chem. Soc. 2013, 136, 321-329. 22. Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L., Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983, 79, 926-935. 23. Gedeon, P. C.; Thomas, J. R.; Madura, J. D., Accelerated molecular dynamics and protein conformational change: a theoretical and practical guide using a membrane embedded model neurotransmitter transporter. In Molecular Modeling of Proteins, Springer: 2015; pp 253287. 24. Dickson, C. J.; Madej, B. D.; Skjevik, Å. A.; Betz, R. M.; Teigen, K.; Gould, I. R.; Walker, R. C., Lipid14: the amber lipid force field. J. Chem. Theory Comput. 2014, 10, 865-879. 25. Kirschner, K. N.; Yongye, A. B.; Tschampel, S. M.; González‐Outeiriño, J.; Daniels, C. R.; Foley, B. L.; Woods, R. J., GLYCAM06: a generalizable biomolecular force field. Carbohydrates. J. Comput. Chem. 2008, 29, 622-655. 26. Maier, J. A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K. E.; Simmerling, C., ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 2015, 11, 3696-3713. 27. Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A., Development and testing of a general amber force field. J. Comput. Chem. 2004, 25, 1157-1174. 28. Wang, J.; Wang, W.; Kollman, P. A.; Case, D. A., Antechamber: an accessory software package for molecular mechanical calculations. J. Am. Chem. Soc 2001, 222, U403. 29. Jakalian, A.; Bush, B. L.; Jack, D. B.; Bayly, C. I., Fast, efficient generation of high‐quality atomic charges. AM1‐BCC model: I. Method. J. Comput. Chem. 2000, 21, 132-146. 30. Jakalian, A.; Jack, D. B.; Bayly, C. I., Fast, efficient generation of high‐quality atomic charges. AM1‐BCC model: II. Parameterization and validation. J. Comput. Chem. 2002, 23, 1623-1641. 31. Joung, I. S.; Cheatham III, T. E., Determination of alkali and halide monovalent ion parameters for use in explicitly solvated biomolecular simulations. J. Phys. Chem. B. 2008, 112, 9020-9041. 32. Kumar, S.; Rosenberg, J. M.; Bouzida, D.; Swendsen, R. H.; Kollman, P. A., The weighted histogram analysis method for free‐energy calculations on biomolecules. I. The method. J. Comput. Chem. 1992, 13, 1011-1021. 33. Schiøtt, B.; Iversen, B. B.; Madsen, G. K. H.; Larsen, F. K.; Bruice, T. C., On the electronic nature of low-barrier hydrogen bonds in enzymatic reactions. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12799-12802. 34. Dapprich, S.; Komáromi, I.; Byun, K. S.; Morokuma, K.; Frisch, M. J., A new ONIOM implementation in Gaussian98. Part I. The calculation of energies, gradients, vibrational frequencies and electric field derivatives. J. MOL. STRUC-THEOCHEM 1999, 461, 1-21. 35. Frisch, M.; Trucks, G.; Schlegel, H.; Scuseria, G.; Robb, M.; Cheeseman, J.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G., Gaussian 09, revision B. 01. Gaussian Inc., Wallingford, CT 2010. 18 ACS Paragon Plus Environment

Page 19 of 20 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


36. Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P. A., A second generation force field for the simulation of proteins, nucleic acids, and organic molecules. J. Am. Chem. Soc. 1995, 117, 5179-5197. 37. Kehres, D. G.; Maguire, M. E., Structure, properties and regulation of magnesium transport proteins. BioMetals 2002, 15, 261-270. 38. Black, C.; Huang, H.; Cowan, J., Biological coordination chemistry of magnesium, sodium, and potassium ions. Protein and nucleotide binding sites. Coord. Chem. Rev. 1994, 135, 165-202. 39. Zhao, Y.; Schultz, N. E.; Truhlar, D. G., Design of density functionals by combining the method of constraint satisfaction with parametrization for thermochemistry, thermochemical kinetics, and noncovalent interactions. J. Chem. Theory Comput. 2006, 2, 364-382. 40. Petersson, G.; Al‐Laham, M. A., A complete basis set model chemistry. II. Open‐shell systems and the total energies of the first-row atoms. J. Chem. Phys. 1991, 94, 6081-6090. 41. Petersson, G.; Bennett, A.; Tensfeldt, T. G.; Al‐Laham, M. A.; Shirley, W. A.; Mantzaris, J., A complete basis set model chemistry. I. The total energies of closed‐shell atoms and hydrides of the first‐row elements. J. Chem. Phys. 1988, 89, 2193-2218. 42. Release, S., Maestro, version 10.1. Schrödinger, LLC, New York, NY 2015. 43. DeLano, W. L., Pymol: An open-source molecular graphics tool. CCP4 Newsletter On Protein Crystallography 2002, 40, 82-92. 44. Asensio, J. L.; Ardá, A.; Cañada, F. J.; Jiménez-Barbero, J. s., Carbohydrate - aromatic interactions. Acc. Chem. Res. 2012, 46, 946-954. 45. McManus, J. B., Mechanistic studies into bacterial cellulose synthesis.; The Pennsylvania State University, 2017.



Page 20 of 20

TOC Graphic


Simulations of Cellulose Synthesis Initiation and Termination in

Recommend Documents