Processive Degradation of Crystalline Cellulose by a Multimodular

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Processive Degradation of Crystalline Cellulose by a Multimodular Endoglucanase via a Wirewalking Mode Kun-Di Zhang, Wen Li, Yefei Wang, Yan-Lin Zheng, Fang-Cheng Tan, Xiao-Qing Ma, Lishan Yao, Edward A. Bayer, Lushan Wang, and Fuli Li Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00340 • Publication Date (Web): 04 Apr 2018 Downloaded from http://pubs.acs.org on April 8, 2018

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Biomacromolecules

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Processive Degradation of Crystalline Cellulose by a

2

Multimodular Endoglucanase via a Wirewalking

3

Mode

4

Kun-Di Zhanga,b, Wen Lib, Ye-Fei Wanga, Yan-Lin Zhengc, Fang-Cheng Tana, Xiao-Qing Mae,

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Li-Shan Yaoa, Edward A. Bayerd, Lu-Shan Wangb,*, Fu-Li Lia,*

6

a

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Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 266101

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Qingdao, P. R. China; bState Key Laboratory of Microbial Technology, Shandong University,

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Jinan, 250100, P. R. China; cCollege of Mathematics and Systems Science, Shandong University

Shandong Provincial Key Laboratory of Energy Genetics, Key Laboratory of Biofuels, Qingdao

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of Science and Technology, Qingdao, 266590, P. R. China; dDepartment of Biomolecular

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Sciences, The Weizmann Institute of Science, Rehovot, 76100, Israel; eShandong Provincial Key

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Laboratory of Synthetic Biology

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KEYWORDS: GH9, CBM3, Open cleft, Cellotetraose, Clostridium cellulosi

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ABSTRACT: Processive hydrolysis of crystalline cellulose by cellulases is a critical step for

16

lignocellulose deconstruction. The classic Trichoderma reesei exoglucanase TrCel7A, which has

17

a closed active-site tunnel, starts each processive run by threading the tunnel with a cellulose

18

chain. Loop regions are necessary for tunnel conformation, resulting in weak thermostability of

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fungal exoglucanases. However, endoglucanase CcCel9A, from the thermophilic bacterium

2

Clostridium cellulosi, comprises a glycoside hydrolase (GH) family 9 module with an open cleft

3

and five carbohydrate-binding modules (CBMs) and hydrolyzes crystalline cellulose

4

processively. How CcCel9A and other similar GH9 enzymes bind to the smooth surface of

5

crystalline cellulose to achieve processivity is still unknown. Our results demonstrate that the C-

6

terminal CBM3b and three CBMX2s enhance productive adsorption to cellulose, while the

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CBM3c adjacent to the GH9 is tightly bound to 11 glucosyl units, thereby extending the catalytic

8

cleft to 17 subsites, which facilitates decrystallization by forming a supramodular binding

9

surface. In the open cleft, the strong interaction forces between substrate-binding subsites and

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glucosyl rings enable cleavage of the hydrogen bonds and extraction of a single cellulose chain.

11

In addition, subsite −4 is capable of drawing the chain to its favored location. Cellotetraose is

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released from the open cleft as the initial product to achieve high processivity, which is further

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hydrolyzed to cellotriose, cellobiose and glucose by the catalytic cleft of the endoglucanase. On

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this basis, we propose a wirewalking mode for processive degradation of crystalline cellulose by

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an endoglucanase, which provides insights for rational design of industrial cellulases.

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Introduction

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Cellulose is the earth’s most abundant biopolymer and is responsible for the structural

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integrity of plants 1. Processive cellulases degrade crystalline cellulose and are defined

19

by their ability to remain bound to their substrates and continuously repeat cycles of

20

catalysis before dissociating

21

reacting with adjacent sites without dissociating from the cellulose chain

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two types of processive cellulases: exoglucanases and processive endoglucanases.

23

Generally, their structures are similar and consist of a glycoside hydrolase (GH) module

2-3

. They do so by binding and sliding along substrates and 4-6

. There are


2

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Biomacromolecules

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and one or more carbohydrate-binding modules (CBMs). The cellulases attach to the

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hydrophobic surface of the crystalline substrate by the CBMs, thus forming an enzyme–

3

substrate complex that produces cello-oligosaccharides in a successive stepwise

4

manner as the enzyme slides along a single cellulose chain. However, there is a

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distinguishing structural feature of active-site architecture between the two processive

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enzyme types – exoglucanases have a closed tunnel while endoglucanases have an

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open cleft.

8

One of the best studied exocellulases is Cel7A of the fungus, Trichoderma reesei

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(TrCel7A). It is composed of a GH7 catalytic module and a CBM1. An early molecular

10

dynamics study suggested that the enzyme–substrate interactions produced forces that

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enabled the enzyme to slide along the cellulose surface with a single substrate chain

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channeled through its catalytic tunnel, which was referred to as threading mode

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visual study using high-speed atomic force microscopy suggested that the movement

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was accompanied by catalytic activity and that the CBM enhanced the concentration of

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enzyme molecules on the substrate 10. Other studies have highlighted the importance of

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hydrophobic interactions between amino acid residues in the tunnel and the cellulose

17

chain

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processivity of GHs is directly related to oligosaccharide binding free energy, and a

19

mathematical model has been derived to quantify the binding free energy of the enzyme

20

to the cello-oligosaccharide 6, 13-17.

7-9

.A

10-12

. Recently, more detailed theories have been proposed that suggest the

21

It has been reported that deletions in the loop regions of the catalytic tunnel in Cel7D

22

from Phanerochaete chrysosporium (PcCel7D) created a long cellulose-binding groove,

23

which resulted in more binding sites compared to that of TrCel7A

18

. In addition,


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Page 4 of 38

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cellobiohydrolase Cel6A (E2) from the bacterium Thermobifida fusca (TfCel6A) had

2

enhanced endoglucanase activity after deletion of a surface loop

3

temperatures protein loops tend to be denatured; hence the loops are considered

4

unstable “hotspots” on proteins. As a result, the reported thermostability of

5

cellobiohydrolases, in which the catalytic tunnel is rich in loops, was relatively low

6

However, the surface of crystalline cellulose was described as so smooth that the

7

salient points could only be observed at near-atomic resolution by atomic force

8

microscopy

9

become the challenge for cellulose hydrolysis, especially at high temperatures, and

10

novel modes for crystalline cellulose degradation are currently being studied. A

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subsequent advancement in this field of research is the discovery of processive

12

endoglucanases that can hydrolyze crystalline cellulose.

13

21

19

, and it was thus difficult for cellulases without loops to bind

. At elevated

20

.

22-23

. This has

The first processive endoglucanase TfCel9A was identified from the filamentous soil 24

14

bacterium T. fusca, and it had the ability to degrade cellulose

15

GH9 catalytic module with a family 3c CBM fused to its C-terminal end, which is

16

essential for processivity, and a family 3b CBM at the C-terminus of the intact protein,

17

which is essential for binding to crystalline cellulose. TfCel9A-68, generated from

18

TfCel9A by limited proteolysis, contained only the GH9 and CBM3c modules. Its crystal

19

structure revealed that these two modules provided an elongated flat face, to which

20

linear cellulose chains can adhere. When binding to a cellopentaose molecule, there

21

was clear electron density for six glucosyl units in two clearly separate segments in its

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active-site architecture: a four-glucosyl segment with a reducing end that had an α-

23

configuration and a two-glucosyl segment. The six glucosyl binding sites in the cleft

. The enzyme contains a


4

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Biomacromolecules

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were numbered from −4 to +2 from the nonreducing to the reducing end. Dozens of

2

amino acid residues that interacted with glucosyl subsites −4 to +2 of the active site

3

were mutated to analyze the mechanism of action of TfCel9A. It was found that E424

4

and D58 were the catalytic acid and base, respectively 25-26. The processivity of mutants

5

R378K and D261A, as well as the double mutant D261A/R378K, was examined and

6

found to be improved, reduced, and even more reduced, respectively. This suggests

7

that processivity requires a precise balance between the catalytic cleft binding on the

8

two sides of the cleavage site, as R378 interacted with subsites +1 and +2, while D261

9

interacted with subsites −1 to −4

26

. Interestingly, at the nonreducing end of the active-

10

site cleft of TfCel9A, there is a blockage formed by residues 245–255. This block of

11

residues leaves insufficient space for a hypothetical subsite −5 to bind in a linear cleft,

12

which leads to product yields no longer than cellotetraose

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TfCel9A was deduced whereby the enzyme is bound to the surface of the crystalline

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substrate via CBM2, whereas CBM3c together with GH9 adheres to the extracted single

15

chain

16

have been reported, most from cellulosome-producing Clostridium spp. (Fig. 1). For

17

example, Cel9G from C. cellulolyticum consists of GH9, CBM3c, and a dockerin domain

18

instead of a cellulose-targeting CBM3b. This and other dockeirn-bearing enzymes rely

19

on the CBM3a or CBM3b of the cellulosomal scaffoldin subunit for the cellulose-

20

targeting property. The crystal structure of C. cellulolyticum Cel9G is very similar to that

21

of TfCel9A, and further analysis revealed complementary surfaces between the flat

22

surface of crystalline cellulose and Cel9G

23

belong to noncellulosomal cellulases and cellulosomal components of this species,

24

. The productivity state of

27

. In addition to TfCel9A, several other GH9/CBM3c processive endoglucanases

28

. Cel9I and Cel9R from C. thermocellum


5


Page 6 of 38

29-30

. Both of them exhibit the GH9/CBM3c modular scheme, and both

1

respectively

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release cellotetraose as their initial product.

3

Clostridium cellulosi is a thermophilic, Gram-positive obligate anaerobe, which can

4

ferment a broad range of carbon sources and generate ethanol, acetate, CO2, and

5

hydrogen

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cell-free extracts of Geobacillus toebii DSM 14590 to the medium of the bacterium

7

utilizing lignocellulose as a sole carbon source at an optimal temperature of 60°C.

8

Zymography analysis revealed that two GHs, ORF3880 and ORF3883, play key roles in

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the hydrolysis of lignocellulose. ORF3880 and ORF3883 are both multimodular

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endocellulases that consist of one GH9 module and several CBMs (Fig. 1), and were

11

thus named CcCel9A and CcCel9B, respectively

12

understanding how CcCel9A with an open cleft maintains processivity by analyzing its

13

multimodular structure and active-site architecture.

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Experimental Section

31

. C. cellulosi CS-4-4 was isolated from decayed corn stalk by addition of

32

. In this study, we focus on

15

Cloning of the gene encoding CcCel9A and its truncated mutants-Genomic DNA

16

of C. cellulosi CS-4-4 was extracted according to Andreou et al (2013) 33. The ORF3880

17

gene product (GenBank accession number LM995447) was designated CcCel9A. The

18

coding sequences for wild-type CcCel9A and its truncated mutants (Fig. 2, TM1–TM5)

19

were amplified from the genomic DNA of C. cellulosi CS-4-4 by PCR using Pyrobest

20

DNA polymerase (Takara, Dalian, Japan). Genes of mutants TM14, TM15 and TM16

21

were obtained by fusing two crude fragments amplified from the wild-type CcCel9A

22

gene. The PCR products were cloned into the pEASY-E2 vector according to the


6

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Biomacromolecules

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protocols described by the manufacturer (TransGen Biotech, Beijing, China), and the

2

correct colonies were selected based on DNA sequencing.

3

Assay of enzymatic activity-The specific enzyme activities for carboxymethyl

4

cellulose (CMC), regenerated amorphous cellulose (RAC), and microcrystalline cellu-

5

lose (Avicel) were assayed as previously described

6

mM CaCl2 to the reaction buffer. Avicel PH-101 and CMC were purchased from Sigma

7

(Beijing, China); RAC was prepared according to Zhang et.al (2006)

8

values of CcCel9A and its mutants toward CMC, RAC, and Avicel were determined by

9

fitting the initial velocities at different substrate concentrations using the Michaelis–

10

Menten equation. The assays were performed in triplicate with at least five substrate

11

concentrations, varying from approximately 1/10 to 20-fold of the Km values, under

12

optimal conditions.

34

with a modification of adding 1

36

. The Km and kcat

13

Measurement of processivity-Processivity was determined by using a protocol

14

described by Ozdemir, et al (2012) 37 with modifications. Single discs of Whatman No. 1

15

filter paper (GE Healthcare, China) with a radius of 5 mm and total weight of 3 mg were

16

incubated with 9 µM purified proteins at 60°C in PC buffer (50 mM phosphate, 12 mM

17

citrate, 1 mM sodium azide, pH 6.5) for 16 h except for TM4 and TM5 which were

18

incubated in 50 mM sodium acetate buffer, pH 5). Then the supernatant fluids were

19

transferred to another tube, and 150 µL were taken for measuring the amount of

20

reducing sugars. The filter paper discs were washed with PC buffer or acetate buffer

21

three times. After washing, 150 µL of the same buffer were added to the discs. Two

22

hundred µL of 3,5-Dinitrosalicylic acid (DNS) reagent were added to the supernatant

23

fluids and to the filter paper tubes. Reducing sugars were determined as described in


7


Page 8 of 38

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the enzyme assay methods. The ratio of soluble sugars (found in the supernatant

2

fraction) to insoluble sugars (found on the filter paper) was calculated as processivity. All

3

of the reactions were performed in triplicate.

4

Binding of CcCel9A and its truncated mutants to cellulose-The potential of wild-

5

type and mutant CcCel9A to bind to cellulose was quantitatively measured at 60°C as

6

previously described 32.

7

Thermostability assay-The thermostability of CcCel9A and its mutants was

8

determined by incubating 1 µM of each enzyme at temperatures between 50°C and

9

75°C for 1 h. Residual activity was expressed as the ratio of specific activity on Avicel to

10

the activity of proteins without incubation measured at optimal temperature.

11

Computational simulation-In order to discern the three-dimensional structure of

12

CcCel9A, homology modeling was conducted using the X-ray crystal structure of Cel9G

13

from Clostridium cellulolyticum as the template. We searched the Protein Data Bank

14

(PDB) at National Center for Biotechnology and Information using BLAST with the

15

amino acid sequence of CcCel9A. The results showed that C. cellulolyticum Cel9G had

16

the highest sequence identity (63%) with the query sequence. Its X-ray crystal structure

17

(PDB ID: 1KFG) was selected as the template for homology modeling and the three-

18

dimensional structure of CcCel9A was constructed using Discovery Studio 2.5 based on

19

the MODELER program. The structure of the modeled protein was viewed using PyMol

20

1.5. The cellohexaose structure was extracted from the crystal structure of TfCel9A

21

(PDB ID: 4TF4) and docked into the cleft of CcCel9A according to the hotspots of the

22

substrate and enzyme using Libdock. Amino acid residues within 5 Å to cellohexaose


8

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Biomacromolecules

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were considered capable of interacting with the substrate. Then, the residues of

2

CcCel9A were aligned with TfCel9A

3

and CcCel9B as previously described 38. All highly conserved residues were selected as

4

targets in the site-directed mutagenesis test.

5

24

, CtCel9I

29

, CtCel9R

30

, CpCel9

35

, CtCel9N

29

,

Molecular dynamics (MD) simulations-MD simulations were performed using 39

40

6

Gromacs 4.5

7

force field

8

adding 12.5 Å TIP3P water in a cubic box, and sodium ions were used to neutralize the

9

system. Before free energy calculations, 5,000 steps of energy minimization followed by

10

1 ns MD simulation at constant pressure (1 atm) and temperature (300 K) were

11

performed to equilibrate the system. The pressure was regulated using the extended

12

ensemble Parrinello–Rahman approach

13

modified Berendson thermostat

14

evaluate the contribution of the long-range electrostatic interactions. A non-bonded pair

15

list cutoff of 12.0 Å was used, and the non-bonded pair list was updated every five steps.

16

All bonds involving hydrogen in proteins were constrained using the LINC algorithm

17

whereas bonds and angles of water molecules were forced by the SETTLE algorithm 49,

18

allowing a time step of 0.002 ps.

41

, with the Amber ff99SB force field

for the cello-oligomer in TIP3P water

for the protein and GLYCAM06

42

. The protein was solvated by

43-44

, and the temperature was controlled by a

45

. The Particle Mesh Ewald Method

46-47

was used to

48

,

19

Site-directed mutagenesis-Several aromatic amino acid residues located in each

20

subsite were selected (Table S2) for the deletion of their functional side chain, because

21

they had been previously identified as crucial for substrate interactions

22

directed mutagenesis, 100 ng of plasmid encoding CcCel9A was used as a template for

23

PCR amplification, and the steps were performed using KAPA HiFi HotStart polymerase

50-52

. For site-


9


Page 10 of 38

1

(Kapa Biosystems, MA) as follows: initial denaturation at 94°C for 3 min, followed by 18

2

cycles of 98°C for 30 s, 63°C for 30 s, and 72°C for 2.5 min (Primers are listed in Table

3

S3). The PCR product was digested using DpnI (New England Biolabs, MA) at 37°C for

4

4 h to degrade the template plasmid DNA. Further, the digestion product was

5

transformed into E. coli DH5α competent cells, and the competent cells were plated on

6

Luria-Bertani agar containing 100 µg/mL of ampicillin, using the spread-plate technique,

7

and the plates were incubated overnight at 37°C. Single colonies were propagated to

8

extract plasmids. The inserts were then sequenced (Genwiz Inc., Beijing, China) to

9

confirm the presence of the correct mutation. Gene expression and protein purification

10

were performed as previously described 32.

11

Analysis of hydrolysis products- The hydrolysis reaction mixture (150 µL)

12

consisted of 0.2 µM enzymes and 100 µL of 1% RAC in PC buffer. Aliquots of the

13

reaction mixture were removed at intervals and boiled for 10 min to inactivate the

14

enzyme. The enzymatic hydrolysis products of soluble cello-oligosaccharides were

15

analyzed by fluorescence-assisted carbohydrate electrophoresis (FACE). The gray

16

values of each band were acquired from the peaks generated using ImageJ k 1.45 and

17

extracted quantitatively53.

18

Calculation of affinity constants-The binding abilities of the wild-type and mutant 54

19

enzymes were evaluated using fluorescence spectra as previously described

20

(0.1 mg/mL) was mixed with 0–1.0% (w/v) CMC (0.1% interval) in 50 mM PBS buffer

21

(pH 6.5) for 30 min, which was then used to test the quenching of fluorescence using a

22

fluorescence spectrophotometer (Hitachi F-4600, Japan). All samples were excited at

. Protein


10

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Biomacromolecules

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295 nm and scanned from 300–500 nm to integrate the spectra. CMC without protein

2

was used as control. Data were fit with non-linear regression: y = 1 / (a + bx)

3 (Eq. 1)

4 5

where y and x represent fluorescence intensity and CMC concentration (mM),

6

respectively. Ka (affinity constant) was calculated by b/a.

7

Results

8

Enzymatic characterization of truncated mutants of CcCel9A-Nine different

9

constructs containing the Cel9A catalytic module were cloned and expressed in

10

Escherichia coli (Fig. 2), using the primers listed in Table S1. The catalytic efficiencies of

11

mutants TM1, TM2, and TM3 that do not contain the cellulose-targeting CBM3b showed

12

higher activities towards CMC than those of CcCel9A, whereas those of mutants TM14,

13

TM15, and TM16, which possess the CBM3b, were higher on RAC and Avicel. The

14

processivities of mutants TM14, TM15 and CcCel9A with 1–3 CBMX2s were similar,

15

while the processivity of TM16 that lacks the CBMX2 was significantly lower. The Km

16

values on insoluble substrates (RAC and Avicel) of CBM3b-lacking mutants were

17

increased as the CBMX2 was deleted one by one from TM1–TM3 (Table S4).

18

Binding affinity of CcCel9A and its CBMX2-lacking mutants-To test whether the

19

CBMX2 is necessary for binding of the wild-type CcCel9A to cellulose, the binding

20

isotherms of the wild-type and CcCel9A mutants TM14, TM15, and TM16 were

21

compared. The dissociation constant (Kd) of wild-type CcCel9A was the lowest and the

22

maximum binding (qmax) was the highest, compared to the those of the mutants (Fig.


11


Page 12 of 38

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3A). The partition coefficient (α), which reflects the binding ability of an enzyme, showed

2

that CcCel9A exhibited the highest binding to Avicel, whereas the binding of mutants

3

TM14 and TM15 was similar but 25% higher than that of TM16, suggesting that the

4

three CBMX2s contributed approximately 50% to the binding of insoluble substrates

5

(Fig. 3A).

6

Thermostability of CcCel9A and its CBMX2 mutants-To test whether the CBMX2 is

7

crucial for the thermostability of wild-type CcCel9A, the relative activities of each protein,

8

after incubating at different temperatures for 1 h, were determined (Fig. 3B). At

9

temperatures below 60°C, the relative activity of wild-type CcCel9A was 30%–100%

10

higher than those of the mutant TM14, TM15, and TM16 enzymes. In contrast, above

11

60°C, wild-type CcCel9A exhibited lower activity, and the thermostabilities of mutant

12

enzymes were indistinguishable from one another. These results suggest that the

13

CBMX2 does not play a key role in maintaining the thermostability of CcCel9A.

14

Key amino acid residues in the active-site architecture-A structural model of 24

and C.

15

CcCel9A TM4 was obtained by homologous modeling with TfCel9A

16

cellulolyticum Cel9G

17

catalytic cleft by molecular docking (Fig. 4A). Six glucosyl units, located in the active-

18

site architecture, were categorized as substrate-binding subsites +1/+2 and product-

19

releasing sites −1 to −4, according to their electron density. Aromatic amino acid

20

residues interacted with each catalytic subsite, and several highly conserved residues

21

were selected and ex-changed with alanine in order to remove the functional group (Fig.

22

5 & 4B). Overall, the product-releasing subsite-directed mutants exhibited similar or

23

improved activity on soluble substrates, compared to the wild type. All of the mutants

28

, with a 17-glucosyl unit single cellulose chain embedded in its


12

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Biomacromolecules

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lost some activity on insoluble substrates, yet the substrate-binding subsite-directed

2

mutants exhibited a greater loss of activity (Table S5). However, an exception to these

3

observations was the specific activity of the −1 subsite-directed mutant on Avicel, which

4

was low, indicating that the −1 subsite was essential for degrading the crystalline

5

substrate. Importantly, though the activity and processivity of mutant Y253A remained at

6

the same level as those of the wild-type enzyme, their values decreased significantly in

7

the double mutant Y253A/D259A, indicating a sophisticated architecture of subsite −4

8

that is crucial for processivity. Analysis of the kinetic parameters revealed that the kcat

9

values of most mutants at subsites −1 to −4 were either improved or equal to that of wild

10

type, owing to their higher product-releasing velocity.

11

The processivity of double mutants at subsites −1 and +1/+2, which nearly lost activity

12

on Avicel, were particularly low, demonstrating that processivity was directly linked to

13

the ability of a cellulase to act on the crystalline substrate 6. The affinity constant for

14

Y253A was the lowest, followed by Y415A, indicating stronger interaction between the

15

substrate and subsites −4 and +1/+2 (Fig. 4D). The interaction forces between the

16

substrate and subsites (Fig.4C) suggest that subsites +1/+2 and −4 bind more tightly

17

than the others, confirming their key role in processivity.

18

Product-releasing

patterns

of

various

mutants

demons-


13


Page 14 of 38

1

trate that those mutated at subsites −1 to −4 were unable to secure relevant lengths of

2

cello-oligosaccharides (Fig. 6). The concentrations of released glucose (G1) and

3

cellotriose (G3) were higher than that of cellobiose (G2) for the subsite −2 mutant,

4

whereas for the subsite −3 mutant, the concentration of cellobiose (G2) was higher than

5

those of glucose (G1) and cellotriose (G3), which showed a similar pattern to that of the

6

subsite −1 mutant (Table S6). In Fig. 6E, no cellotetraose (G4) was detected after

7

hydrolysis by the subsite −4 mutant. As for the substrate-binding site mutants, G4

8

accumulated increasingly with extended reaction times, because it could not be further

9

hydrolyzed by these mutants.

10

Discussion

11

C. cellulosi is a thermophilic cellulolytic microbe that produces a series of extracellular GHs

12

and is capable of fermenting some difficult substrates that other thermophilic Clostridium spp.

13

are unable to utilize, including glycogen, inulin, mannitol, and sucrose, to produce

14

acetylmethylcarbinol

15

cellulosi CS-4-4, comprising a family 9 GH module and five CBMs 32. Recombinant CcCel9A is

16

active on CMC, RAC, Avicel, and xylan at 60°C.

31

. CcCel9A is one of the most abundant GH enzymes secreted by C.

17

Structural studies of processive glycoside hydrolases revealed that the polysaccharide-binding

18

sites form a groove or ring, suggesting that partial or complete substrate enclosure is a

19

prerequisite for processivity 5. Many cellulases contain large grooves, and, in general,

20

exoglucanases have evolved loops that fold over their active sites, resulting in an enclosed

21

substrate-binding tunnel

22

elevated temperatures. Instead, family 9 processive endoglucanases like TfCel9A do not possess

55-57

. However, these loops create structural instability for enzymes at


14

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Biomacromolecules

24

1

mobile loops, but their active sites reside in a long, open cleft

. It is assumed that a cellulose

2

chain binds to the long cleft, where the initial cleavage occurs. CcCel9A, a homologue of

3

TfCel9A, possesses higher processivity at 60°C. The absence of the loops serves to improve

4

thermostability, but in exchange, the basic element for forming a closed tunnel is lost. In order to

5

remedy the loss of affinity to the substrate in the cleft, CcCel9A has developed a unique

6

supramodular structure to achieve processivity on a crystalline substrate.

7

CcCel9A contains an unusually high number of five CBMs, each serving a different function.

8

Molecular docking results show that, in addition to the catalytic subsites +2 to −4, CBM3c

9

extends the catalytic cleft to subsite +13, in order to stabilize the single cellulose chain in the

10

open cleft (Fig. 4A). Intriguingly, several conserved residues in the CBM3c of TfCel9A were

11

also found to be well aligned to interact with the cellulose chain, leading into the catalytic cleft,

12

which would increase the potential binding sites along CBM3c to render the binding in the cleft

13

more favorable 58. Biochemical data revealed that the processivity and activity on Avicel of the

14

CBM3c-deletion mutant (TM5) were undetectable, indicating that CBM3c is crucial for

15

hydrolyzing crystalline substrates. It has been proposed that CBM3c could feed a single cellulose

16

chain into the active-site architecture of TfCel9A 59. It was also confirmed that there was a strong,

17

selective non-covalent binding interaction between GH9 and CBM3c via physical association

18

and co-crystallization of the two modules60-61.

19

The processivity of CcCel9A was improved by 40% after adding a CBMX2 to the

20

GH9/CBM3c unit and by 2.36 times for further addition of CBM3b. Adding a second CBMX2 to

21

these proteins resulted in an increase of 84% and 4.73 times in the absence and the presence of

22

the substrate-targeting CBM3b module, respectively (Fig. 2). Furthermore, the activity on

23

crystalline substrates and processivity of enzymes with the CBM3b, including the wild type and


15


Page 16 of 38

1

mutants TM14, TM15, and TM16, were significantly higher than related enzymes without the

2

CBM3b. The characteristics of TM1 and the wild type revealed that the CBM3b contributed

3

25%–45% toward the maintenance of processivity, demonstrating that the CBM3b is necessary

4

for processivity and hydrolysis of insoluble substrates (Fig. 2). Subfamilies of family 3 CBMs

5

had been verified to play an eminent cellulose-binding role in bacterial cellulases from early on,

6

as CBM3a and 3b were found associated with cellulosomes and/or free cellulases, respectively62-

7

66

8

lacks all three CBMX2, decreased by 70%. However, the processivity of the wild type or

9

mutants lacking one or two CBMX2s were similar, thus indicating little or no cumulative effect

10

of CBMX2 on processivity. Nevertheless, with three CBMX2s a cumulative effect is observed as

11

the binding to Avicel was improved by 64% from TM14 to the wild-type enzyme (Fig. 3A). The

12

gradual increase in Km values of CBM3b-lacking mutants (TM1–TM3) on RAC and Avicel

13

indicates the contribution of CBMX2 to substrate affinity (Table S4).

. The CBMX2 was also necessary for processivity, because the processivity of TM16, which

14

Site-directed mutants designed for each catalytic subsite were constructed and characterized.

15

The data revealed that the substrate-binding subsites play a vital role in processivity. Regardless

16

of specific activity, the processivity or catalytic efficiency of subsite +1/+2 mutants were all

17

decreased significantly, compared to those of the wild type and other subsite-directed mutants

18

(Fig. 5A and Table S5). The activities of the mutants on Avicel as substrate, including

19

Y203A/Y424A, Y253A/D259A, Y415A, H372A/Y415A, H123A, and R374A, decreased more

20

than 90%, while in each case their processivity was almost completely lost, thus revealing the

21

relationship between processivity and ability to degrade crystalline cellulose. Double mutants

22

Y203A/Y424A, Y253A/D258A, and R374A/Y415A of subsites −1, −4, and +1/+2, respectively,

23

had a clear additive effect on the characteristics of CcCel9A, because they comprise highly


16

Page 17 of 38 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

Biomacromolecules

1

conserved amino acids residues: Y203, D258, and R374. Most of the amino acid residues that

2

interacted with subsites −2 and −3 were not conserved, except for the aromatic residues such as

3

W257, Y202, and Y315.

4

Interestingly, the specific activity and processivity of M384A was similar or slightly higher

5

than that of the wild type, because of its weak binding to the glucan chain (Table S5). Although

6

the affinity between Y253 and the glucan chain was the strongest among the aromatic amino acid

7

residues distributed in the glucosyl subsites (Fig. 4D), especially those forming hydrogen bonds

8

with subsite −3 and stacking interactions with subsite −4 (Fig. 4B), the characteristics of Y253A

9

were barely altered (Fig.4A & Table S5). It is possible that some water molecules exist within

10

the site of Y253, as the cleft opening was relatively near to the exit, which would help retract the

11

cellulose chain, and therefore, maintain high processivity. The Ka of Y415A also decreased,

12

which highlighted the importance of substrate-binding subsites +1/+2. Taken together with the

13

strong interactive forces observed between the substrate and subsites +1/+2 (Fig. 4C), we can

14

conclude that both subsites +1 and +2 form strong hydrophobic interactions with the cellulose

15

chain and play a role similar to that of W38 and W40, which are located at the entrance of the

16

active-site tunnel of TrCel7A7, 10-11, 67.

17

The hydrolysis product patterns revealed that CcCel9A initially produced cellotetraose (G4)

18

(Fig. 6A). Compared to TrCel7A, four glucosyl units bound more tightly than cellobiose to the

19

enzyme, which prevented the cellulose chain from drawing back. Following this, G4 was

20

subsequently hydrolyzed to glucose (G1), cellobiose (G2), and cellotriose (G3) as secondary

21

reaction products. Additionally, the ability to produce smaller oligosaccharides consistent with

22

subsite −4 to −1 mutants was reduced, whereas G4 was accumulated by subsite +1/+2 mutants


17


Page 18 of 38

1

(Fig. 6F). Since G4 is a competitive inhibitor of CcCel9A 27, the increasing amounts of G4 could

2

not be further hydrolyzed, and therefore, repressed catalysis. In summary, subsites +1/+2 are

3

instrumental in disrupting the hydrogen bonds between the substrate and CBM, whereas subsites

4

−4 to −1 are responsible for drawing the single chain and moving it through the catalytic cleft.

5

These two processes are cooperative, allowing the enzyme to slide along the cellulose chain

6

processively.

7

Hydrolyzing recalcitrant crystalline cellulose has posed a general bottleneck for cellulase

8

application. Other than the cellobiohydrolases that possess a structural advantage for processivity,

9

the thermophilic processive endoglucanase, CcCel9A, has evolved a novel mode to accomplish

10

this process. In this context, multiple CBMs (3 × CBMX2 + CBM3b) improve the affinity to the

11

flat surface of crystalline cellulosic substrates. In addition, CBM3c extends the binding cleft of

12

GH9 module during decrystallization, and the strong interactive forces between subsites +1/+2

13

and the glucosyl rings of the substrate enable disruption of hydrogen bonds and physical

14

extension of the single cellulose chain. Then the chain is drawn to subsite −4 via interaction

15

between the glucosyl rings and the product-releasing subsites, and finally cellotetraoses are

16

released from the catalytic cleft as an initial product in order to achieve high processivity. In

17

contrast to the tunnel-threading mode of cellobiohydrolases, we propose a “wirewalking mode”

18

as a descriptive term for the GH9 processive endoglucanases that underscores their open cleft-

19

like structure at the active center (presented schematically in Fig. 7).

20

Conclusion

21

CcCel9A recruits multiple modules, whereby the GH9 and CBM3c together form an open

22

cleft and four product-releasing glucosyl sites, consisting of up to 17 glucosyl binding subsites,


18

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Biomacromolecules

1

to achieve processivity. Additional modules coordinate separate functions, whereby CBM3b

2

serves as a targeting agent that also promotes decrystallization of the cellulosic substrate, while

3

the CBMX2s assist in the stripping out of the single cellulose chain from the crystalline region.

4

In the open cleft, the substrate chain is first bound tightly by 13 subsites (i.e., subsites +1 through

5

+13) and is then drawn forwards to the location of subsite −4 by the product-releasing subsites.

6

Consequently, the enzyme “walks” 4 glucosyl units along the cellulose chain, which constitutes a

7

proposed “wirewalking mode” of action. The two sides of the cleavage site work synergistically,

8

producing cellotetraose in assembly-line fashion. Because of the open cleft formed in CcCel9A,

9

cellotetraose is further hydrolyzed to cellotriose, cellobiose, and glucose, by working in a manner

10

similar to that of a typical endoglucanase. Multiple CBMs, the long binding cleft, and larger

11

product size are all unique and advantageous features of endoglucanases to achieve processivity.

12

These findings provide a potential platform for construction of potent industrial cellulases.


19


Page 20 of 38

1

ASSOCIATED CONTENT

2

Supporting Information. This material is available free of charge via the Internet at

3

http://pubs.acs.org.

4

Figure S1. Amino acids sequence alignment of GH9 cellulases from different strains shown in

5

Figure 1. Table S1. PCR primers used for amplification of truncated mutants of CcCel9A.

6

Underlined portions are overlapping regions for isothermal assembly. Table S2. The amino acids

7

that interacted with the glucosyl subsites in the active-site architecture. Table S3. PCR primers

8

used for construction of site-directed mutants. Table S4. Kinetic parameters of wild-type

9

CcCel9A and its truncated mutants on CMC, RAC and Avicel. Table S5. Characteristics of wild-

10

type CcCel9A and its active-site mutants. Table S6. Gray values of the cellooligosaccharide

11

bands in Figure 6.

12

AUTHOR INFORMATION

13

Corresponding Author

14

* [email protected]

15

* [email protected]

16

ORCID

17

Kun-Di Zhang: 0000-0003-0320-5979

18

Lu-Shan Wang：0000-0002-9625-0359

19

Fu-Li Li: 0000-0003-0381-2234

20

Notes


20

Page 21 of 38 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

Biomacromolecules

1

The authors declare no competing financial interest.

2

ACKNOWLEDGMENT

3

We thank Dr. Ely Morag from the Weizmann Institute of Science, Israel, for helpful discussions

4

and suggestions. This work was supported by the National Science Foundation of China

5

(31600050, 31600051, 31770077 and 31770054), the Shandong Province Natural Science Funds

6

for Distinguished Young Scholar (JQ201507), and the Key Scientific and Technological Project

7

of Shandong province (2015ZDXX0403A01).

8

ABBREVIATIONS

9

GH, glycoside hydrolase; CBM, carbohydrate-binding module; TrCel7A, Cel7A from

10

Trichoderma reesei; CcCel9A, Cel9A from Clostridium cellulosi; TfCel9A, Cel9A from

11

Thermobifida fusca; CtCel9I&CtCel9R, Cel9I&Cel9R from Clostridium thermocellum; CpCel9,

12

Cel9 from Clostridium phytofermentans; CMC, carboxymethyl cellulose; RAC, regenerated

13

amorphous cellulose; DNS, 3,5-Dinitrosalicylic acid; TM, truncated mutant; MD, molecular

14

dynamics;

FACE,

fluorescence-assisted

carbohydrate

electrophoresis.


21


1 2

Page 22 of 38

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Biomacromolecules

Figure 1. The schematic structures of GH9+CBM3c-bearing cellulases from different strains. Cc, Clostridium cellulosi, this study; Tf, Thermobifida fusca24; Ct, Clostridium thermocellum29-30; Cp, Clostridium phytofermentans35. SP, signal peptide; GH, glycoside hydrolase; CBM, carbohydrate-binding module; Doc, dockerin. 172x78mm (300 x 300 DPI)



Figure 2. Schematic modular architectures and characteristics of the wild-type (WT) CcCel9A and its truncated mutants (TMs). Processivities and specific activities towards Avicel and CMC were all shown as % of those of the wild-type enzyme. 173x79mm (300 x 300 DPI)


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Biomacromolecules

Figure 3. Characteristics of wild-type and mutants of CcCel9A. Quantitative studies of the binding of CcCel9A and truncated mutants TM14, TM15 and TM16 to Avicel at 60°C. Avicel was mixed with various concentrations of the designated proteins, and the binding activities were estimated as described in Experimental procedures. The graphs showed the binding isotherms between bound proteins (nmol/g of Avicel) and free proteins (A). Thermostability of wild-type (WT) and mutant (TM14, TM15 and TM16) proteins. Proteins were incubated for 1 h at the indicated temperatures, and the residual enzyme activity was assayed. Results represent the means and standard deviations from three independent experiments (B). 76x33mm (300 x 300 DPI)



Figure 4. Modelled crystal structure of CcCel9A mutant TM4 with the docking of a cellulose chain in the catalytic cleft. Molecular surface of TM4 (A). The megascopic active-site architecture of the GH9 module. The cellulose chain is colored green, and amino acid residues that interact with the glucosyl ring are colored red. The 17 glucosyl binding sites in the cleft, are numbered from subsite −4 to +13, from the non-reducing to the reducing end (A), and subsites −4 to +2 constitute the active-site architecture (B). Interaction forces between substrate and subsites in the active-site architecture by molecular dynamics (C). Affinity constants (Ka) of aromatic amino acid-directed mutants compared to the wild type determined for CMC (D). 173x110mm (300 x 300 DPI)


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Biomacromolecules

Figure 5. Processivities (% of wild type) of site-directed mutants (A), related to 6 subsites, and analysis of the conserved sites based on the amino acid sequences of enzymes shown in Fig. 1 using WebLogo (B). CS, cleavage sites. 115x77mm (300 x 300 DPI)



Figure 6. Hydrolysis patterns of wild-type CcCel9A (A), site-directed mutants of subsites −1 (B), −2 (C), −3 (D), −4 (E) and +1/+2 (F) on RAC. Enzymes (0.3 µM) were incubated with 5 mg/ml RAC at 60°C, and samples were collected at the indicated time intervals. The subsite −1, −2, −3, −4 and +1/+2 mutants represent Y203A/Y424A, Y202A/W257A, Q308A/Y315A, Y253A/D258A and H372A/Y415A, respectively. 173x120mm (300 x 300 DPI)


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Biomacromolecules

Figure 7. Working model of the “wirewalking” mode of CcCel9A action. The CBM3b module recognizes the gap on the surface of crystalline cellulose (A); CBM3b binds to the non-reducing end of the gap (B); CBM3b decrystallizes the substrate with the aid of the CBMX2s (C); A single cellulose chain is extracted and fed into the catalytic cleft by CBM3c (D); Subsites +1/+2 enable extension of the cellulose chain into the active-site cleft (E); The non-reducing end of the chain is drawn to the position of subsite −4 (F); Cleavage occurs between subsites +1 and −1 (G); Cellotetraose is produced and released through the exit of cleft (H). 173x116mm (300 x 300 DPI)



Table of contents use only 70x35mm (300 x 300 DPI)


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Processive Degradation of Crystalline Cellulose by a Multimodular

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