Comparison of Enzymes Catalyzing the Hydrolysis of Insoluble

Chapter 1

Comparison of Enzymes Catalyzing the Hydrolysis of Insoluble Polysaccharides David B. Wilson, Mike Spezio, Diana Irwin, Andrew Karplus, and Jeff Taylor

Downloaded by 5.189.206.246 on October 14, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0618.ch001

Department of Biochemistry, Molecular and Cell Biology, Cornell University, 458 Biotechnology Building, Ithaca, NY 14853

Amylases, cellulases, chitinases, lysozymes and xylanases are all insoluble polysaccharide hydrolases. Many of these enzymes possess a separate substrate binding domain that is pointed to the catalytic domain by a hinge peptide. A l l of them contain multiple binding sites for sugar monomers (from four to eight) in their active sites and either two or three carboxyl side chains that function in catalysis. There are three amylase families, nine cellulase families, two chitinase families, five lysozyme families and two xylanase families. The enzymes in some families invert the conformation of the glycoside oxygen during hydrolysis while the enzymes in the other families retain the conformation. The structure of an inverting endocellulase, Thermomonospora fusca E2, has been determined by X-ray crystallography at 1.18 Åresolution. It has an open active site cleft, while CBHII which is an exocellulase with the same polypeptide chain fold has its active site in a tunnel. Several E2 mutants have been isolated and characterized to help determine its catalytic mechanism.

Insoluble polysaccharides function either as storage polymers or as the major structural components of bacteria, Crustacea, insects, fungi and plants. Because of their specific functions and the abundance of these polymers, many organisms produce enzymes which can degrade them. Most of these enzymes only hydrolyze one type of polysaccharide but some hydrolyze more than one. This either results from a single active site that can bind polymers with related structures such as cellulose and xylan (1) or chitin and bacterial cell walls (2) or because of the presence of two or more different catalytic domains in one protein (3). A given type of enzyme can play quite different roles in different organisms. Thus plants produce cellulases to modify their cell walls to allow growth (4) or to ripen fruits (5) while plant pathogens produce cellulases to allow entry through plant cell walls (6). Many bacteria and fungi produce cellulases to utilize cellulose as a carbon source and they are essential for recycling plant cell walls in nature. Few animals make cellulases; so that ruminants, shipworms and termites utilize the

cellulases produced by symbiotic bacteria and fungi to digest cellulose (7-9). Chitinases are produced by Crustacea, fungi and insects to allow the organisms to

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Saddler and Penner; Enzymatic Degradation of Insoluble Carbohydrates ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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ENZYMATIC DEGRADATION OF INSOLUBLE CARBOHYDRATES

grow; since chitin is a major structural material in these organisms (10). Chitinases also are produced by bacteria that utilize chitin as a carbon source. Interestingly, chitinases are produced by many plants as part of a defense mechanism against fungal pathogens (11). There are several properties that are shared by many insoluble polysaccharide hydrolases. One is the presence of a separate domain that binds tightly to the insoluble polymer substrate. These domains have been found in amylases (12), cellulases (13), chitinases (14) and xylanases (15). Surprisingly all of the xylanase binding domains that have been studied so far bind cellulose while some of them bind xylan (16) and some do not (15). A Streptomyces chitinase was also found to contain a cellulose binding domain (17). Binding domains are C-terminal to the catalytic domain in some hydrolases and N-terminal in others. For the six Thermomonospora fusca cellulases we have studied the binding domain is Cterminal in three ( E l , E2, and E4) and N-terminal in the others (E3, E5 and E6) (18). In all of the hydrolases containing binding domains it appears that the binding domain is joined to the catalytic domain by a flexible peptide called a hinge or linker region (19). In many cases these peptides are easily recognised because they are rich in proline, and either threonine, serine or glutamine and often have a repeating sequence such as x-Pro. The presence of a cellulose binding domain increases the activity of the cellulase on crystalline substrates such as Avicel, cotton or filter paper but usually does not effect the activity of the enzyme on soluble substrates like carboxymethyl cellulose or oligodextrins (20). We have constructed two mutants that alter the hinge region of T. fusca endocellulase E2. The amount of insoluble and total reducing sugar produced from filter paper by the E2 catalytic domain, a mutant lacking the linker peptide and a mutant with a linker that is twice as long as the native linker were measured. The catalytic domain has about half of the activity of the native enzyme and produces a higher percentage of insoluble reducing sugar (48% versus 31%). Interestingly, addition of pure binding domain does not stimulate the catalytic domain, showing that the binding domain has to be joined to the catalytic domain to effect activity. The enzyme lacking the hinge peptide was more active than the E2 catalytic domain but less active than E2. The double linker enzyme is slightly more active than the native enzyme. These results support a model in which the enzyme is bound to its substrate by the binding domain and the hinge region allows the catalytic domain to catalyze a number of cleavages due to its flexibility. Domains with amino acid sequence similarity to fibronectin type III domains are present in a number of bacterial hydrolases including an amylase, several cellulases and several chitinases. However the function of these domains is not known and most hydrolases do not contain them (10). A l l insoluble polysaccharide hydrolyases appear to have active sites that contain multiple binding sites for sugar monomers with a range of from four to as many as eight subsites. In addition these enzymes appear to utilize catalytic mechanisms that involve two carboxyl side chains with different pKs, as was first proposed for egg white lysozyme (21). One of these carboxyl groups is thought to be protonated and donates a proton to the leaving sugar while the other is thought to be ionized and either stabilizes the carbonium ion present in the transition state or forms a covalent linkage with the rest of the substrate. In some enzymes, a bound water may replace one of the carboxyl groups in this mechanism. The strongest evidence for this mechanism comes from studies in which the three dimensional structure of enzyme-inhibitor complexes have been determined by xray crystallography and the carboxyl side chains identified in the structure have been changed to other residues by site directed mutagenesis. Such studies have been carried out on both hen egg white (22) and T4 lysozyme (23), the exocellulase T. reesei CBHII (24), T. fusca endoglucanase E2 (25), an amylase (26) and a xylanase (27). In each structure, carboxyl side chains were identified


1.

WILSON ETAL.

Hydrolysis of Insoluble Polysaccharides

that were correctly positioned to function in catalysis and mutants lacking these residues had greatly reduced activity. Additional evidence for the presence of essential carboxyl groups comes from experiments in which hydrolases have been reacted with carbodiimides which usually modify carboxyl groups and which inhibit most of the hydrolases that have been studied (28). Another common inhibitor of most of these enzymes is H g , which normally reacts with sulfhydrol groups but since most of the insoluble polysaccaride hydrolases lack free sulfhydrol groups, Hg++ must be inhibiting in some other fashion, possibly by binding to the active site carboxyl groups. For each class of enzyme, many different genes from a number of different organisms have been cloned and sequenced. These genes can be grouped into a number of families based on the amino acid sequences of their catalytic domains (19,29). It is very likely that all members of a given family have a similar threedimensional structure and it is also likely that members of different families have different structures, but this is less certain since some proteins with very little sequence identity have similar three-dimensional structures. The number of families that have been proposed for each class of these enzymes is shown in Table I. Most cellulolytic organisms contain cellulases from different families and where they contain enzymes from the same family the enzymes are often quite divergent. Usually one is an endocellulase and one is an exocellulase. This is true for T. reesei where CBHI and Endo I both belong to family C and for T. fusca where E2, an endocellulase, and E3, an exocellulase, belong to family B while E l , an endocellulase, and E4, an exocellulase, belong to family E (30). One of two stereochemically different mechanisms is utilized by each polysaccaride hydrolase. Some enzymes catalyze hydrolysis with inversion of the glycoside linkage, while the others catalyze hydrolysis with retention of the stereochemistry of the glycoside linkage. It appears that enzymes that give inversion catalyze a direct attack of water in an Sn2 reaction on the glycoside linkage as shown in Figure la. Enzymes that give retention appear to catalyze a two step process in which an active site caboxyl group attacks the glycoside linkage with inversion, usually forming an enzyme bound intermediate (Fig. lb). Then the intermediate is attacked by water, again inverting the conformation, with the overall result of two inversions being retention of the original stereochemistry. Most retaining enzymes can catalyze transglycosylation by reacting the enzyme bound intermediate with a sugar molecule rather than water (31). However, transglycosylation only occurs when there is a high concentration of an acceptor species to complete with water. Enzymes that invert do not catalyze transglycosylation. Studies have shown that all members of a given family appear to utilize the same mechanism (32). Both inventory and retaining enzymes have been demonstrated for amylases, with (J amylases causing inversion and a amylases giving retention. Cellulases in family A and family C give retention while cellulases in family B and E give inversion. In the case of xylanases, the two major families both give retention of the (i linkage (32). This is also true for lysozymes while the stereochemistry of chitinases has not been reported. However it has been reported that a bacterial chitinase can catalyze transglycosylation which indicates that it probably gives retention (33). An important property of cellulases is their ability to give synergism in hydrolysis of crystalline cellulose. No cellulase acting alone has a specific activity on filter paper that is greater than one p.mole/min/u; mole of enzyme while the best synergistic mixture of three cellulases has an activity of nearly 16 \i moles/min/p, mole of enzyme (20). We define synergism as the ratio of the activity of a mixture of enzymes divided by the sum of the activities of the enzymes in the mixture activity acting alone. Synergism is seen in most mixtures containing an endocellulase and an exocellulase and in some mixtures containing two different


+ +


3

4


Table I Hydrolase Families (29,34) Families

Amylase Cellulase Chitanase Lysozyme Xylanase

13,14,15 5,6,7,8,9,12,44,45,48 18,19 21,22,23,24,25 10,11


Enzyme

(General Acid)

HOCH

2

Figure la.

Mechanism of hydrolysis of cellulose with inversion.




1. WILSON ETAL.

H O HOCH

2

OH HOCH

2

O H

o-

JL Figure lb.

Mechanism of hydrolysis of cellulose with retention.


5


6


exocellulases. It is very rare to see synergism between endocellulases. The optimum mixture contains two different exocellulases plus an endocellulase with the endocellulase making up only 20% of the total enzyme. A major goal of our research on i. fusca cellulases is to engineer a cellulase mixture that has a higher activity in degrading insoluble cellulose then T. reesei crude cellulase. An important step forward toward this goal was the determination of the three-dimensional structure of the catalytic domain of endoglucanase E2 at 1.18A resolution which was carried out by Mike Spezio, in collaboration with Dr. Andrew Karplus at Cornell. We first tried to crystallize the native enzyme but as I believe is always the case for proteins that contain hinge peptides we could not get crystals of the multidomain enzyme; so we then tried to crystallize the isolated catalytic domain which contains 286 amino acids and has a molecular weight of 30,000. Crystals were obtained from ammonium sulfate and by seeding we can reproducibly obtain crystals that diffract to l.OA (25). The structure was solved by multiple isomorphous replacement using four derivatives at 2.6 A and then refined first to 1.8 A and then to 1.18 A. The R value for the refined structure including all reflections from 66 to 1.18 A is 23.2% while the RMS deviation from ideality in the bond lengths is 0.008 A and in the bond angles is 1.4°. The structure contains 230 water molecules and two bound sulfate ions. A n example of the quality of the electron density that we obtain at our highest resolution (1.6A) is shown in Figure 2 which shows the electron density map for Tip 41. At this level of resolution side chains that adopt two different conformations can be detected and about 15 of the 286 side chains show alternate conformations. The overall model of E2cd that we have determined is shown in Figure 3 and it is a globular protein with a pronounced cleft at the top. The active site is in the cleft as was shown by the fact that when the structure of a cocrystal of E2cd and the competitive inhibitor, cellobiose, was determined, the cellobiose bound into the cleft. This is shown in Figure 4 which is a model of this structure. The basic chain fold of E2cd is an unusual parallel (3 barrel which is similar to the fold determined for the exocellulase, CBHII, from Trichoderma reesei (24) (Figure 5). The major difference between these two enzymes is that the open active site cleft in E2 is a tunnel in CBHII. The peptide backbones of the two enzymes are shown in Figure 6, where the thick line is the E2 a carbon trace while the thin line is the a carbon trace for CBHII. The closed cleft in CBHII results from an extra 20 amino acid loop on the left which is missing in E2 and from the alternate position of another loop in CBHII which completes the roof of the tunnel. Some of the residues in the active site of E2 are shown in Figure 7. We believe that A s p and A s p probably provide the catalytically important carboxyl groups. A s p is likely to be ionized as it appears to form a salt bridge with Arg while A s p is likely to be protonated as it is close to A s p C We have isolated mutants in which A s p was changed to either a Phe or a Val residue and find that they have greatly reduced activity. Work with other family B cellulases including CBHII have shown that the residue equivalent to A s p is an essential residue, as changes in that residue reduce the activity to 1% or less (24). We have also isolated a mutant in which G l u was changed to Gly. Glu^63 appears to participation substrate binding but the mutant has wild type activity on all cellulosic substrates. It does show lower activity on cellotetraose and that appears to be caused by a higher Km value. The fact that the G l u mutant has normal activity provides indirect evidence that the low activity found for the A s p and A s p mutants does not result from the inability of E2 to fold properly due to the loss of a carboxyl group in the active site. We are trying to use the information about the structure of E2 to first understand how it catalyzes the hydrolysis of crystalline cellulose and then to design more active endocellulases. 1 1 7

2 6 5

2 6 5

2 2 1

1 1 7

1 5

2 6 5

1 1 7

2 6 3

2 6 3

1 1 7

2 6 5



Electron density of Trp41 in cellulase E2 at l.OA resolution.


8



Figure 3.

Model of E2 showing the peptide chain and accessible surface.



1. WILSON ET AL.


Figure 4.

Ribon diagram of the E2 structure with bound cellobiose (side view).

Figure 5.

Ribon diagram of the E2 structure with bound cellobiose (top view).


9


Figure 6.

Comparison of the peptide chains of E2 (thick line) and CBHII (thin line).



1. WILSON ETAL.

Figure 7.


11

Potential catalytic residues in the active site of E2.

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13. Vantilbeurgh, H., Tomme, P., Claoeyssens, M., Bikhabhai, R., and Petterson, G. Limited proteolysis of the cellbiohydrolase I from Trichoderma reesei: separation of functional domains. FEBS Lett. 204, 223-227 (1986). 14. Lucas, J.; Henshen, A.; Lottspeich, F.; Voegeli, U. and Boiler, T. FEBS Lett.1985, 193, pp.208-210. 15. Hall, J.; Hazelwood, G.P.; Huskisson, N.S.; Durrant A.J. and Gilbert, H.J. Mol. Microbiol. 1989, 3, pp.1211-1219. 16. Irwin, D.; Jung, D.D. and Wilson, D.B. Appl. Environ.Microbiol.1994, 60, pp.763-770. 17. Fujii, T. and Miyashita, K. J. Gen.Microbiol.xxxx, 139, pp.677-686. 18. Wilson, D.B. Crit. Rev. Biotechnol. 1992, 12, pp.45-63. 19. Gilkes, N.R.; Henrissat, B.; Kilburn, D.G.; Miller, R.C. Jr. and Warren, R.A.J. Microbiol. Rev. 1991, 55, pp.303-315. 20. Irwin, D.C.; Spezio, M.; Walker, L.P. and Wilson, D.B. Biotechnol. and Bioengin. 1993, 42, pp.1002-1013. 21. Jolles, P. and Jolles, J. Mol. Cell. Biochem. 1984, 63, pp.165-189. 22. Malcolm, B.A.; Rosenberg, S.; Corey, M.J.; Allan, J.S.; Baetselier, A. and Kirsh, J.F. Proc.Natl.Acad. Sci USA 1989, 86, pp.133-137. 23. Anand, N.N.; Stephen, E.R. and Narang, S.A. Biochem. Biophys. Res. Commun. 1988, 153, pp.862-880. 24. Rouviner, J.; Bergfors, T.; Teeri, T.; Knowles, J.; and Jones, A. Science 1990, 249, pp.380-385. 25. Spezio, M.; Wilson, D.B. and P. A. Karplus. Biochemistry 1993, 32, pp.99069916. 26. Matsuura, Y.; Kusonoki, M.; Harada, N. and Kakudo M. J. Biochem. 1984, 95, pp.697-702. 27. Ko, E.P.; Akatsuka, H.; Moriyama, H.; Shinmyo, A.; Hata, Y.; Katsobe, Y.; Urabe, L.; Okada, H. Biochem. J. 1992, 288, pp.117-121. 28. McGinnis, K.; Wilson, D.B. Biochemistry 1993, 32, pp.8151-8156. 29. Henrissat, B. and Bairoch, A. Biochem. J. 1993, 293, pp.781-788. 30. Jung, E.D.; Lao, G.; Irwin, D.; Barr, B.K.; Benjamin, A. and Wilson, D.B. Appl. and Env.Microbiol.1993, 59, pp.3032-3043. 31. Matsui, L.; Yoneda, S.; Ishikawa, K.; Miyaira, S.; Fukui, S.; Umeyama, H., and Honda, K. Biochemistry 1994, 33, pp.451-458. 32. Gebler, J.; Gilkes, N.; Claeyssens, M.; Wilson, D.B.; Beguin, P.; Wakarchuk, W.; Kilburn, D.; Miller, Jr. R., Warren ; R.A. and Withers, S.G. J. Biol. Chemistry 1992, 267, pp.12559-12561. 33. Nanjo, F.; Sakai; Ishikawa, M.; Isobe, K. and Usui, T. Agric. Biol. Chem. 1989, 53, pp.2189-2195. RECEIVED August 18, 1995


Comparison of Enzymes Catalyzing the Hydrolysis of Insoluble

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