Polaina J., MacCabe A.P. (ed.). Industrial Enzymes. Structure, Function and Applications

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CHAPTER 32

TRANSGLUTAMINASES

MARÍA JESÚS ARRIZUBIETA

∗

Departamento de Ciencias del Medio Natural, Universidad Pública de Navarra, Pamplona, Spain

∗

mjesus.arrizubieta@unavarra.es

1. INTRODUCTION

Transglutaminases (TG: EC 2.3.2.13, protein glutamine:amine -glutamyl-

transferase) are a group of thiol enzymes that catalyse the post-translational modifi-

cation of proteins mainly by protein to protein cross-linking, but also through

the covalent conjugation of polyamines, lipid esterification, or the deamidation of

glutamine residues (Folk and Chung, 1973; Lorand and Conrad, 1984; Aeschlimann

and Paulsson, 1994; Nemes et al., 1999). Transglutaminases are widely distributed

among bacteria, plants and animals. Mammalian transglutaminases comprise a

group of structurally and phylogenetically related multidomain enzymes strictly

dependent on calcium for their activity (Grenard et al., 2001). These enzymes are

related to different physiological processes and diseases and consequently have

raised medical and pharmacological interests in their potential as therapeutic targets

or diagnostic aids (Griffin et al., 2002). In contrast to mammalian transglutami-

nases, the microbial transglutaminase from Streptomyces is a monomeric enzyme

that does not require calcium for activity. Phylogenetically this enzyme is not

related to mammalian transglutaminases and presents a novel catalytic mechanism

(Kashiwagi et al., 2002). Its discovery has enabled a diversity of industrial appli-

cations in the food and textile industries. Transglutaminase activity has also been

found in higher and lower plants (Del Duca and Serafini-Fracassini, 2005), and

the available data indicate that plant transglutaminases are similar in overall

structure and catalytic mechanism to those of mammals (Villalobos et al., 2004).

2. ENZYME ACTIVITY AND MECHANISM OF CATALYSIS

TGs catalyse the acyl-transfer reaction between the -carboxyamide group of a

peptide-bound glutamine residue and a primary amine. In this reaction the glutamine

side chain serves as the acyl donor, whereas the primary amine functions as acceptor.

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568 ARRIZUBIETA

The outcome is the generation of a covalent bond between the two substrates and the

release of ammonia. In most cases described, the primary amine is the -amino group

of a lysine residue, and the reaction results in the formation of -(-glutamyl)lysine

linkages. Depending on the nature of the substrates (proteins or peptides), the reaction

may result in protein cross-linking or peptide conjugation to proteins. When primary

amines are not available, water can function as the acyl group acceptor, with the

consequent deamidation of the glutamine residue (Folk and Chung, 1973; Lorand and

Conrad,1984).Inaddition, thehydroxylgroupsof hydroxyceramidescanalsofunction

as acyl acceptor substrates resulting in ester bond formation between the glutamine

side chains of proteins and hydroxylipids (Nemes et al., 1999).

The transglutaminase reaction is reversible and proceeds via a modified double-

displacement mechanism in which an acyl-enzyme intermediate is formed between

the acyl portion of the glutamine substrate and the sulfhydryl group of the catalyti-

cally active cysteine residue on the enzyme. An acyl acceptor, like water or a primary

amine,reactswiththisintermediateresultingindeamidationorcross-linkingreactions,

respectively(Folk,1969;FolkandChung,1973).ThefirstcrystalstructureofaTG,that

of blood coagulation factor XIII (FXIII), revealed striking similarities to cysteine-

proteases at the active site, leading to the proposal of a similar catalytic mechanism

for the TG-catalysed cross-linking reaction (Pedersen et al., 1994). According to this

mechanism, Cys-314 acts as the nucleophile and His-373 as the acid/base catalyst.

Asp-396 is proposed to play a secondary role, stabilizing the protonated form of His-

373 and providing a favourable orientation of this residue. Based on the structural

and mechanistic similarities between FXIII-like TGs and cysteine-proteases, it has

been proposed that these two groups of enzymes, catalysing almost opposite reactions,

evolved from a common ancestor (Pedersen et al., 1994).

TGs are synthesized as inactive zymogens that require activation before exhibiting

their transamidating activity (Lorand and Conrad, 1984). The mechanisms for

controlling the transamidating activity varies from enzyme to enzyme and involves

activation by proteolytic cleavage, calcium binding, GTP binding, substrate seques-

tration, and often combinations of two or more of these mechanisms (Lorand and

Conrad, 1984; Griffin et al., 2002).

Different enzymes display specificity differences towards substrate proteins. This

constitutes an important aspect of their biological function and it is also relevant to

their biotechnological applications. In general, there is more stringent specificity for

the glutamine substrate whereas the specificity for the primary amine substrate is

broader. The nature of the amino acids surrounding the sensitive glutamine residues

and the degree of the latters’ exposure to solvent appear to be the main determinants

of substrate specificity (Coussons et al., 1992).

3. ENZYME TYPES

3.1. Mammalian Transglutaminases

Nine different transglutaminase encoding genes have been characterized in

mammals (Ichinose et al., 1990; Aeschlimann and Paulsson, 1994; Aeschlimann

TRANSGLUTAMINASES 569

et al., 1998). The products of eight of them are active enzymes whereas the

remaining one is a non-catalytic protein, the erythrocyte protein band 4.2. These

genes present a high degree of sequence similarity and share the same genetic

organization.

All characterized mammalian TGs bind calcium and function as metal-

enzyme complexes in different biological processes (Aeschlimann and Paulsson,

1994). Blood coagulation factor XIII catalyses covalent cross-linking between

fibrin molecules during blood clot formation (Pisano et al., 1968; Ichinose

et al., 1990). There are six different TGs expressed during epidermal differ-

entiation. These enzymes cross-link the structural proteins that constitute the

protective cornified cell envelop of differentiating keratinocytes (Nemes and

Steinert, 1999; Candi et al., 2005). Tissue transglutaminase, or TG2, besides

being a cross-linking enzyme functions also as a GTPase, and participates in

processes as diverse as apoptosis, development, intracellular signalling, cell-

matrix interactions and cell migration (Akimov et al., 2000; Piacentini et al.,

2000; Mangala et al., 2005; Sarang et al., 2005). Prostate transglutaminase,

or TG4, is an extracellular TG secreted by the prostate gland into the

seminal fluid and is responsible for the production of the rodent vaginal plug

(Aeschlimann and Paulsson, 1994).

3.2. Microbial Transglutaminases

The first characterized microbial transglutaminase (MTG) was that of the bacterium

Streptomyces mobaraensis (previously termed Streptoverticillium mobaraensis)

(Ando et al., 1989). This enzyme is secreted as a zymogen that is sequen-

tially processed by two endogenous enzymes to yield the mature form (Zotzel

et al., 2003). The mature enzyme is a monomeric protein 331 amino acids

long and contains a single cysteine (Cys-64) which is the catalytic residue

(Kanaji et al., 1993; Pasternack et al., 1998). The amino acid sequence of MTG

bears little significant similarity to the FXIII-like TGs or to any other known

sequence. MTG does not require calcium for activity, shows broad substrate

specificity and can be produced at relatively low cost. These properties are

advantageous for industrial applications. Currently the enzyme is produced by

fermentation using a Streptomyces strain; however, a lot of effort has been

directed towards achieving production of recombinant MTG in a heterologous host

(Yokoyama et al., 2004).

TG activity has also been found in Bacillus species (Kobayashi-K et al.,

1996). The enzyme purified from B. subtilis catalyses the cross-linking of the

spore coat protein GerQ thereby contributing to the physical and chemical resis-

tance of the bacterial spore (Ragkousi and Setlow, 2004). In addition, bacterial

virulence factors such as Escherichia coli cytotoxic necrotizing factor and Borde-

tella dermonecrotic toxin present transglutaminase activity. They exert their

cytotoxic effects by activating the small intracellular GTPases of the Rho family by

570 ARRIZUBIETA

deamidation of specific Gln residues or by cross-linking the GTPases with

polyamines (Horiguchi, 2001).

3.3. Plant Transglutaminases

Plant transglutaminases are present in various compartments such as chloro-

plasts, mitochondria, cell walls and cytoplasm. Their functions are related to plant

growth, cell division, differentiation, programmed cell death, fertilization and stress

(Serafini-Fracassini et al., 1995). So far only three TG have been characterized at

the molecular level, one in Arabidopsis (Della Mea et al., 2004) and two proteins

of 39 and 58 kDa in the chloroplast of Zea mays (Della Mea et al., 2004; Villalobos

et al., 2004). The expression and activity of the corn enzymes is light dependent

and it has been proposed that they might play a role in the light-harvesting process

in thylakoids (Della Mea et al., 2004).

4. ENZYME STRUCTURE

4.1. Factor XIII

The structure of the zymogen of the A subunit of factor XIII (FXIIIA) has been

published (Yee et al., 1994). The solved structures correspond to a homodimer. The

region corresponding to the mature enzyme is composed of four domains. Starting

from the N-terminus, there is a -sandwich domain (residues 43–184) followed by

a catalytic core (residues 185–515) and two -barrels - barrel 1 (residues 516–628)

Figure 1. A) 3D structure of factor XIIIA. Only one subunit of the dimer is represented. The catalytic

cysteine is represented in space-filling mode. B) Detail of the catalytic triad

TRANSGLUTAMINASES 571

and barrel 2 (residues 629–727) - at the C-terminus (Fig. 1). The core domain

includes 10 segments of -helix arranged around a twisted -sheet of six antiparallel

strands. The two C-terminal barrels have a seven-stranded -barrel fold, with a

fibronectin-like structure. Residues Cys-314, His-373 and Asp-396 constitute the

catalytic triad of factor XIIIA and they are located at the base of a cavity bounded by

the core and barrel 1 domains. The catalytically active cysteine, Cys-314, sits at the

N-terminus of a long helix in the core domain and is hydrogen bonded to His-373,

which in turn forms a hydrogen bond with Asp-396. Both His-373 and Asp-396 sit

on two -strands of the same sheet. In addition, Cys-314 is also hydrogen bonded

to Tyr-560 located in one of the loops of the barrel 1 domain.

In the zymogen structure the active site of the molecule is inaccessible to solvent,

and thus to substrate, due to both intra- and inter-subunit interactions. Access to

the catalytic Cys-314 residue is blocked by Tyr-560, and movement of the -barrel

1 domain is required to allow the substrate to approach the nucleophilic Cys-314.

In addition, the structure of the dimer shows that the activation peptide of each

A-subunit crosses the dimer interface and partially occludes the opening of the

active site in the catalytic core of the other subunit. Only after removal of this

pro-peptide is the active site accessible to substrates. Crystallographic studies have

shown that neither calcium binding (Fox et al., 1999) nor pro-peptide cleavage

alone (Yee et al., 1995) causes the large conformational changes required to expose

the active site. Based on these results it has been postulated that binding of both

and the acyl donor substrate is required to trigger the conformational change

necessary to expose the active site of the enzyme.

4.2. Other Animal Transglutaminases

Besides human FXIIIA, the X-ray structures of three other animal TGs have been

solved: human tissue transglutaminase, TG2 (Liu et al., 2002), human epithelial

transglutaminase, TG3 (Ahvazi et al., 2002) and the fish-derived TG, fTG (Noguchi

et al., 2001). All these TGs present the same overall four domain structure as

FXIIIA: -sandwich, / catalytic core, barrel 1 and barrel 2. The position of the

catalytic triad Cys-His-Asp is conserved in the three enzymes, and in all of them

the active site is shielded from contact with the solvent thus requiring major struc-

tural changes for enzyme activation. The conservation of these features indicates

a common mechanism of catalysis and a common phylogenetic origin for the

members of this family of enzymes. Nevertheless, the solved structures of these

additional TGs have revealed the structural basis for the biochemical peculiarities

of these enzymes, such as the GTP regulation of TG2 activity (Liu et al., 2002)

and the mechanism of calcium activation of TG3 (Ahvazi et al., 2002).

4.3. Streptomyces mobaraensis Transglutaminase

The crystal structure of the microbial transglutaminase from Streptomyces

mobaraensis (Kashiwagi et al., 2002) revealed a completely different structure

572 ARRIZUBIETA

from that of FXIIIA and related TGs. Furthermore, the structure of MTG seems to

present a novel fold since it does not resemble any structure published to date. The

configuration of the catalytic residues also suggests a distinct catalytic mechanism

that might account for the peculiar biochemical characteristics of this enzyme.

In contrast to the multidomain structure of the FXIII-like TGs, MTG presents a

single domain having a disk-like shape (Fig. 2). The active site is located at the

bottom of a deep cleft at the edge of the disk. The structure of the MTG belongs to

the  + folding class, containing 11 -helices and 8 -strands. These secondary

structure elements are arranged so that a central -sheet with seven antiparallel

strands is surrounded by the -helices.

The size of MTG (331 amino acids) is similar to that of the core domain of FXIIIA

(330 amino acids). Although they both belong to the  + folding class, their

overall folding patterns are considerably different. Nevertheless, the arrangements

of the secondary structure elements around their active sites are very similar. Both

active site cysteines occur near the N-termini of -helices, and in each TG this

-helix is flanked by a four-stranded -sheet that contains the other two residues

of the catalytic triad.

Unlike the catalytically active cysteine groups of animal TGs, the sulfhydryl

group of Cys-64 is partially exposed to the solvent and can readily react with

substrates, which may explain the higher reaction rate of MTG. Besides Cys-64,

residues Asp-255 and His-274 constitute the catalytic triad. The most striking

difference between MTG and the FXIII-like TGs is that the positions occupied

by Asp-255 and His-274 in MTG superimpose well on the positions of His-373

and Asp-396 in FXIIIA, respectively. In other words, in the MTG molecule the

relative positions of the catalytically important His and Asp residues are reversed

Figure 2. A) 3D structure of the microbial TG from Streptomyces mobaraensis. The catalytic cysteine

is represented in space-filling mode. B) Detail of the catalytic triad

TRANSGLUTAMINASES 573

Figure 3. Comparison of the active centres of factor XIIIA (dark grey) and MTG (light grey). Organi-

zation of secondary structure elements and catalytic residues at the active site

relative to the Cys residue (Fig. 3). These results have been taken to indicate that in

the catalytic mechanism of MTG, Asp-255, and not His-274, acts as the acid/base

catalyst. It has been suggested that the negatively charged state of Asp-255 during

the reaction might be responsible for the weaker deamidation activity of MTG

compared to the FXIII-like TGs since this would favour positively charged groups

acting as acyl acceptors instead of neutral species such as water.

The similarities between the active site structures and the differences in the

overall structures between MTG and the FXIII-like TGs suggest that the relationship

between these enzymes is a special case of convergent molecular evolution.

5. INDUSTRIAL APPLICATIONS OF TRANSGLUTAMINASES

TG-mediated cross-linking of proteins has dramatic effects on their physical and

chemical properties. This has triggered the use of these biocatalysts in a wide range

of industrial sectors, from cosmetics to the food industry. In addition, and arising

from the involvement of this group of enzymes in many physiological and patho-

logical processes, they have found important applications in the pharmaceutical

industry. Their biotechnological potential is best reflected in the rapidly growing

number of patent applications regarding this group of enzymes (Griffin et al., 2002).

The first assays to test possible industrial uses of TG were carried out to modify

food proteins using mammalian enzymes (Matheis and Whitaker, 1987). Despite

the interest in these enzymes for modulating food rheological properties, limited

574 ARRIZUBIETA

supply hindered their commercial utilization. The situation changed in 1989 with the

purification of the microbial TG from Streptomyces mobaraensis (Ando et al., 1989)

which could be produced relatively cheaply by fermentation methods. Subsequently,

the application of TG in industry, and especially in the food industry, began to

increase at a steady rate. It is remarkable that sixteen years later, the microbial

transglutaminase obtained from Streptomyces is still the only commercial source of

MTG. The use of microbial transglutaminase in food processing has been covered

in a series of reviews (Nielsen, 1995; Motoki and Seguro, 1998; Kuraishi et al.,

2001; Yokoyama et al., 2004).

5.1. Meat and Fish Products

Muscle proteins, especially myosin, are polymerised by TG (Huang et al., 1992) and

the resulting cross-links strengthen the protein network in muscle-derived products.

The addition of the TG during the setting process of surimi results in a gel with

greater breaking stress and improved elasticity (Sakamoto et al., 1995), both of

which are principle determinants of the value of the product. Fish species with

high proteolytic activity were traditionally not capable of being used for surimi

production. In these under utilized fish species the addition of TG has been reported

to improve the gel characteristics of fish paste (Haejung et al., 1996) thereby

allowing their utilization in surimi production.

In the case of sausages, hams and other meat products the effect of TG addition

is improved texture with higher breaking strength or firmness, and increased defor-

mation and elasticity of the gel. This leads to an increase in manufacturers’ profits

due to reduced product loss during processing and slicing. On the other hand, the

use of TG results in the production of reduced salt/phosphate meat products with

improved water-holding capacity and texture, which constitutes a valuable health

benefit for the consumer. The covalent nature of the isopeptide bonds created by TG

determines that the changes in structure are resistant to both heating at high temper-

atures and freezing. Thus, manufacturing of canned or frozen products benefits

from the utilization of this enzyme. One of the most successful applications of

TG in the meat industry is probably the elaboration of restructured meat products.

Although other methods had been previously described and used for this purpose,

the combination of TG and caseinate has emerged as a very convenient and practical

method to obtain restructured meat products of high quality (Motoki and Seguro,

1998; Kuraishi et al., 2001).

5.2. Dairy Industry

Of the proteins present in milk, caseins, which have an open conformation, react

readily with TG, while globular whey proteins react only under conditions that

favour unfolding (Matsumura et al., 1996; Lorenzen et al., 1998). In yoghurt

manufacturing, TG treatment of milk results in an increase in firmness and viscosity

and reduced syneresis. In cheese manufacturing, TG treatment of milk results in an

TRANSGLUTAMINASES 575

increase in curd yield, a less dry texture and reduced whey separation. Processed

cheese products treated with the enzyme present higher heat stability, maintaining

viscosity when melting. In ice-cream manufacturing, TG application allows the

production of low calorie, sugar-free, ice-cream which is softer, smoother and easier

to scoop (Motoki and Seguro, 1998; Kuraishi et al., 2001). TG also has potential

commercial applications as a food-grade additive capable of improving the heat

stability of milk (Osullivan et al., 2002), thus conferring resistance to coagulation

at sterilization temperatures or gelation during storage. Enzymatic cross-linking has

also potential applications in controlling the stability of milk protein-containing

emulsions and foams (Dickinson and Yamamoto, 1996).

5.3. Bakery and Noodles

Gluten, the protein of wheat flour, can be cross-linked by TG and the action of the

enzyme reinforces the protein network structure changing the viscoelastic properties

of the dough (Larre et al., 2000). Consequently, pasta manufactured from TG-treated

dough has a higher breaking strength, or firmness. In addition, treated pasta releases

fewer solids into the boiling water, retaining its firmness and elasticity for a longer

period after cooking (Kuraishi et al., 2001). TG has also found use in the baking

industry as an improver of dough stability and loaf volume (Gerrard et al., 1998),

as well as an improver of the lift of puff pastry and the volume of yeasted croissants

(Gerrard et al., 2000). These effects are retained even after freezing offering a

potential solution to the problem of dough deterioration during frozen storage. In

addition, deep-fried dough products such as doughnuts absorb 25% less oil when the

dough has been treated with transglutaminase, with the concomitant health benefit

for the consumer (Kuraishi et al., 2001).

5.4. Soy Derivatives

TG cross-links soy globulins resulting in modification of the gelation and textural

characteristics of soybean products. In the case of tofu, a soybean curd product

obtained by coagulation of soybean proteins, TG treatment increases its water-

holding properties and results in tofu with a smoother and firmer texture (Nonaka

et al., 1994). In addition, treatment with the enzyme allows for better control of

the coagulation reaction and a reduction in weight loss during retort cooking. Soy

proteins are added to many processed foods such as sausages, ham and surimi to

improve their textural and nutritional characteristics. Thus, TG offers the potential

to modulate textural properties of those foods to which soy proteins are added

(Motoki and Seguro, 1998).

5.5. Bioavailability of Cross-linked Proteins

The wide application of MTG in food production has raised concerns about

the potential effects of the cross-linked proteins on consumer’s health and the