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

Подождите немного. Документ загружается.

576 ARRIZUBIETA

bioavailability of the cross-linked residues. Glutamine-lysine isopeptide bonds are

widely distributed in natural products and foodstuffs (with the exception of milk)

and cooking itself results in glutamine-lysine isopeptide bond formation (Motoki and

Seguro, 1998). Mankind has therefore been ingesting food containing glutamine-

lysine isopeptide bonds since prehistoric times, suggesting that their consumption

does not pose a health risk. Mammalian digestive enzymes do not cleave this

isopeptide bond. However, two kinds of enzymes have been found in kidney and

in intestinal brush border membranes that degrade the bond in vivo (Seguro et al.,

1995; Yokoyama et al., 2004). Furthermore, nutritional studies have indicated that

the glutamine-lysine moiety in cross-linked proteins can be metabolised and that

the lysine is incorporated into proteins (Seguro et al., 1996). Thus, TG catalysed

cross-linking does not reduce the nutritional value of the residues implicated and/or

proteins. The allergenic risk of added MTG in food has been assessed and no

safety concerns with regard to the allergenic potential of MTG have been identified

(Pedersen et al., 2004).

5.6. Medical Applications

As mentioned above, TGs participate in a myriad of physiological processes.

Therefore, it is not surprising that these enzymes have been implicated in different

pathological states. Some of these conditions are alleviated by supplementation with

exogenous TG activity but in many cases, related molecules, such as TG inhibitors

or antiTG-specific antibodies, find application as therapeutic or diagnostic agents.

The first medical application of a TG was the use of FXIII in blood clotting

products to control bleeding during surgery and as a general tissue adhesive.

In addition, FXIII has found application in substitutive therapy of congenital or

acquired deficiencies in FXIIIA that result in severe bleeding due to lack of fibrin

clot stabilization. Tissue transglutaminase, or TG2, has also received commercial

interest as a tissue adhesive and as a cell adhesion protein in medical implants

(Collighan et al., 2002).

Celiac disease is a gluten sensitive enteropathy that is characterized by atrophy

of the intestinal villi and results in malabsorption and malnutrition. The causative

protein is gliadin, a component of wheat gluten, and the only satisfactory treatment

involves a lifelong gluten-free diet. IgA auto-antibodies against endomysium are

often found in sufferers and have been a useful screening method, avoiding the

need for intestinal biopsy. The main endomysial auto-antigen has been shown to be

the tissue TG, TG2 (Dieterich et al., 1997). An ELISA method has been developed

based on recombinant human tissue TG2 (Sardy et al., 1999) that allows early

identification of celiac patients.

Hypertrophic scarring occurs in a significant number of patients following surgery

or serious burns. A higher percentage of type III collagen due to elevated levels of

TG activity is associated with these scars. The use of putrescine as a TG inhibitor

has been patented for treatment of the scar tissue (Dolynchuk and Bowness, 1999).

TRANSGLUTAMINASES 577

This patent led to a topical cream which has been proven to significantly reduce

hypertrophic scarring in clinical trials.

TGs have been implicated in other pathological processes such as tumour growth

and metastasis, arteriosclerosis, neurodegenerative diseases and skin pathology

(Griffin et al., 2002). Clarification of the role of TG in these processes is a very

active area of research and there is strong evidence that TG enzymes or their

inhibitors offer big potential as therapeutic agents for these processes, and in some

cases medical applications have already been patented (Steinert et al., 2000).

5.7. Cosmetic Industry

The important roles that TGs play in epidermal tissue differentiation determines

that modulation of the activity of these enzymes will affect the structure of the skin

and can be used to improve skin appearance. Topical preparations consisting of

TG and one or more of the corneocyte proteins are proposed to form a protective

layer on the surface of hair, skin and nails (Green and Dijan, 1996). It has been

suggested that appearance of wrinkles associated with ageing can be retarded by the

potentiation of TG1 expression by the topical application of plant extracts (Megata,

2004). In addition, TG can be used to covalently bind various primary amine-

containing compounds, such as antimicrobials, UV-absorbers, anti-inflammatory

substances, antioxidants, colouring agents, perfumes and insect repellants to skin,

nails and hair surfaces (Richardson et al., 1996). A method has been describe for

curl retention in hair and lashes based on the action of the enzyme TG (Mammone

and Popescu, 2004).

5.8. Textile and Leather

Treatment of leather with TG, preferably together with a glutamine and/or lysine

containing polymer such as keratin or casein, has a beneficial effect on the subse-

quent dyeing and colour properties of leather (Collighan et al., 2002). Transglu-

taminase treatment is also shown to improve resistance to abrasion and collagenase

activity (Addy et al., 2005).

The treatment of wool textiles with TG has been shown to improve the qualities

of finished garments: shrink resistance, handle, wettability, reduction of felting

tendency, improved softness, tensile strength retention, improved stretch, improved

dye uptake, dye wash-fastness, and protection from proteolytic detergents (McDevitt

and Winkler, 1999; Cortez et al., 2005). Using a similar methodology to that applied

to human skin, hair and nail treatments, TG can be used to covalently bind primary

amine-containing active compounds or proteins to fibrous textiles such as animal

wool (Griffin et al., 2003).

5.9. Other applications

Many authors have described applications of this enzyme as an enzymatic cross-

linking agent useful for the generation of analytical tools for immunoassays or

578 ARRIZUBIETA

biosensors. In this context, it can be used to conjugate proteins (Tanaka et al., 2004)

and create hapten-antibody conjugates (Meusel, 2004), labelling of proteins (Josten

et al., 2000), or to immobilize proteins onto solid surfaces (Kamiya et al., 2005).

REFERENCES

Addy, V., Griffin, M., Collighan, R. and Clara, S. (2005) Enzymatic treatment of hides and leather.

World patent: WO2005007897.

Aeschlimann, D. and Paulsson, M. (1994) Transglutaminase: Protein cross-linking enzyme in tissues

and body fluids. Thromb. Haemost. 71, 402–415.

Aeschlimann, D., Koeller, M.K., Allen-Hoffmann, B.L. and Mosher, D.F. (1998) Isolation of a cDNA

Encoding a Novel Member of the Transglutaminase Gene Family from Human Keratinocytes.

Detection and identification of transglutaminase gene products based on reverse transcription-

polymerase chain reaction with degenerate primers. J. Biol. Chem. 273, 3452–3460.

Ahvazi, B., Kim, H.C., Kee, S.-H., Nemes, Z. and Steinert, P.M. (2002) Three-dimensional structure

of the human transglutaminase 3 enzyme: binding of calcium ions changes structure for activation.

EMBO J. 21, 2055–2067.

Akimov, S.S., Krylov, D., Fleischman, L.F. and Belkin, A.M. (2000) Tissue transglutaminase is an

integrin-binding adhesion coreceptor for fibronectin. J Cell Biol. 148, 825–838.

Ando, H., Adachi, M., Umeda, K., Matsuura, A., Nonaka, M., Uchio, R., Tanaka, H. and Motoki, M.

(1989) Purification and characteristics of a novel transglutaminase derived from micro-organisms.

Agric Biol Chem. 53, 2613–2617.

Candi, E., Schmidt, R. and Melino, G. (2005) The cornified envelope: a model of cell death in the skin.

Nat Rev Mol Cell Biol. 6, 328–340.

Collighan, R., Cortez, J. and Griffin, M. (2002) The biotechnological applications of transglutaminases.

Minerva Biotecnologica. 14, 143–148.

Cortez, J., Bonner, P.L. and Griffin, M. (2005) Transglutaminase treatment of wool fabrics leads to

resistance to detergent damage. J. Biotechnol. 116, 379–386.

Coussons, P.J., Price, N.C., Kelly, S.M., Smith, B. and Sawyer, L. (1992) Factors that govern the

specificity of transglutaminase-catalysed modification of proteins and peptides. Biochem J. 282,

929–930.

Del Duca, S. and Serafini-Fracassini, D. (2005) Transglutaminases of higher, lower plants and fungi.

Prog Exp Tumor Res. 38, 223–247.

Della Mea, M., Caparros-Ruiz, D., Claparols, I., Serafini-Fracassini, D. and Rigau, J. (2004) AtPng1p.

The first plant transglutaminase. Plant Physiol. 135, 2046–2054.

Della Mea, M., Di Sandro, A., Dondini, L., Del Duca, S., Vantini, F., Bergamini, C., Bassi, R. and

Serafini-Fracassini, D. (2004) A Zea mays 39-kDa thylakoid transglutaminase catalyses the modifi-

cation by polyamines of light-harvesting complex II in a light-dependent way. Planta. 219, 754–764.

Dickinson, E. and Yamamoto, Y. (1996) Rheology of milk protein gels and protein-stabilized emulsion

gels cross-linked with transglutaminase. J. Agr Food Chem. 44, 1371–1377.

Dieterich, W., Ehnis, T., Bauer, M., Donner, P., Volta, U., Riecken, E. and Schuppan, D. (1997)

Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med. 3, 797–801.

Dolynchuk, K.N. and Bowness, J.M. (1999) Use of transglutaminase inhibitor for the treatment of scar

tissue. US Patent: US5885982.

Folk, J.E. (1969) Mechanism of action of guinea pig liver transglutaminase. VI. Order of substrate

addition. J Biol Chem. 244, 3707–3713.

Folk, J.E. and Chung, S.I. (1973) Molecular and catalytic properties of transglutaminases. Advances in

Enzymology. 38, 109–191.

Fox, B.A., Yee, V.C., Pedersen, L.C., Le Trong, I., Bishop, P.D., Stenkamp, R.E. and Teller, D.C.

(1999) Identification of the Calcium Binding Site and a Novel Ytterbium Site in Blood Coagulation

Factor XIII by X-ray Crystallography. J. Biol. Chem. 274, 4917–4923.

TRANSGLUTAMINASES 579

Gerrard, J.A., Fayle, S.E., Wilson, A.J., Newberry, M.P., Ross, M. and Kavale, S. (1998) Dough

properties and crumb strength of white pan bread as affected by microbial transglutaminase. J. Food

Sci. 63, 472–475.

Gerrard, J.A., Newberry, M.P., Ross, M., Wilson, A.J., Fayle, S.E. and Kavale, S. (2000) Pastry lift and

croissant volume as affected by microbial transglutaminase. J. Food Sci. 65, 312–314.

Green, H. and Dijan, P. (1996) Cosmetic containing corneocyte proteins and transglutaminase, and

method of application. US Patent: US5525336.

Grenard, P., Bates, M.K. and Aeschlimann, D. (2001) Evolution of transglutaminase genes: identi-

fication of a transglutaminase gene cluster on human chromosome 15q15. Structure of the gene

encoding transglutaminase X and a novel gene family member, transglutaminase Z. J Biol Chem. 276,

33066–33078.

Griffin, M., Casadio, R. and Bergamini, C.M. (2002) Transglutaminases: nature’s biological glues.

Biochem J. 368, 377–396.

Griffin, M., Cortez, J.M. and Bonnes, P. (2003) Method for enzymatic treatment of textiles such as

wool. US Patent: US2003154555.

Haejung, A., Peters, M.Y. and Seymour, T.A. (1996) Roles of endogenous enzymes in surimi gelation.

Trends Food Sci and Tech. 7, 321–327.

Horiguchi, Y. (2001) Escherichia coli cytotoxic necrotizing factors and Bordetella dermonecrotic toxin:

the dermonecrosis-inducing toxins activating Rho small GTPases. Toxicon. 39, 1619–1627.

Huang, Y.P., Seguro, K., Motoki, M. and Tawada, K. (1992) Cross-linking of contractile proteins from

skeletal muscle by treatment with microbial transglutaminase. J Biochem (Tokyo). 112, 229–234.

Ichinose, A., Bottenus, R.E. and Davie, E.W. (1990) Structure of transglutaminases. J Biol Chem. 265,

13411–13414.

Josten, A., Haalck, L., Spener, F. and Meusel, M. (2000) Use of microbial transglutaminase for the

enzymatic biotinylation of antibodies. J Immunol Methods. 240, 47–54.

Kamiya, N., Doi, S., Tominaga, J., Ichinose, H. and Goto, M. (2005) Transglutaminase-mediated protein

immobilization to casein nanolayers created on a plastic surface. Biomacromolecules. 6, 35–38.

Kanaji, T., Ozaki, H., Takao, T., Kawajiri, H., Ide, H., Motoki, M. and Shimonishi, Y. (1993) Primary

structure of microbial transglutaminase from Streptoverticillium sp. strain s-8112. J Biol Chem. 268,

11565–11572.

Kashiwagi, T., Yokoyama, K., Ishikawa, K., Ono, K., Ejima, D., Matsui, H. and Suzuki, E. (2002)

Crystal Structure of Microbial Transglutaminase from Streptoverticillium mobaraense. J. Biol. Chem.

277, 44252–44260.

Kobayashi K, Kumazawa, Y., Miwa, K. and Yamanaka, S. (1996) epsilon-(gamma-glutamyl)lysine

cross-links of spore coat proteins and transglutaminase activity in Bacillus subtilis. FEMS Microbiol.

Let. 144, 157–160.

Kuraishi, C., Yamazaki, K. and Susa, Y. (2001) Transglutaminase: its utilization in the food industry.

Food Rev. Internat. 17, 221–246.

Larre, C., Denery Papini, S., Popineau, Y., Deshayes, C., Desserme, C. and Lefebvre, J. (2000)

Biochemical analysis and rheological properties of gluten modified by transglutaminase. Cereal Chem.

77, 121–127.

Liu, S., Cerione, R.A. and Clardy, J. (2002) Structural basis for the guanine nucleotide-binding activity

of tissue transglutaminase and its regulation of transamidation activity. Proc Natl Acad SciUSA.

99, 2743–2747.

Lorand, L. and Conrad, S. (1984) Transglutaminases. Mol. Cell. Biochem. 58, 9–35.

Lorenzen, P.C., Schlimme, E. and Roos, N. (1998) Crosslinking of sodium caseinate by a microbial

transglutaminase. Nahrung. 42, 151–154.

Mammone, T. and Popescu, L. (2004) Method for curl retention in hair and lashes. World Patent

Application WO 2004/052279A3.

Mangala, L.S., Arun, B., Sahin, A.A. and Mehta, K. (2005) Tissue transglutaminase-induced alterations

in extracellular matrix inhibit tumor invasion. Mol Cancer. 4, 33.

Matheis, G. and Whitaker, J.R. (1987) A review: enzymatic cross-linking of proteins applicable to foods.

J Food Biochem. 11, 309–327.

580 ARRIZUBIETA

Matsumura, Y., Chanyongvorakul, Y., Kumazawa, Y., Ohtsuka, T. and Mori, T. (1996) Enhanced

susceptibility to transglutaminase reaction of alpha- lactalbumin in the molten globule state. Biochim

Biophys Acta. 1292, 69–76.

McDevitt, J.P. and Winkler, J. (1999) A method of treating wool, wool fibers or animal hair with a

proteolytic enzyme and a transglutaminase. World patent: WO9960200.

Megata, H. (2004) Epidermis keratinization normalizing agent and skin care preparation for external use

for skin containing the same. Japan patent: JP2004091376.

Meusel, M. (2004) Synthesis of hapten-protein conjugates using microbial transglutaminase. Methods

Mol Biol. 283, 109–123.

Motoki, M. and Seguro, K. (1998) Transglutaminase and its use for food processing. Trends Food Sci

and Tech. 9, 204–210.

Nemes, Z., Marekov, L.N., Fesus, L. and Steinert, P.M. (1999) A novel function for transglutaminase

1: Attachment of long-chain omega -hydroxyceramides to involucrin by ester bond formation. Proc

Natl Acad SciUSA.96, 8402–8407.

Nemes, Z. and Steinert, P.M. (1999) Bricks and mortar of the epidermal barrier. Exp Mol Med. 31, 5–19.

Nielsen, P. (1995) Reactions and potential industrial applications of transglutaminase. Review of liter-

ature and patents. Food Biotech. 9, 119–156.

Noguchi, K., Ishikawa, K., Yokoyama, K., Ohtsuka, T., Nio, N. and Suzuki, E. (2001) Crystal Structure

of Red Sea Bream Transglutaminase. J. Biol. Chem. 276, 12055–12059.

Nonaka, M., Toiguchi, S., Sakamoto, H., Kawajiri, H., Soeda, T. and Motoki, M. (1994) Changes caused

by microbial transglutaminase on physical properties of thermally induced soy protein gels. Food

Hydrocolloids. 8, 1–8.

Osullivan, M., Kelly, A. and Fox, P. (2002) Effect of transglutaminase on the heat stability of milk: a

possible mechanism. J Dairy Sci. 85, 1–7.

Pasternack, R., Dorsch, S., Otterbach, J.T., Robenek, I.R., Wolf, S. and Fuchsbauer, H.-L. (1998)

Bacterial pro-transglutaminase from Streptoverticillium mobaraense. Purification, characterisation and

sequence of the zymogen. Eur J Biochem. 257, 570–576.

Pedersen, L.C., Yee, V.C., Bishop, P.D., Le-Trong, I., Teller, D.C. and Stenkamp, R.E. (1994) Trans-

glutaminase factor XIII uses proteinase-like catalytic triad to crosslink macromolecules. Protein Sci.

3, 1131–1135.

Pedersen, M.H., Hansen, T.K., Sten, E., Seguro, K., Ohtsuka, T., Morita, A., Bindslev-Jensen, C. and

Poulsen, L.K. (2004) Evaluation of the potential allergenicity of the enzyme microbial transglutaminase

using the 2001 FAO/WHO Decision Tree. Mol Nutr Food Res. 48, 434–440.

Piacentini, M., Rodolfo, C., Farrace, M. and Autuori, F. (2000) “Tissue” transglutaminase in animal

development. Int J Dev Biol. 44, 655–662.

Pisano, J., Finlayson, J. and Peyton, M. (1968) Cross-link in fibrin polymerized by factor 13: epsilon-

(gamma-glutamyl)lysine. Science. 160, 892–893.

Ragkousi, K. and Setlow, P. (2004) Transglutaminase-mediated cross-linking of GerQ in the coats of

Bacillus subtilis spores. J Bacteriol. 186, 5567–5575.

Richardson, N.K., Schilling, K.M., Polcalyko, D.J. and Bailey, P.L. (1996) Covalent bonding of active

agents to skin, hair or nails. US Patent: US5490980.

Sakamoto, H., Kumazawa, Y., Toiguchi, S., Seguro, K., Soeda, T. and Motoki, M. (1995) Gel strength

enhancement by addition of microbial transglutaminase during onshore surimi manufacture. J. Food

Sci. 60, 300–304.

Sarang, Z., Molnar, P., Nemeth, T., Gomba, S., Kardon, T., Melino, G., Cotecchia, S., Fesus, L. and

Szondy, Z. (2005) Tissue transglutaminase (TG2) acting as G protein protects hepatocytes against

Fas-mediated cell death in mice. Hepatology. 42, 578–587.

Sardy, M., Odenthal, U., Karpati, S., Paulsson, M. and Smyth, N. (1999) Recombinant human tissue

transglutaminase ELISA for the diagnosis of gluten-sensitive enteropathy. Clin Chem. 45, 2142–2149.

Seguro, K., Kumazawa, Y., Ohtsuka, T., Ide, H., Nio, N., Motoki, M. and Kubota, K. (1995) epsilon-

(gamma-glutamyl)lysine: hydrolysis by gamma-glutamyltransferase of different origins, when free or

protein bound. J. Agr Food Chem. 43, 1977–1981.

TRANSGLUTAMINASES 581

Seguro, K., Kumazawa, Y., Kuraishi, C., Sakamoto, H. and Motoki, M. (1996) The epsilon-(gamma-

glutamyl)lysine moiety in crosslinked casein is an available source of lysine for rats. J Nutr. 126,

2557–2562.

Serafini-Fracassini, D., Del Duca, S. and Beninati, S. (1995) Plant transglutaminases. Phytochemistry.

40, 355–365.

Steinert, P., Marekov, L. and Nemes, Z. (2000) Methods for ameliorating icthyosiform skin diseases.

World patent: WO0078937.

Tanaka, T., Kamiya, N. and Nagamune, T. (2004) Peptidyl linkers for protein heterodimerization

catalyzed by microbial transglutaminase. Bioconjug Chem. 15, 491–497.

Villalobos, E., Santos, M., Talavera, D., Rodriguez-Falcon, M. and Torne, J.M. (2004) Molecular cloning

and characterization of a maize transglutaminase complementary DNA. Gene. 336, 93–104.

Yee, V., Pedersen, L., Le Trong, I., Bishop, P., Stenkamp, R. and Teller, D. (1994) Three-dimensional

structure of a transglutaminase: human blood coagulation factor XIII. Proc Natl Acad SciUSA.91,

7296–7300.

Yee, V.C., Pedersen, L.C., Bishop, P.D., Stenkamp, R.E. and Teller, D.C. (1995) Structural evidence that

the activation peptide is not released upon thrombin cleavage of factor XIII. Thrombosis Research.

78, 389–397.

Yokoyama, K., Nio, N. and Kikuchi, Y. (2004) Properties and applications of microbial transglutaminase.

Appl Microbiol Biotechnol. 64, 447–454.

Zotzel, J., Pasternack, R., Pelzer, C., Ziegert, D., Mainusch, M. and Fuchsbauer, H.-L. (2003) Activated

transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the

final step. Eur J Biochem. 270, 4149–4155.

CHAPTER 33

PENICILLIN ACYLASES

DAVID W. SPENCE

∗

AND MARTIN RAMSDEN

GlaxoSmithKline, North Lonsdale Road, Ulverston, United Kingdom

∗

dave.w.spence@gsk.com

1. INTRODUCTION

The production of penicillin and cephalosporin antibiotics is a multi thousand

tonne industrial operation. Benzyl and phenoxymethyl penicillins (penicillin G and

penicillin V respectively) are fungal fermentation products and the precursors to a

wide range of semi-synthetic antibiotics (amoxicillin, ampicillin etc.). The chemical

modification of the fermentation product is initiated by removal of the natural acyl

group leaving the 6-aminopenicillanic acid (6-APA) penicillin nucleus. Alternative

synthetic acyl groups can then be added to confer novel properties to the antibiotic

such as resistance to stomach acid, a certain degree of penicillinase resistance or

an extended range of antibiotic activity. The method of choice for the conversion

to 6-APA at the industrial scale is the use of penicillin acylase. Penicillin acylases

or amidases (EC 3.5.1.11) are a group of enzymes which can cleave the acyl chain

of penicillins to yield 6-amino penicillanic acid (6-APA) and the corresponding

organic acid (Shewale and Sivaraman, 1989), and in a number of cases the same

enzyme can be used to direct the synthesis of the new antibiotic by the addition of

the novel acyl group (Fig. 1). Apart from their use in modifying -lactams, these

enzymes also have applications in peptide synthesis and the resolution of racemic

mixtures of chiral compounds.

2. BIOLOGICAL SOURCES OF PENICILLIN ACYLASES

Penicillin acylases (PAC) are microbial enzymes and are found in a wide range of

bacteria, actinomycetes, yeasts and fungi (Suddhakaran and Borkar, 1985 a,b). Based

on their substrate specificities (side chain preferences), they have been classified into

three groups: penicillin G acylases, penicillin V acylases and ampicillin acylases.

583

J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 583–597.

584 SPENCE AND RAMSDEN

Antibiotic R

Penicillin G

Penicillin V

Ampicillin

Penicillin K

Penicillin dihydro F

Penicillin F

(CH

)

COOH

Penicillin acylase

COOH

R -COOH

6-amino penicillanic acid (6-APA)

COOH

COOMe

6-APA

Penicillin

acylase

COOMe

7-ADCA

Penicillin

acylase

COOH

Ampicillin

(X=H)

Amoxicillin

(X=OH)

Cephalexin Cefadroxil

(X=H) (X=OH)

(CH

)

Reactions of Penicillin Acylase

Figure 1. Reactions of Penicillin Acylase

The most abundant, the penicillin G acylases, are found in species including

Escherichia coli (Cole, 1969), Alcaligenes faecalis (Verhaert et al., 1997) and

Arthrobacter viscosus (Ohashi et al., 1989). Penicillin V acylases are found in species

including Bacillus subtilis (Olsson et al., 1985) and Fusarium oxysporium (Lowe

et al., 1986), whilst ampicillin acylases are found in Streptomyces lavendulae (Torres

et al., 1999) and Pseudomonas melangenum (Kim and Byun, 1990). It should be noted

however, that the enzymes from very few of the producing strains are actually used as

commercial biocatalysts.

PENICILLIN ACYLASES 585

3. ORGANISATION, EXPRESSION AND REGULATION

OF THE ACYLASE GENE (PAC)

The regulation of penicillin G acylase synthesis has been extensively studied in

E. coli. This is a good model organism since it is believed that penicillin acylases

from diverse sources are processed in a similar manner. There are three major

regulatory mechanisms involved in acylase synthesis: induction by phenylacetic

acid (PAA), catabolite repression and thermoregulation.

The expression of the wild type E. coli acylase gene (pac) is induced by PAA

and repressed by glucose, these effects being exerted at the transcriptional level

(Merino et al.,, 1992). It had been suggested that a pac-specific repressor protein,

encoded by a putative pacR gene transcribed in the opposite direction to pac from

within the pac coding sequence, is the mediator of the induction (Jiang et al., 1997).

However, lacZ fusion studies have shown that an intact pac gene is not required for

PAA induction (Rao and Garcia, 1999). Catabolite repression has been implicated

by the presence of putative cAMP receptor protein (CRP)–binding sites upstream

of the pac gene (Valle et al., 1986). It was also reported that the CRP is directly

involved in PAA induction and gene expression (Rao and Garcia, 1999).

PAC is normally produced at temperatures between 20



C and 30



C. It is not

produced at 37



C. It is not thought that lack of activity is due to thermal inactivation

of the protein since the optimal enzyme activity is 50



C (Savidge and Cole, 1975).

It is more likely that temperature–sensitive proteolytic processing is involved in the

maturation of the active enzyme (Oh et al., 1987). In addition, improper folding

at the higher temperature results in enzyme inactivity (Lindsay and Pain, 1991).

The processing steps involved in the formation of the active peptide subunits are:

1) synthesis of the precursor polypeptide (prepro PAC) which contains a signal

peptide,  subunit, spacer peptide and  subunit all encoded by the pac gene;

2) transport of the precursor peptide to the periplasmic space and signal peptide

removal (pro PAC); 3) proteolytic digestion of the spacer by cleavage at the N

terminus of the  peptide; 4) removal of the spacer peptide by C terminal proteolytic

processing. A process model has recently been proposed (Ignatova et al., 2005) from

in vitro folding studies which suggests that an initial fast phase of global burial of

hydrophobic residues leads to a stable molten globule–like intermediate state which

contains substantial secondary structure with bound calcium ions. This calcium ion,

which is found in the mature native enzyme, is responsible for stabilisation rather

than contributing directly to protein folding. In the final protein the calcium is

found at the junction of the two chains which coordinate it (Kasche et al., 2003).

Western blot analysis (Dai et al., 2001) has shown that at elevated temperatures

> 30



C the pac gene is transcribed and translated but the precursor polypeptide

is incorrectly folded resulting in an inactive enzyme. The catabolite repression and

PAA induction detailed for E. coli can vary in other organisms. For example, in

the enterobacteria Providencia (Proteus) rettgeri the pac gene is not induced by

PAA and is repressed by succinate and fumarate rather than glucose (Daumy et al.,

1982); in Alcaligenes faecalis the gene is induced by PAA but not regulated by

586 SPENCE AND RAMSDEN

catabolite repression. However, the temperature–dependence of the synthesis of the

enzyme appears to be an invariable parameter in all organisms.

4. PROTEIN STRUCTURE AND REACTION MECHANISM

Penicillin acylase is a member of the N terminal nucleophilic (Ntn) hydrolase

structural superfamily of proteins, a class of enzymes which share a common fold

around the active site and contain a catalytic serine, cysteine or threonine at the

N–terminal position (Brannigan et al., 1995). PAC has an acyl-binding site which

accommodates a PAA group and a nucleophile-binding site which can bind 6-APA

or another nucleophile (Fig. 2). It acts through the  chain terminal serine alone

to bring about its reaction. The reaction mechanism involves the formation of a

covalent intermediate and is similar to that of the serine proteases. After an attack

COOH

PAA

6-APA

Proposed mechanism of Penicillin Ac

lase catal

sed h

drol

sis of Penicillin G

Figure 2. Proposed mechanism of Penicillin Acylase catalysed hydrolysis of Penicillin G