University of Groningen

Production and utilization of peptides in Lactococcus lactisFang, Gang

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:2002

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):Fang, G. (2002). Production and utilization of peptides in Lactococcus lactis. s.n.

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 30-04-2022

1

CHAPTER I

Production and Utilization of Peptides in Lactococcus lactis

Gang Fang, Bert Poolman, and Wil N. Konings

INTRODUCTION

General introduction Lactic acid bacteria, defined by the characteristic production of lactic acid as a majorproduct of carbohydrate fermentation, are widely used in the food industry. Lactococci,which belong to the lactic acid bacteria, play crucial roles in mesophilic starters in the dairyindustry. These starters are used in the production of Dutch-type cheeses, Cheddar, blue andwhite mould cheeses, cottage cheese, butter, sour cream, and dour milk. One essentialrequirement for industrial fermentation of milk is the rapid lowering of the pH by theproduction of lactic acid by the starter organisms. This can be achieved by rapid growth ofthe starter bacteria in milk to high cell densities. However, the amounts of free amino acidsand peptides in milk are only sufficient to support 20% of the maximal growth yield (Millsand Thomas, 1981). For optimal growth, the lactococcal cells depend on their proteolyticsystem which provides them with peptides and amino acids, mainly from the hydrolysis ofmilk proteins such as caseins (Juillard et al., 1995b). Proteolysis is the most complex and perhaps the most important biochemical event duringthe maturation of most cheese varieties. The extent of proteolysis varies from very limited(e.g., Mozzarella) to very extensive (e.g., blue mold varieties), and the products range fromlarge polypeptides, comparable in size to intact caseins, through a range of medium andsmall peptides, to free amino acids. Proteolysis is responsible for softening of the texture ofcheese during the early stages of ripening. It also influences to some extent the developmentof cheese flavour via the formation of amino acids and peptides. Amino acids can alsoindirectly affect the flavour development by serving as precursors for numerous flavourcompounds. Proteolysis also affects the mouthfeel of cheese and the release of flavourcompounds from cheese during mastication. The inability of lactic acid bacteria to synthesizemany of the amino acids required for protein synthesis necessitates the active functioning ofa proteolytic system in environments where proteins constitute the main nitrogen source.Biochemical and genetic analysis of the pathway by which exogenous proteins supplyessential amino acids for growth has been one of the most actively investigated aspects ofthe metabolism of lactic acid bacteria, especially in those species that are of importance inthe dairy industry, such as the lactococci. Much information has now been accumulated onindividual components of the proteolytic pathway in lactococci: the cell envelope-boundproteinase, amino acid and peptide transport systems and a range of intracellular peptidases.In this introduction, an updated overview of the physiology and biochemistry of theproteolytic system is given. It mainly focuses on the stepwise degradation of caseins, the(molecular) mechanisms of peptide transport and the substrate specificities of the

2

transporters involved. More detailed information on the genetics of the proteolytic systemand the structural properties of the proteolytic enzymes can be found in a series of reviews(Kok and de Vos, 1994; Poolman et al., 1995; Kunji et al., 1996; Konings et al., 2000;Detmers et al., 2001).

Proteolytic system of Lactococcus lactis Several metabolic activities of lactic acid bacteria, i.e., fermentation and depletion of themilk sugar lactose, reduction of the redox potential, citrate fermentation and degradation ofmilk proteins (caseins), serve special functions which directly or indirectly have an impacton processes such as flavour development and ripening of dairy products. In milk most ofthe amino acids and peptides are present in free form at low concentrations. Nitrogen istherefore a growth limiting factor and the production of lactic acid is slow unless starter cellshave proteolytic enzymes to degrade the milk proteins (caseins) to peptides and free aminoacids (Poolman et al., 1995). The process of casein degradation and utilization requires theconcerted actions of proteinase, peptidases, and amino acid and peptide transport systems.This whole set of enzymes is termed the proteolytic system (Konings et al., 1989). In the proteolytic system of Lactococcus lactis (Fig. 1), the proteinase PrtP is involved inthe initial degradation of caseins, yielding a large number of different oligopeptides. Toutilize amino acids for biosynthesis, these degradation products have to be transported acrossthe cell membrane. There are a number of transport systems involved in these translocationprocesses. The lactococci possess at least 10 different amino acid transport systems, oneoligopeptide transporter (Opp) and two di- and tripeptide transporters (DtpP and DtpT).Following casein breakdown by the extracellular proteinase PrtP and uptake via peptidetransporters, the casein-derived peptides are hydrolysed further into amino acids byintracellular peptidases.

Proteinases of Lactic acid bacteria The enzymes involved in the degradation of milk proteins (caseins) can be divided into twoclasses: (i) the extracellular-cell wall bound proteinase (PrtP) and (ii) the intracellularpeptidases. The proteinase (PrtP) of lactic acid bacteria is the first enzyme in the pathwayof casein utilization (Laan et al., 1989; Smid et al., 1991; Kunji et al 1998). PrtP issynthesized as an inactive pre-pro-protein. The maturation protein PrtM catalyses theactivation after the excretion of the pro-protein across the cell membrane. Different cell wall-associated proteolytic activities are found in different lactococcus strains. These enzymesappear to be large proteins of approximately 180-190 kD with pH optima of around 5.5 to6.5 and isoelectric points around 4.5 (Hugenholtz et al., 1987; Laan and Konings, 1989).Ca2+ stabilizes or activates (at low concentrations) the enzymes, whereas specific serineproteinase inhibitors (phenylmethanesulphonylfluoride (PMSF) or di-isopropylfluorophosphate (DFP)) effectively inhibit the enzymes. On the bases of theirbiochemical properties, especially the degradation patterns towards caseins, two types ofproteinases have been initially distinguished in lactococci, which are designated as PI andPIII (Visser et al., 1986). The PI-type enzymes preferentially degrade β-casein and to a lesserextent κ-casein, while PIII-type enzymes degrade mainly αsI-, β- and κ-casein (Pritchard andCoolbear, 1993). The products resulting from the degradation of caseins by various proteinases have beenstudied in great detail (Monnet et al., 1986, 1989; Visser et al., 1988; Exterkate et al., 1990;Yamamoto et al., 1993; Juillard et al.,1995a; b; Kunji et al., 1998). The peptides obtained

3

Fig. 1: Model for the proteolytic system of Lactococcus lactis. The cell wall-boundproteinase (PrtP), three peptide transport proteins (Opp, DtpT, and DtpP), and 17intracellular peptidases are represented at their locations in the cell. Peptidases are classifiedon the basis of their cleavage specificity. (Data from Guedon et al., 2001).

from β-casein by the digestion of proteinases from L. lactis AM1 and SK11, NCDO763 andWg2, HP and H2 have been identified with HPLC-MS techniques (Juillard et al., 1995a;Kunji et al., 1998). The results show that β-casein is degraded into different oligopeptidesranging from 4 to 30 residues and that hardly detectable amounts of di- and tripeptides orfree amino acids are produced. The major peptide fractions in the hydrolysates are formedin the beginning of the degradation process, and these fractions originate from the C-terminalpart of β-casein. Although it is not possible to define unequivocally a consensus cleavagesite, thirteen bonds are split systematically by all lactococcal proteinases studied to date, andan additional six bonds, in particular the bonds next to glutamine and serine residues, arepreferentially hydrolyzed. The degradation by various proteinases of κ-, αsI- and αsII-caseins have also been studied, but only the major products have been identified so far(Monnet et al., 1992; Reid et al., 1994; Visser et al., 1986; 1994; Bockelmann et al., 1989).

4

These identified products are all oligopeptides and are derived mainly from the C-terminalregion of these caseins. Again no significant amounts of free amino acids and di- andtripeptides are detected in the hydrolysates.

Peptide and amino acid metabolism of lactic acid bacteria The casein-derived large peptides released by the proteinases need to be hydrolyzed furtherinto smaller peptides and amino acids. Considerable information indicates that this processis carried out intracellularlly and involves a number of peptidases. The peptidases isolatedso far from lactococci are aminopeptidases, proline-specific peptidases or endopeptidases.The possible pathways for the further degradation of the relatively large casein oligopeptidessupplied by proteinases can be: (i) cleavage of the oligopeptides by endopeptidases intosmaller peptides, and (ii) systematic cleavage of amino acids or dipeptides from the N-terminus of the oligopeptide by amino-peptidases or proline-specific peptidases. Thepeptidases can be divided according to their substrate specificities in a number of classes:general amino peptidases, proline-specific peptidases etc. These will be discussed below.

General aminopeptidasesAminopeptidase N The aminopeptidase N (PepN) is mainly involved in the metabolism of small oligopeptidesand is capable of cleaving N-terminal amino acids from a wide range of peptides differingboth in size and composition (Tan et al., 1990; 1993; Baankreis 1992). Generally, substrateswith a N-terminal lysine, leucine or arginine are cleaved preferentially, substrates with a N-terminal alanine, phenylalanine or methionine are considerably less favoured, and substrateswith a N-terminal glycine, proline and glutamine are hardly or not cleaved (Tan et al., 1990;1993; Miyakawa et al., 1992; Niven et al., 1995).Aminopeptidase C The aminopeptidase C (PepC) has a specificity profile different from aminopeptidase N.PepC has significant activity towards substrates with basic (Arg, His, and Lys), acidic (Gluand Asp), hydrophobic/uncharged (Ala and Leu) and aromatic (Phe) residues (Klein et al.,1994). Hydrolysis has been also reported for a variety of di- and tripeptides with unchargedor basic residues in the amino terminal position. The hydrolytic efficiency is highlydependent on the length of the peptide, optimal for tetrapeptides and further enhanced by thepresence of hydrophobic residues in the P1 positions of the substrate (Mistou and Gripon1998).Dipeptidases There are two types of dipeptidases: dipeptidase V (PepV) and dipeptidase D (PepD).PepV has a distinctly different pH optimum from PepD (8 vs 6), quaternary structure(monomer vs octomer) and catalytic properties (metallopeptidase vs thiolpeptidase). Thespecificity of PepV is strictly confined to dipeptides, since none of the tested tri- andoligopeptides are hydrolysed. Some preference of PepV for N-terminal hydrophobic residueshas been observed, whereas Pro-, Glu-, Gly- or Asp-containing dipeptides are hardly or notat all hydrolysed (van Boven et al., 1988; Tan et al., 1995). The dipeptidase PepD from Lb.helveticus has a substrate specificity similar as PepV (Dudley et al., 1996; Vesanto et al.,1996). However, PepD from L.lactis cannot hydrolyze Val-X (-Arg, -Gly, -Leu) and Ile-X(-Gln, -Val) dipeptides, while other dipeptides with hydrophobic/uncharged N-terminalresidues are hydrolyzed. Growth studies and activity measurements indicate that PepD doesnot play a significant role in the liberation of essential amino acids from milk-derived

5

peptides (Vesanto et al., 1996).Tripeptidases Tripeptidase T (PepT) is capable of cleaving a wide range of tripeptides composed ofhydrophobic/uncharged, aromatic, basic, acidic, and sulphur-containing residues (Bosmanet al., 1990; Bacon et al., 1993). Although PepT can hydrolyze tripeptides with Pro in firstor third position, it cannot cleave peptides containing Pro in second position. In accordancewith the strict substrate size specificity of PepT, the enzyme does not hydrolyze di-, tetra-,or larger oligopeptides.

Proline-specific peptidases A group of peptidases capable of hydrolyzing Pro-containing sequences have beenidentified. These peptidases are very important for the degradation of casein-derived peptidesbecause of the high content of proline in casein.Aminopeptidase P The aminopeptidase P (PepP) can liberate the N-terminal amino acids from oligopeptidesranging from three to nine residues containing X-Pro-Pro-(Y)n sequences (Mars and Monnet,1995). No hydrolysis of X-Pro dipeptides occurs.Prolidase The prolidase (PepQ) activity is defined by specificity for X-Pro dipeptides. The preferredX-Pro substrates contain N-terminal residues that are hydrophobic/uncharged (Ala-, Ile-,Leu-, Val-), basic (His-), aromatic (Phe-, Tyr-) or sulphur containing (Met-) (Kaminogawaet al., 1984; Booth et al., 1990; Morel et al., 1999). However, not all prolidases areexclusively X-Pro dipeptidases, nor do they hydrolyze all X-Pro substrates.Proline iminopeptidases Proline iminopeptidase (PepI) activity is defined by specificity for peptides with N-terminal proline. Hydrolytic activity has been reported for Pro-X substrates when the C-terminal residue is hydrophobic/uncharged (-Ala, -Gly, -Ile, -Leu, -Val), acidic (-Glu) oraromatic (-Phe, -Tyr) (Gilbert et al., 1994). In addition, this enzyme liberates proline fromthe N-terminus of a tripeptide, but apparently not from tetra- or penta-peptides (Baankreisand Exterkate 1991).Prolinase Prolinase (PepR) activity is defined by specificity for Pro-X dipeptides as well as by someactivities towards larger peptides and Pro-(ρNA, βNA) substrates. Hydrolysis of Pro-Xsubstrates has been reported for C-terminal residues that are hydrophobic/uncharged (-Ala,-Ile, -Leu, -Val), aromatic (-Phe) or sulphur containing (-Met) ( Shao et al., 1997).Hydrolysis of X-Pro dipeptides has not been reported.X-prolyl-dipeptidyl aminopeptidase The X-prolyl-dipeptidyl aminopeptidase (PepX) liberates X-Pro dipeptides from the N-terminus of peptides. The highest activities reported for PepX with X-Pro-ρNa substrates arewith those where the N-terminal residues are uncharged (Ala-, Gly-) or basic (Arg-). PepXis also capable of hydrolyzing Pro-Pro-(X)n substrates, but little or no hydrolysis is observedfor X-Pro-Pro (including when X is Pro) (Booth et al., 1994b; Miyakawa et al., 1991; 1994;Sanz and Toldra, 2001). In addition to being a peptidase, PepX has amidase and esteraseactivity (Houbart et al., 1995; Yoshpe-Besancon et al., 1994).

Unique aminopeptidasesGlutamyl aminopeptidase

6

The glutamyl aminopeptidase (PepA) is capable of hydrolyzing N-terminal acidic aminoacid residues from di-, tripeptides and oligopeptides consisting of up to ten amino acidresidues (Exterkate and de Veer, 1987c; Niven, 1991; Bacon et al., 1994). Hydrolysis of Glu-and Asp-X dipeptides is observed when the C-terminus amino acid is basic (-Lys), uncharged(-Gly), hydrophobic/uncharged (-Ala, -Leu), polar/uncharged (-Ser), or aromatic (-Phe, -Tyr).Pyrrolidone carboxylyl peptidase The pyrrolidone carboxylyl peptidase (PCP) activity is defined by its specificity forcleaving N-terminal pyrrolidone carboxylyl residues of peptides and proteins (pGlu-(X)n)(Exterkate, 1977).Leucyl aminopeptidase The leucyl aminopeptidase (PepL) hydrolyzes preferentially N-terminal leu-containingdipeptides and some tripeptides (Klein et al., 1995).

EndopeptidasesEndopeptidase E The endopeptidase E (PepE) is capable of hydrolyzing Met-enkephalin (Tyr-Gly-Gly-Phe-Met) at the Gly-Phe- bond and Bradykinin (Arg-Pro-Gly-Phe-Ser-Pro-Phe-Arg) at the Gly-Phe- bond (Fenster et al., 1997). No activity was reported with β-casomorphin, n-benzoyl-Val-Gly-Arg-ρNa, or with Arg-, Phe-, Pro-, Lys-, Gly-, or Val-ρNa.Endopeptidase O The endopeptidase O (PepO) is reported to hydrolyze oligopeptides ranging in length fromfive to thirty five residues (Tan et al., 1991; Pritchard et al., 1994; Stepaniak and Fox 1995;Lian et al., 1996). Although PepO hydrolyzes several large casein derived fragments, thenative caseins are not detectable degraded.Endopeptidase F The endopeptidase F (PepF) is reported to hydrolyze oligopeptides ranging in length fromseven to seventeen residues (Monnet et al., 1994). However, there is a clear preference forsubstrates of about eight or nine residues as indicated by the rates of hydrolysis. Nohydrolysis was observed for ACTH f1-24, glucagon (29 residues) or insulin β chain (30residues), suggesting the upper size limit for substrates is less than twenty-four residues.Additionally, native caseins are not detectable hydrolyzed.

Carboxypeptidases No carboxypeptidases have been identified so far in lactic acid bacteria.

Location and physiological role of peptidases The proteinase PrtP is located at the external surface of the cells, while all peptidasesidentified in LAB seem to be located intracellularly. Signal membrane anchor sequences arenot found in any of the peptidases which is consistent with an intracellular location. Thecapacity of the Opp transport system to transport peptides ranging from penta-peptides topeptides with more than 35 residues indicates that there is no need for the further degradationof the PrtP released peptides by extracellular peptidases (Kunji et al., 1998; Detmers et al.,1998; Lanfermeijer et al., 1999; 2000). After transport of the casein-derived peptides further hydrolysis results in the productionof amino acids which can be utilized for processes such as protein synthesis, generation ofmetabolic energy, and recycling of reduced cofactors. The physiological role of individual

7

peptidases has been investigated in numerous single and multiple peptidase mutants(Christensen and Steele 1996; Mierau et al., 1996; Matos et al., 1998; Yuksel and Steele1999). The growth rate of peptidase mutants in milk has been measured and found todecrease especially when more than one peptidase is inactivated. However, the basis for thisreduction in growth rate has not been analysed. In addition, little information is availableregarding the potential role of the peptidases in cellular protein turnover modification.

Amino acid catabolism The catabolism of amino acids in LAB contributes significantly to the quality and safetyof fermented foods. Amino acid catabolism by anaerobic/fermentative microorganisms alsoplays an important role in the conservation of metabolic energy in nutrient-limitedenvironments. Although the catabolic pathways for many amino acids are only poorlycharacterized, the relationships between these pathways and the production of compoundsbeneficial for human health have received intensive attention from industry.Histidine catabolism Histidine decarboxylase in L. helveticus plays an essential role in the conversion ofhistidine to histamine. This process has an important physiological function in the generationof metabolic energy and the regulation of the intracellular pH (Molenaar et al., 1993). Theproduction of the biogenic amine, histamine in fermented food can cause food poisoning andtherefore has significant implications for health and food safety (Doelglas et al., 1967;Taylor et al., 1982).Arginine catabolism In LAB there are two pathways for the catabolism of arginine. The first one produces nitricoxide and citrulline from arginine by a nitric oxide synthase (Morita et al., 1997). In thesecond pathway the catabolism of arginine occurs via the arginine deiminase (ADI) pathway(Manca de Nadra et al., 1982; Konings et al., 1995). This pathway results in the conversionof arginine into ammonia, ornithine, carbon dioxide, and leads to the production of 1 ATPper arginine converted. The arginine deiminase pathway is the only known catabolic pathwayfor amino acids in LAB that produces ATP via substrate level phosphorylation. Theproduction of ATP from arginine can supply the energy for growth in the absence ofcarbohydrate, while the production of ammonia raises the pH and thereby enhances thesurvival at low medium pH-values (Crow and Thomas 1982; Marquis et al., 1987). Argininecatabolism during wine production can result in the production of precursors of ethylcarbonate which is a known carcinogen and affects human health (Liu and Pilone 1998).Glutamate catabolism Three enzymes are known to be involved in Glu catabolism in LAB. The first twoenzymes, Glu-aminotransferase and Glu-dehydrogenase, result in the production of α-ketoglutarate from Glu. The third enzyme, Glu-decarboxylase, can produce γ-aminobutyrate(GABA) and this conversion results in the generation of metabolic energy and increase ofthe intracellular pH. Glu-dehydrogenase also plays a crucial role in the eye formation incheese ( Zoon and Allersma 1996; Sanders et al., 1998).Aspartate catabolism Three aspartate catabolic pathways are found in LAB. The Asp-aminotransferase catalyzesAsp transmission with α-ketoglutarate and is most likely involved in the last step of aspartatebiosynthesis (Paulus 1993; Christensen et al., 1999). Asp-decarboxylase is involved in thedecarboxylation of Asp to Ala and this pathway yields metabolic energy and increases theintracellular pH (Konings et al., 1995; Abe et al., 1996). Lastly, aspartase catalyses the

8

reversible reaction: Asp⇔ fumarate + NH+4. The physiological role of this enzyme is

unknown (Rollan et al., 1985).Threonine catabolism Threonine catabolism in LAB is initiated by threonine aldolase, which converts Thr toacetaldehyde plus Gly (Lees and Jago 1976; Marshall and Cole 1983). It is generallybelieved that the physiological role of the threonine pathway lies in the production of Gly(Reiter and Oram 1962). The production of acetaldehyde contributes to the development oftypical yogurt flavour (Law 1981; Tamine and Seeth 1980).Branched-chain amino acid catabolism The catabolic pathways for branched-chain amino acids (BCaas) in LAB are involved inthe conversion of Leu, Ile and Val to their corresponding α-ketoacids by aminotransferase,which subsequently are converted into aldehydes by decarboxylases, alcoholes bydehydrogenases or fatty acids by unknown enzymes (Nakae and Elliott 1965;Tucker andMorgan 1967; Hummel et al., 1985). The physiological role of the catabolism of branched-chain amino acids is unclear. The major concerns of dairy industry relate to the effects ofBCaa catabolism on the production of aroma or flavour (Sheldon et al., 1971; Griffith andHammond 1989; Brennand et al., 1989).Aromatic amino acid catabolism The catabolism of aromatic amino acids is in most LAB initiated by an aminotransferase,converting Trp, Phe and Tyr to indole-pyruvate, phenyl-pyruvate and p-OH-phenyl-pyruvate,respectively (Nakazuma et al., 1977; Hummel et al., 1984; Gao et al., 1997). Furtherdegradation of these pyruvates to skatole, lactate, acetate or aldehyde compounds is carriedout by unknown enzymes (Fei et al., 1997). Tyr and Trp can also be converted bydecarboxylases to tyramine and tryptamine, respectively. These pathways have somephysiological roles in the regulation of intracellular pH and the generation of metabolicenergy (Konings et al., 1989; Straub et al., 1995; Masson et al., 1996; Roig-sagues et al.,1997). Aromatic amino acid catabolism is responsible for the occurrence of objectionable(unclean) flavours in the production of fermented dairy products (Dunn and Lindsay 1985;Milo and Reineccius 1997). The production of biogenic amines, such as tyramine, has humanhealth implications, as tyramine is the common cause of monoamine intoxication. This ischaracterized by an increase in blood pressure that results in palpitations, headaches,hypertension, nausea, vomiting and prostration (Voight et al., 1978; McCabe 1986).

Amino acid transport systems There are at least 10 amino acid transport systems in LAB. Growth experiments in minimalmedium supplemented with amino acids indicated that these transport systems are activeenough to allow growth of L.lactis on amino acids as sole source of nitrogen. Three typesof transport systems have been identified on the basis of the energy requirement (Koningset al., 1989). The amino acid transport systems for Ala/Gly, Ser/Thr, Leu/Val/Ile and of Met,His and Lys are driven by the proton motive force (Otto et al., 1982; Driessen et al., 1987;Driessen et al., 1989). The amino acid transport systems for Glu/Gln, Asn and Pro/Glycine-Betaine are driven by hydrolysis of ATP (Konings et al., 1989; Poolman 1993). The thirdtype of amino acid transport system is the antiporter for arginine and ornitine. Thiselectroneutral Arg/Orn exchange is driven by the concentration gradient of both solutes(Konings et al., 1989).

Oligopeptide transport system (Opp)

9

The oligopeptide transport system (Opp) in L. lactis belongs to the superfamily of ATP-Binding Cassette (ABC) transporters, which typically have an architecture of one bindingprotein, two integral membrane domains and two ATP-binding domains (Higgins, 1992).Genetic and biochemical analysis revealed that there are five open reading frames encodingthe five polypeptides of Opp, namely OppA (peptide binding protein), OppB and OppC(integral membrane domains), OppD and OppF (ATP-binding domains) (Tynkkynen et al.,1993). In contrast to the limited knowledge on the structure-function relationships in themembrane domains of ABC-transporters, considerable information is available for thestructure and specificity of the substrate binding proteins (Tame et al., 1994; Dunten andMowbray, 1995; Kunji et al., 1998; Detmers et al., 1998; Lanfermeijer et al., 2000). On thebasis of the tertiary structure of the homologous OppA protein from S. typhimurium, a three-domain structure is very likely for OppA from L. lactis. Domain I and III would be involvedin the binding of substrates, while domain II is typical for binding proteins belonging to theclass of peptide-ABC transporters but its function is unknown (Tame et al., 1994). Opp of S. enterica can transport tri-, tetra- and penta-peptides. The 3-D structure of thecorresponding OppA has indicated that the peptide backbone binds through hydrogen bondsand salt bridges, while the side chains of the peptide are accommodated in voluminoushydrated pockets with limited contribution to the binding affinity. The water molecules playroles as flexible adapters, matching the hydrogen-binding requirements of the protein andthe substrate, and shielding charges on the buried substrate (Tame et al., 1994). The N-terminal residue of the tri-, tetra- or penta-peptide is bound to a negatively charged Asp419by forming a hydrogen bond, while the C-terminal residue of a tri-, tetra- and penta-peptideare bound to the positively charged Arg413, His371 or Lys307, respectively. A similarprinciple applies to the binding of dipeptide to the dipeptide binding protein DppA of E. coli.The main difference between OppA from S. typhimurium and DppA from E.coli is thereplacement of His371 and Lys307 (interacting with tetra- and penta-peptides, respectively)in OppA by Ala361 and Tyr299 in DppA, respectively (Dunten and Mowbray, 1995). Thepositive charged residues important for peptide binding to OppA and DppA of entericbacteria are not conserved in OppA and DppA from L. lactis (Sanz et al., 2000; Detmers etal., 2001). OppA from S. typhimurium does not seem to bind peptides longer than five or sixresidues. The binding of even longer peptides by OppA from L.lactis and the transport ofthese long peptides by the Opp system has been studied extensively (Detmers et al., 2000;Lanfermeijer et al., 1999; 2000). In vivo studies indicate that Opp in E. coli and S.typhimurium can transport peptides of 3 to 6 amino acids. Transport studies with peptidesreleased from β-casein by the action of the proteinase PrtP demonstrated that peptides withup to at least 10 amino acid residues can be transported in vivo by Opp from L.lactis (Kunjiet al., 1998). Further studies, using synthetic peptides, showed that Opp of L. lactistransports peptides of at least 18 amino acid residues (Detmers et al., 1998). Moreover,binding studies with purified OppA showed that peptides with up to 35 amino acid residuesstill interact strongly with the protein (Detmers et al., 2000; Lanfermeijer et al., 2000). Thebinding of peptides to OppA from L. lactis involves the enclosure of the first six N-terminalresidues in the binding pocket, whereas the remaining C-terminal residues of the peptidestick out and interact with the surface of the binding protein. OppA has low binding affinityfor peptides of 3 to 5 residues. The affinity increases with increasing length of the peptide(optimal peptide length is nine residues), indicating that the interactions of the C-terminalresidues of the peptide contribute significantly to the affinity (Detmers et al., 2000;Lanfermeijer et al., 2000). Peptide binding to OppA from L. lactis has some resemblance to

10

that of OppA from S. typhimurium, but the following differences are notable: (i) OppA ofL. lactis binds peptides up to at least 35 residues; (ii) a free N-terminal amino group is notso critical for binding in OppA from L. lactis as in OppA from S. typhimurium or E.coli, orin DppA from E. coli or L. lactis; (iii) the first six N-terminal residues are enclosed withinOppA whereas the remaining C-terminal residues interact with the surface of the bindingprotein; and (iv) peptide binding to OppA from L. lactis lacks the interaction of the carboxylgroup of the peptide with a positively charged residue (Detmers et al., 1998; 2000;Lanfermeijer et al., 2000).

Di/tripeptide transport system (DtpT) In addition to the ABC-type Opp systems, a new family of proton motive force-drivenpeptide transport systems, called the PTR-family has been discovered (Steiner et al., 1995).These newly identified secondary peptide transport proteins are not only found in bacteria(Hagting et al., 1994; Nakajima et al., 1997), but also in rabbit, chicken, rat, hamster, pig andhuman (Daniel et al., 1992; Thamotharan et al., 1996; Liang et al., 1995; Perry et al., 1994;Jamai et al., 1994). PTR- transporters are, without exceptions, encoded by a single gene withthe signature motif, F- (S,T,N,M)-X-F-(V,Y,F)-h-X-I-N-h-G-(S,A)-h (h indicates ahydrophobic residues), present in the fifth putative membrane spanning segment. On thebases of hydropathy analysis in combination with the ‘positive inside rule’, the PTR-transporters are predicted to have 12 α-helical transmembrane segments (TMS). Membranetopology studies on DtpT from L. lactis confirmed this structural model and indicated furtherthat both the N- and C-termini are located intracellularly (Hagting et al., 1997; Covitz et al.,1997). On the bases of pairwise comparisons of the amphipathicity of PepT1 from human,a model for this protein was proposed in which the TMSs with the highest amphipathicityare forming a central channel (Lee et al., 1999). A key result emerging from these studieswas the arrangement of the polar residues containing TMSs 7, 8, 9 and 10 (Fig. 2). All threepairwise comparisons (7-8, 8-9, and 9-10) resulted in defined orientations of the helices withthe polar residue R282 (TMS7), D341 (TMS8) and E595 (TMS10) orienting towards thecenter of a ‘half-channel’ created by the four TMSs. The remaining helices are organized inpart on the basis of their hydrophobic character. The solvent excluded surface created by thetwelve helices of PepT1 (Ferrin et al., 1988) form a cavity in the center of the putativechannel that would be large enough to accommodate a dipeptide molecule. The entry fromthe extracellular side to this channel is restricted by W294, Y588, and E26. In the center ofthe channel are Y12, E595, D341, and Y91. At the cytoplasmic side of the channel are Y167and R282, which may regulate the exit of the peptide from the channel (Fig. 3). In contrast to the limited information on the tertiary structure of the proteins, significantknowledge has been obtained on the substrate specificities of these peptide transporters(Döring et al., 1998; Fang et al., 2000). DtpT from L. lactis has the following specificitycharacteristics: (i) the affinities constants for peptides vary from µM to mM; (ii) both freeamino and carboxyl terminus are important for high affinity interaction with DtpT; (iii) anamino group and peptide bond nitrogen located in the α position are important for highaffinity interaction with the protein; (iv) peptides with a trans peptide bond interact withhigher affinities than those in the cis configuration; (v) the highest affinities are observedwith L-α-amino acid isomers at both amino- and carboxyl- termini; (vi) hydrophobic aminoacids located at the P1 position of the dipeptide increase the affinity; (vii) charged aminoacids located at the P2 position of a dipeptide or at the P2 or P3 position of a tripeptidedecrease the affinity; (viii) dipeptides have higher affinities than analogous tripeptides; (ix)

11

Fig. 2: Model of the arrangement of the twelve transmembrane segments of PepT1 (topview). The area in the center represents the putative channel (Data from Lee et al., 1999).

hydrophobic peptides have higher affinities than hydrophilic peptides; (x) in most cases,cationic peptides have significantly lower affinities than neutral or anionic ones; (xi) Phe orPhe-containing di- or tripeptides with a carbobenzoxy group at the amino terminus competeeffectively for uptake with Pro-[14C]Ala; (xii) free amino acids and peptides with a backboneof more than three amino acid residues are not accepted as substrates. (xiii) ω-amino fattyacid compounds are not substrates. The substrate specificity of DtpT from L. lactis is morerestricted than that of the mammalian transporters PepT1 or PepT2; for instance, PepT1 andPepT2 accept ω-amino fatty acids (ω-AFA) as substrates. Other differences between DtpT

12

Fig. 3: Model of the putative transmembrane channel of PepT1 (side view). Only the keyresidues are shown (Data from Lee et al., 1999).

and Pep T1/T2 are: (i) in DtpT the basic and acidic amino acids at the C-terminus of thepeptide strongly reduce the affinity, whereas no significant reduction in affinity is observedwhen these charged amino acids are located at the N-terminus; (ii) DtpT is more selectivefor L-isomers at the N-terminal position than PepT2; (iii) DtpT is more selective fordipeptides than for tripeptides, whereas PepT2 has very similar affinities for both.

Di/tripeptide transport system (DtpP) A third peptide transport system (DtpP) has been discovered in L. lactis by mutant analysis(Foucaud et al., 1995). Inhibitor studies indicated that ATP drives this transport system.Recent genome analysis revealed an operon coding for a putative peptide transporter, whichbelongs, like Opp, to the ABC superfamily. The gene encoding the binding protein (DppA)

13

of this system shares only 30%, 25%, and 30% identity with OppA from S. typhimurium,OppA from L.lactis, and DppA from E.coli, respectively. Instead of transportingoligopeptides as Opp, this system only accepts di- and tripeptides. Kinetic and substratespecificity studies (Sanz et al., 2000) revealed the following properties for the bindingprotein DppA: (i) a high affinity for di- and tripeptides; (ii) a requirement for a free N-terminal α-amino group and an α-peptide bond contiguous with the N-terminal amino group;(iii) stereospecificity for L-isomers; (iv) a preference for dipeptides containing methionineor arginine and hydrophobic tripeptides consisting of leucine or valine residues. In contrastto DtpT, DtpP does not show a clear preference for either di- or tripeptides.

Outline of this thesis Lactococci have a limited capacity to synthesize amino acids and are therefore dependenton the utilization of exogenous nitrogen sources for optimal growth. When growing in milk,these organisms degrade milk proteins (caseins) to fulfil their needs for amino acids. Thefirst step in the utilization of milk proteins is the degradation of these proteins tooligopeptides by extracellular proteinase (PrtP). Next, peptides are taken up by theoligopeptide transport system (Opp). In the third step, the intracellularly accumulatedpeptides are degraded by a multitude of peptidases. In this thesis, some of the unresolvedissues regarding the proteolytic system in lactococci have been addressed via in vivo and invitro approaches.Following breakdown by the proteinase and/or uptake, the casein-derived peptides need tobe hydrolysed further by peptidases. The degradation efficiency of these peptides bypeptidases will affect the growth of the lactococci in milk. The sequence of events in theproteolytic pathway has been studied by analyzing the degradation of β-casein in vivo,following the uptake of the formed peptides from the extracellular medium as well as theappearance of peptides and amino acid intracellularly (Chapter II). The di- and tripeptide transport protein DtpT is unique among bacterial peptidetransporters, as it is encoded by a single gene and uses the proton motive force to drive thetransport of di- and tripeptides. DtpT of L. lactis is used as a model system to study thestructure-function relationship of this type of peptide transporters. As a first step towardscharacterization of the DtpT protein, we developed an efficient single step purificationmethod for this protein. Ni2+-affinity chromatography of histidine-tagged DtpT is followedby reconstitution into preformed, detergent destabilised liposomes. The functional propertiesof the purified protein in the liposomal membranes are presented (chapter III). The substrate specificity of bacterial peptide transporters has been analysed in growthexperiments with amino acid auxotrophs and using peptides with different sizes, but also intransport studies with a reporter substrate and competing peptides. The disadvantage of thetransport studies in whole cells is that the transported peptides are instantly broken down bythe peptidases, and efflux of amino acids takes place. After successful reconstitution of theDtpT protein into detergent-destabilized preformed liposomes, this type of studies can bedone more accurately. In the proteoliposomes, various parameters can be modified andstudied selectively without being influenced by other enzymatic and transport activities. Inthe present studies, we have analyzed the kinetics and substrate specificity of the bacterialpeptide transporter DtpT in proteoliposomes using Pro-[14C]Ala as reporter peptide. Theinhibition constant (IC50) was determined for peptides with different side-chains at the 1st,2nd or 3rd position and/or configuration of the peptide backbone as well as peptidemimetics(Chapter IV). Also in vivo transport assays were carried out with well-defined mutants to

14

explore the possible role of DtpT in the proteolytic pathway. The results have contributedtowards our understanding of the functional mechanism of peptide transport via DtpT(Chapter V).