ELSEVIER FEMS Microbiology Reviews 18(1996) 3 19-344 Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 Bacterial : Properties and functions

Thierry Gonzales, Janine Robert-Baudouy *

Luhorutoire de Ghznttique Mole’culaire des Microorganismes et des Interactions Cellulaires. C.N.R.S. UMR 5577, Institut Natronal des Sciences Applique’eu. B&iment 406. 20 Avenue Albert Einstein. 69621 Villeurbnnne cede.x, France

Received 1 November 1995; revised 18 March 1996; accepted 28 March 1996

Abstract

Aminopeptidases are that selectively release N-terminal amino residues from polypeptides and . Bacteria display several aminopeptidasic activities which may be localised in the cytoplasm, on membranes, associated with the cell envelope or secreted into the extracellular media. Studies on the bacterial aminopeptide system have been carried out over the past three decades and are significant in fundamental and biotechnological domains. At present, about one hundred bacterial aminopeptidases have been purified and biochemically studied. About forty encoding aminopeptidases have also been cloned and characterised. Recently, the three-dimensional structure of two aminopeptidases, the methionine from Escherichiu coli and the leucine aminopeptidase from Aeromorzas proteo/uica, have been elucidated by crystallographic studies. Most of the quoted studies demonstrate that bacterial aminopeptidases generally show Michaelis-Menten kinetics and can be placed into either of two categories based on their specificity: broad or narrow. These can also be classified by another criterium based on their catalytic mechanism: metallo-, - and -aminopeptidases, the former type being predominant in bacteria. Aminopeptidases play a role in several important physiological processes. It is noteworthy that some of them take part in the catabolism of exogenously supplied and are necessary for the final steps of turnover. In addition, they are involved in some specific functions, such as the cleavage of N-terminal methionine from newly synthesised chains (methionine aminopeptidases), the stabilisation of multicopy ColEI based plasmids (aminopeptidase A) and the pyroglutamyl aminopeptidase (Pep) present in many bacteria and responsible for the cleavage of the N-terminal pyroglutamate.

Keywords: Aminopeptidase; Classification; Catalytic mechanism; Location; Function

Contents

1. Introduction...... 320

2. Biochemical properties of bacterial aminopeptidases...... 320 2.1. Classification ...... 320 2.2.Structure ...... 321 2.2.1. Quatemary structure...... 321 2.2.2. Crystallographic studies ...... 32 1

* Corresponding author. Tel: + 33 72 43 83 31; Fax: + 33 72 43 87 14: E-mail: [email protected] .fr

0168.6445/96/$32.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V PI/ SOl68-6445(96)00020-4 320 T. Gonzales, J. Robert-Baudouy/FEMS MicrobiologyReviews 18 (1996) 319-344

2.3. Enzymatic mechanisms ...... 326 2.3.1. The metallo-aminopeptidases ...... 326 2.3.2. Cysteine and serine aminopeptidases ...... 327 2.4. Enzymatic properties ...... 328 2.5. Substrate specificity ...... 328

3. Cellular location and regulation of bacterial aminopeptidases ...... 329 3.1. Cellular location ...... 329

3.2. Regulation of synthesis ...... 330 Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 3.2.1. Transcriptional control ...... 330 3.2.2. Other mechanisms of regulation...... 331

4. Physiological role of bacterial aminopeptidases...... 331 4.1. Utilisation of exogenous peptides as ...... 331 4.2, Degradation of intracellular proteins and peptides ...... 332 4.2.1. Protein turnover ...... 332 4.2.2. The role of peptidases in the degradation of endogenousproteins ...... 333 4.2.3. Recognition of proteins to be catabolised ...... 333 4.3. Protein maturation ...... 334 4.4. Other known functions ...... 335

5. Conclusions...... 335

Acknowledgement...... 336

References ...... 336

1. Introduction 2. Biochemical properties of bacterial aminopepti- dases Aminopeptidases are enzymes that catalyse the cleavage of residues at the N-terminal 2. I. Classification position of peptides and proteins. These enzymes are found widely distributed amongst both procaryotic Peptidases, i.e. enzymes that catalyse the degrada- and eucaryotic types. The first studies on bacterial tion of relatively large peptide fragments, may be aminopeptidases, stimulated by both basic and ap- classified into two groups: and ex- plied interests, were carried out over 30 years ago. opeptidases. The first group comprises enzymes that Some bacterial peptidase systems are of considerable cleave peptide links within the polypeptide chain, interest to agro-industries such as the dairy industry whereas the second consists of enzymes that cleave (for reviews see [ 1-lo]) while others may be studied amino acid residues at the extremities of the in fundamental research (for a review see [l11) or in polypeptide. Enzymes in the latter group may be bacterial taxonomy (for a review see [12]). These either , which free an amino acid studies have allowed the characterisation of a wide residue at the C-terminal end of the polypeptide, or number of aminopeptidases at the biochemical, aminopeptidases, which free an amino acid residue at molecular and physiological levels. the N-terminal end of the polypeptide (Fig. 1). The objective of this review is to summarise The properties of the main bacterial aminopepti- current knowledge concerning both the fundamental dases studied to date are summarised in Table 1. In properties of bacterial aminopeptidases and their po- addition, certain enzymes which are not aminopepti- tential physiological functions. dases in the strict sense of the term, such as dipep- T. Gonzales, J. Robert-Baudouy/ FEMS Microbiology Reriews 18 (1996) 319-344 321

buried within the protein structure while also reduc- ing the contact surface with the medium, thus limit-

H,N--Xl -x2- x3-x4-x5 -x6-coon ing the quantity of necessary to stabilise these

Fig. I Peptidases classification. Endopeptidases cleave peptidic proteins [ 151. bounds inside polypeptides (1). Exopeptidases cleave residues To date, only two examples of bacterial amino- located at the N-terminal position (2, aminopeptidases) or C- peptidase displaying hetero-multimeric structures are terminal position (3, carboxypeptidases) of polypeptide. known. The 397-kDa aminopeptidase APMy of Mv-

coplasma salivarium (Table 1, family 2) is formed Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 by the association of two monomers of 46 and 50 tidy1 aminopeptidases (which cleave a dipeptide) as kDa [ 161. The API aminopeptidase of Bacillus well as di- and tri-peptidases (which react on di- or stearothermophilus (Table 1, family 31 is a 400-kDa tri-peptides only) have been included. While this enzyme which appeared a priori to be composed of work aims to be as complete as possible, only en- 12 identical sub-units of 36 kDa [ 171. However, zymes on which a minimum number of studies have N-terminal amino acid sequence determination re- been carried out are quoted. vealed the existence of two distinct sub-unit types Different classification systems exist for which share 67% homology [ 181. This high degree of aminopeptidases. The most frequently used classifi- homology would suggest that the two monomer types, cation parameters include substrate specificity, cellu- termed cr and p, are coded for by phylogenetically lar location, catalytic function, requirement for co- related genes. In addition, it was demonstrated that factors and pH optimum [ 13,141. In this biblio- three isoforms of this enzyme exist in vivo, distin- graphic study, bacterial aminopeptidases were guished by different o//3 ratios [ 191, and that both grouped together into families using the following types of sub-unit possess catalytic activity [ 181. Such criteria: substrate specificity; physico-chemical and complexity of structure may be due to the substrate enzymatic properties (catalytic function, molecular specificity of the CYand p sub-units: the (Y sub-unit weight, pH optimum, etc.) and peptide sequence is specific for non-charged amino , while the p similarity (where the corresponding genes have been sub-unit preferentially cleaves acid residues [ 181. The charactet-ised and sequenced). Enzymes classed into resulting hetero-multimer therefore possesses a the same family based uniquely on physico-chemical broadened substrate specificity. However, the B. and enzymatic properties are not necessarily phylo- stearothermophilus aminopeptidase appears to be genetically related. atypical of bacterial aminopeptidases.

2.2. Structure 2.2.2. Crystallographic studies Recently crystals of different aminopeptidases 2.2.1. Quaternary structure have been obtained [20-241, and the three-dimen- Almost half (47%) of the 102 bacterial aminopep- sional structures of two bacterial aminopeptidases tidases described to date are monomers (Table 1). resolved. The methionine aminopeptidase from Es- This monomeric structure is found mainly amongst cherichia coli (Table 1, family 7) is a monomeric aminopeptidases secreted into the external environ- metallo-peptidase which contains two ions ment. The remaining 53% display a multimeric struc- (Co’+ 1 and shows a new fold without homology ture (Table 1). In general, the quaternary structure is with any other proteolytic enzymes [25,26]. The two due to the combination of a number of pairs of the co?+ are situated in the backbone of the protein, at same sub-unit type: enzymes of 2, 4 or 6 monomers two double p anti-parallel sheets which partly con- are the most common. Aminopeptidases of this type tain the of the enzyme. The leucine show for the most part Michaelis-Menten kinetics, aminopeptidase of Aeromonas proteolytica (Table 1, however, the advantage of a multimeric structure, as family 2) is also a monomeric metallo-enzyme which opposed to several monomeric proteins, remains ob- is composed of a unique globular a//3 domain scure. It is possible that the formation of a quater- [27,28]. The active site contains two ions (Zn2’) nary structure allows hydrophobic regions to be situated closely together. The two metal ions appear 322 T. Gonzales, J. Robert-Buudouy/ FEMS Microbiology Reviews 18 C19961319-344 Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 x0-x 2

8

Am,“” pept1daw hnrad Zn melalloen7ylnc IS6 (4 x 3x 41 h-7 [IXSI Amano peptidav I broad (A\p. Ala) Ill~l~ll0LWylllC 450 (4 x I 13) 7.5 [IX61 Am,no peptldaw I, broad (Leu) mctnlloenrymc 81 (I x XI) x2 [IXhl Arylamidarc broad metalloen7ymc 170(1 x 170) 7 o-7 2 Cl871 Amino pepttdaw broad (Ala) Vl&lllO~ll~y~lW I70 (‘J) YS ISl.lXXl

broad (Leo. I.y\. (X6. IX‘).I YOI Met) broad x-s [IYII hn,ad 7 5-Y 4 ,nducl,on a, h,gh [17- IY.YX.IYZ] tempcralure (55°C) broad ImrlalloenLymr 100 (2 x 46.S) 7 [4S.l931

hrrrad (Al.,. Leu) mrlalloen7ymc 120 (4 x 80) h.7 7s (521

hmad cy*tc,nr rntyme 300 (6 x SO) 7 JO [66.‘)31

cy\te,nr enzyme 300 I6 x SO) 7 SO cyrtc,nc cnrymc “(N x Sl)

ry\re,ne enlymr ” (N X 49) ?

cyainc cnrymc “(N x 51)

cy\,e,ne en7yme 300 (6 x 49 4) 7.5 ‘! Family 5: Other cy\tetnr amlnoprptldasc\ with hrnad spcc!T~sty XcAP Xarlrko,,ro,,~r

D - AP O< hrohcr~~rra,,,

MAP Es~hrrrchlo ‘,Oli Co metallocnryrne 29 (I x 29) uytoplawl (26.X31 MAP Scrlnwrr~llo rn~lzimrrurtm Co ~metalloen7ymr 35.5 (I x 3S.S) cytopl~wl [I43.IYXl MAP Ba~illrtr .suhrtlir ” (N X 27) [ ,041 Family 8: Aspartameamlnopeptldase Amino peptidar A L<. I<,‘,,,up. ()‘Y,,1,,)‘,1 metallocnrymc I30 (1 x 43) X-Y so-55 mner membrane [I’)‘)]

Amino pcptidarc A L‘. IN< ,,r \\p. I‘,‘,,,, mctellornrymc ?JS(bX41) x 6S wluhle [441

~Wl~ll0~!l~ylllC 43lJ (IO x 43) [ZOO]

cy\,c,ne entynw 41 (2 x 2.3) [21)1~2031 c)*,c,nc enrynw 74(‘) [961

cy*te,ne enlyme 91 (4 x 24) (?OJ-2061

cy\teinr rnryme 4s (2 x 30) [xl] Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 September 24 on guest by https://academic.oup.com/femsre/article/18/4/319/529845 from Downloaded Table I (continued)

Name Bacteria Specificity Catalytic Molecular mass Optimal Location Regulation of References family (kDa) (number condmons synthesis and mass of for acttwty cub-untts) P” temperature (“a

Pyroglutamylpeptidase Bacillus amsloliquefaciun,s pGludX cyateme enzyme 51 or72 6.5-8 0 45-50 soluble , [2 I .207,208] (2-4 X 23) PCP Streprococcus fueriunl pGluJX - cystane enzyme 80 (2 X 42) 76 ? soluble Do91 ‘7 -PCP Srreprococcus pyo@wrs pGluJX - cysteine enzyme 85 (4 X 23) 7 30 soluble [210,2ll] Family 10: Arginine aminopeptidases Arginine aminopeptidase Srreprncoccus songais A&X - cyhteine enzyme ?(N X 170) 7.2 ? nnner mb aa,ociated ? [971 “-p Esrhrrirhiu co/i A&X - ? ‘, ? (N X 37.9) , ? membranes ‘1 [9Ol Family 11: Aminopepttdnse P

PepP-l Eschrrichirr co/i XJPro - metalloenzyme 3507 8 55 cytoplasm ? [84.212]

PepP - II Escherichia co/i XdPro - Zn-metalloenryme 210 (4 x 50) 9 41 cytoplasm ? [84,212,2131 1 PepP Srreppromycu,s lil idrrns XJPro - 100 (2 X 54) ‘1 cytoplasm 0141 Family 12: Prolme lminopeptldaaes PIP Buci//us mrguterium Pr0JX - senne enzyme 6O(l X 60) 7 ‘? \oluble [2l51 PIP Bacillus coagulanr PIOJX serine enzyme 33(1 x 33) 8 40 soluble ‘1 [20,216,217] PIP Arromorros sohrio ProdX renne enzyme 192(4X48) 8.5 55 soluble ‘! [217,21X] PIP Nciswrra ,qmorrhouw ProJX - wine enzyme ? (N X 34,5) ‘, ? soluble u54 promoter [lo61 -. Proltneiminopeptldase Lb. drlhrwckii asp. huly~ricrn ProJX - ProdX - Y xrine enzyme 100 (3 X 33) 6.0-7.0 ‘J cytoplasm and ? [219,2201 cell envelope Prolinelminopeptida*e Lb drlhrueckii ssp. Ir~rrs ProJX - berine enzyme ? (N X 33) , ‘? cytoplasm [741 PepPN Lb hvlr rticw ProdX - ProjX - Y senne enzyme ? (N X 35) I2211 PIP? Brrrihucrrriurn sp. E53 I ProJX - GlydX-AlaJX - ‘P 250 (6 X 43) 7.5-8.0 ? soluble D221 Di- and tnpepttdase Lc. lurris ssp. cremorrr ProJX - Prodx - Y metalloenryme” I IO (2 x SO) 8.5 37 cytoplasm [531 Family 13: Dipeptidyl aminopeptidase X - ProDPAP(PepX) Lc. lacris ssp. lacrrs X-ProJY - X-AladY - sernne enzyme 170 (2 X 88) 6-9 40-45 cytoplarm ‘, [223-2261

X - ProDPAP(PepX) Lc. loctb asp. cremorir X-Pr”,lY X-AlaJY - \erlne enzyme 180 (2 x 88) 6.5-8.0 45-50 cytoplasm. lnductmn [69,94,227]

+/- I” late mner mb exponential ascociated phase X ProDPAP(PepX) Lb. delhrurrku ssp. lrrcrrs X-ProdY - X-AladY swine enzyme 88 (I X 88) 7 46-50 cytoplasm ? [711 X-Pro DPAP Lb. drlhrurckit spp hul~orics\ X-ProJY X-ALadY - sertne enzyme 82 (I x 82) 7 50 cytoplasm constitutive 12281 X-Pro DPAP (PepX) Lb. dulhrueckii FIN. hulgoricus X-Pr”,lY - X-AladY - sertne enzyme 200 (2 or 3 x 95) 6.5 45-50 soluble , [229,230] X-Pro DPAP Lb. acidophilus X-ProJY - X-AladY - erine enzyme 200 (2 x 95) 6.5 45 soluble 1 D291 X-Pro DPAP Lb helrrricus X-ProJY - X-AladY - wine enzyme 72(1 X 72) 7 40 soluble ? I2311 X-Pro DPAP Lb. cusei asp. cosei X-ProJY - X-AlaJY - wine enzyme 79(l x 79) 7 0 & 9.5 50 soluble ? [2321 Dipeptidyl peptldase IV Flu nhuctvrium meninyo- X-ProdY - X-AladY - wine enzyme 160(2X75) 7.4-7.8 45 cytoplasm, ? [IO01 svpticum +/- inner mb associated X-Pro DPAP X-ProJY - X-AladY xnne enzyme 160 (2 X 80) 6.5-8 0 45 soluble [233] Dnpeptldyl peptidax IV X-ProJY - X-AladY rerine enzyme 120 (2 x 53) 6.0-8.7 ? *oluble I2341

Dlpepttdyl peptidaae II x-YjZ - *erine enzyme ? (N X 37) 7 52 extracellulal [235] Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 September 24 on guest by https://academic.oup.com/femsre/article/18/4/319/529845 from Downloaded Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021

[bCZ1 EE 85 [802’66’951 $5 S’L

[LPZ'YPZI i. S’S

d k [ElI‘SSl <, (SPX OSP

[OPZ’ZZI i, S’L-O’L (SZ x I) SZ ChCZl i. (SZ X N) i. [Of] t. S’LPX’Y (i) XSI

IL-S x i) 001 (Lt x Z) t-6 326 T. Gonzales, J. Robert-Baudouy/ FEMS Microbiology Reviews 18 f 19%)319-344 to form a co-catalytic unit and fulfil equivalent func- central part of these polypeptides. These domains tions in . Hence these two metallo-amino- exhibit a high degree of conservation amongst the peptidases possessing very different structures clearly five bacterial Peps and are likely to be involved in belong to two distinct families of proteolytic en- their biological activity. The second domain, contain- zymes [28]. ing a cysteine residue, appears to be functionally By contrast, strong structural homologies between important and may form part of the catalytic site of the E. coli methionine aminopeptidase and a creati- these enzymes. This has been confirmed by site-di-

nase (a catalysing a very different enzy- rected mutagenesis of the B. amyloliquefuciens [21] Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 matic reaction) from Pseudomonas putida, have been and P. fluorescens genes [252]. The comparison of demonstrated [29]. Even though peptide sequence these results could lead to phylogenetic analysis of similarities between these two are very Peps. low, the authors concluded that the genes coding for these enzymes had a common origin. Furthermore, 2.3. Enzymatic mechanisms the mechanisms of catalysis of these two enzymes are also distinct: the aminopeptidase being a Bacterial aminopeptidases may be sub-divided into metallo-enzyme whereas the creatinase is not. Also, three main catalytic groups based on their sensitivity the peptide sequence residues that fix Co’+ in the to various inhibitors as follows: metallo-amino- former enzyme are not conserved in the P. putidu peptidases, whose activity is regulated by the pres- creatinase [29]. Two distinct mechanisms are cur- ence of divalent metallic cations (inhibition by rently proposed to explain how enzymes may acquire chelating agents e.g. EDTA, l,lO-phenanthroline, new functions. The most commonly proposed is that etc.); cysteine aminopeptidases (sensitivity to Hg’+, an active site may evolve in order to catalyse a new iodoacetamide, N-ethylmaleimide, p-chloro- reaction. In this case, the basic principles of the mercuribenzoate) and serine aminopeptidases (sensi- mechanism of catalysis remain more or less un- tivity to phenylmethylsulfonyl fluoride and diiso- changed. The second hypothesis suggests that a new, propyl fluorophosphate). totally different active site can be formed indepen- In addition, sensitivity towards certain inhibitors dently of the first site. The E. coli aminopeptidase of microbial origin may help define sub-groups within and the P. putida creatinase provide the first exam- each catalytic type [31]. These inhibitors include: ple of enzymes possessing a common ancestor but bestatin and (metallo-enzymes); antipaine probably having diverged by the second mechanism and compound E-64 (cysteine enzymes); leupeptine 1301. (cysteine and serine enzymes) and chymostatin For bacterial pyrrolidone carboxyl peptidases (serine enzymes). (Peps), many biochemical and enzymatic studies have been carried out (Table 1). In addition, some of them 2.3.1. The metallo-aminopeptidases have recently been genetically characterised [250]. The metallo-aminopeptidases constitute the largest We cloned the pep genes from two Gram-positive group of aminopeptidases comprising two-thirds of bacteria, [210] and B. sub- these enzymes (Table 11. Even though the ionic tilis [205], and from the Gram-negative bacterium, co-factor has seldom been characterised, Znzt ap- P. fluorescens [203]. Two other pep genes have also pears to be the most frequently associated cation. been cloned, one from B. amyloliquefuciens [21] the Amongst the Zn*+ metallo-aminopeptidases, two other from [25 11. phylogenetically unrelated sub-groups may be distin- The five bacterial pep genes characterised to date guished. appear to have a common structure. These genes The first comprises mainly of the PepN encode polypeptides of 2 15 or 2 13 amino acids with aminopeptidases of E. coli and lactic acid bacteria similar deduced molecular weights. For the complete (Table 1, family 1). These enzymes contain at least analysis of these genes and the very conserved pro- one Zn’+ per monomer and all display the character- tein sequences refer to refs. [250] and [25 I]. istic peptide motif HExxH [32-371. Other enzymes, Two domains of 20 amino acids are located in the such as human N aminopeptidase, and , T. Gonzales, J. Robert-Baudouy/FEMS Microbiology Reviews 18 (1996) 319-344 321 an from B. stearothermophilus, also co-ordination bonds with certain enzyme residues as belong to this sub-group [37-391. Originally the E. well as with the oxygen molecule of the carbonyl coli N-aminopeptidase was classified as a cysteine group of the peptide link target of the substrate. In aminopeptidase, however, due to strong amino acid addition, certain cations can also participate in the sequence similarities it has been re-classed as a Zn*+ stabilisation of the three-dimensional structure of the enzyme displaying the characteristic HExxH motif enzyme. [35l.

The second sub-group of Zn*+ aminopeptidases 2.3.2. Cysteine and serine aminopeptidases Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 (Table 1, family 2) comprises enzymes that show Unlike metallo-aminopeptidases, cysteine and ser- peptide sequence similarities with bovine lens leucine ine aminopeptidases have no ionic co-factor associ- aminopeptidase [ 13,141. Amongst these are the E. ated with their structure. Catalysis requires a highly coli PepA [40] and the leucine aminopeptidase of A. reactive cysteine or serine residue and is found less proteolytica [41]. These enzymes display strong ther- frequently amongst aminopeptidases: each type rep- mostability, possess two closely located Zn*+ per resenting less than 20% of the bacterial aminopepti- monomer, and their activity is strongly inhibited by dases studied to date (Table 1). The characterised bestatin, a compound isolated from Streptomyces cysteine aminopeptidases comprise mainly enzymes oliL~oreticuli [ 13,141. of broad specificity, PepC-type, (cf. references Table A third type of catalysis requiring the presence of 1, family 4) or narrow specificity, Pep-type (cf. divalent cations has been recently identified for the references Table 1, family 9). Serine aminopeptidase E. coli methionine aminopeptidase (Table 1, family activity has only been detected for the D-aminopepti- 7 [26]). In contrast to zinc metallo-enzymes, this dase of Ochrobactrum anthropi ([57,58]; Table 1, enzyme contains two Co*+ ions per monomer. In family 12) the iminopeptidases family (cf. addition, other aminopeptidase enzymes which dis- references Table 1, family 13) and the X-proline play a strong increase in activity in the presence of dipeptidyl aminopeptidase family (cf. references Co’+ may have a similar type of catalysis. Such Table 1, family 13). enzymes include: the prolidase of Luctococcus lactis However, the mechanism of activity of these two ssp. cremoris (Table I, family 14 [43]); aminopepti- enzyme types is better understood than that of met- dase-A of Lc. lactis ssp. lactis (Table 1, family 8 allo-aminopeptidases [59-641. For both types, cat- [MI); aminopeptidase-II of B. stearothermophilus alytic reactions begin with a nucleophilic attack of (Table 1, family 3 [45]) and a from L. the sulphur of the sulphydril group (cysteine sake (Table 1, family 14 [46]). aminopeptidases) or of the oxygen of the hydroxyl It is possible that additional types of catalysis group (serine aminopeptidases), of the of the which depend on other metal cations, in particular carbonyl group involved in the substrate peptide Mn’+, exist. In fact Mn’+ is capable of both restor- bond target. The active site of these enzymes con- ing and/or increasing the enzymatic activity of some tains other residues that complete the metallo-aminopeptidases [47-541. Furthermore, the or tetrad [65] and their function consists mainly of peptide sequences of some metallo-aminopeptidases sufficiently polarising either the S-H link (cysteine (PepD and Iap from E. coli; PepT from Salmonella enzymes) or O-H link (serine enzymes) in order to typhimurium and Lc. lactis ssp. cremoris; carboxy- allow the initial nucleophilic attack. peptidase G2 from Pseudomonas) contain a peptide Like metallo-aminopeptidases, cysteine and serine motif of approximately 40 highly conserved amino aminopeptidases may also be sub-divided into differ- acids. These peptidases show no similarity at the ent sub-groups based on the organisation of their peptide sequence level with the other families of catalytic site. PepC-type cysteine aminopeptidases metallo-enzymes which have been characterised display peptide sequence similarities with regions [55,56]. surrounding the active site of some eucaryotic pro- Although the mechanism of catalysis of metallo- teases, e.g. and . Their aminopeptidases is still poorly understood [37,39, I 31, active sites could be composed of the tetrad ‘Gln + it has been demonstrated that the metal cations form Cys + His + Asn’ [65-681. Thus, Pep-type en- 328 T. Gonzales, J. Robert-Baudouy/ FEMS Microbiology Rel:iews 18 (1996) 339-344 zymes do not possess peptide sequence similarity monomers, each of 41 kDa, and displays a positive with the above enzymes and may belong to another, co-operation at the level of substrate fixation. as yet uncharacterised, class of cysteine enzymes. The optimum temperatures and pH for activity Bacterial serine aminopeptidases do not belong to vary considerably from one aminopeptidase to an- either of the two main serine proteolytic enzyme other (Table 1). pH optima are normally found in the families, represented by and [65]. range pH 6-9 and may in some cases extend over Nevertheless, peptide sequence analysis has revealed several pH units. This characteristic often differenti-

that both proline iminopeptidases and X-proline ates bacterial aminopeptidases from those of eucary- Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 dipeptidyl aminopeptidases contain a catalytic triad otic origin, which normally display a narrow pH composed of ‘Ser + His + Asp’. X-proline dipep- optimum. In addition, for the majority of extracellu- tidy1 aminopeptidases are characterised by the con- lar aminopeptidases the pH optimum is basic 1781. stant peptide motif, GxSYxG [69-711. Like prolyl The optimum temperature of reaction often reflects , proline iminopeptidases are charac- the optimum temperature for growth of the microor- terised by the motif GxSxGG, encompassing the ganism from which the aminopeptidase originated. catalytic serine residue [65,72-741. At the catalytic Aminopeptidases isolated from bacteria that grow at site level, the 0. anthropi D-aminopeptidase appears high temperatures, such as from B. to be even further removed from serine enzymes stearothennophilus or the aminopeptidase from Sul- such as trypsin and subtilisin than the X-proline folobus solfuturicus (cf. references Table 1, family 3) dipeptidyl aminopeptidases or proline iminopepti- generally show good thermostability and an elevated dases. In fact, this enzyme displays peptide sequence optimum temperature for activity. The molecular similarities with other hydrolases such as the DD- basis of this thermostability is still poorly understood carboxypeptidases and the @lactamases [58]. The but it appears that a few minor modifications, such catalytic activity of this category of enzymes seems as the stabilisation of the peptide extremities of the to rely on the tetrad ‘Ser + Lys + Ser + Glu’ par- protein, or the substitution of hydrogen links re- tially organised on the primary sequence on the quired to maintain the structure by higher energy peptide motif SxxK [58,65]. Here, lysine acts as a ionic links, may be responsible [79]. proton acceptor, a function normally carried out by 2.5. Substrate specificity in other families of serine enzymes [75]. Other serine proteins, e.g. the E. coli LexA [74] and Bacterial aminopeptidases may be divided into ‘signal peptidases’ [76,77] also possess a catalytic two categories on the basis of their substrate speci- lysine and may be placed in this family of enzymes. ficity. Aminopeptidases of broad specificity, capable of cleaving several different amino acids at the N- 2.4. Enzymatic properties terminal or P 1-positions (according to Schechter’s Even though numerous bacterial aminopeptidases nomenclature [80]), as well as aminopeptidases that possess a multimeric structure, the majority display cleave a single type of residue at the Pl-position, Michaelian saturation kinetics with regard to their may be distinguished. However, both these cate- substrate. The Michaelis constants (K, > vary con- gories of aminopeptidases display several character- siderably from one enzyme to another. Often K, istics in common: they lack endopeptidase and car- values close to 1 mM are observed, a characteristic boxypeptidase activity, with the exception of the E. of enzymes which have a weak or moderate affinity co/i N-aminopeptidase (Table 1, family 1) which for their substrate. It is also likely that bacterial possesses residual endopeptidase activity [8 1I; they aminopeptidases function in vivo at rates far inferior only hydrolyse residues with a free N-terminal ex- to their maximum rate. tremity, except for proline iminopeptidases and Peps To date the only aminopeptidase shown to ob- (cf. references Table 1, families 9 and 12) and they serve allosteric kinetics is aminopeptidase A from are stereospecific, the vast majority of them cleaving Lc. Zuctis ssp. Zuctis (Table 1, family 8) [44]. This amino acid L-forms only or in the case of the 0. metallo-enzyme, which is specific for acid residues anthropi aminopeptidase, the D-form (Table 1, fam- at the N-terminal position, is made up of six identical ily 6 [57]). This stereospecifity is especially pro- T. Gonzales, J. Robert-Baudouy/ FEMS Microbiology Reuiews 18 (19%) 319-344 329 nounced for the amino acid residue situated in the ciating different combinations of cations at the two Pl-position. fixation sites, may be obtained. The type of cation Broad spectrum aminopeptidases often demon- fixed at these two sites, not only regulates the ther- strate a marked preference for an amino acid or modynamic constants of the reaction (K,, K,,,) but group of amino acids in the Pl-position. For exam- also the primary specificity of the enzyme for its ple, the E. coli N-aminopeptidase hydrolyses pep- substrates. A similar mechanism was also demon- tides more rapidly with an alanine in the N-terminal strated for two other Zn2+ aminopeptidases; the B.

position [Sl] while the A. proteolytica leucine subtilis BSAP (Table 1, family 3 [86]) and the Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 aminopeptidase demonstrates a marked preference leucine aminopeptidase from S. griseus (Table 1, for leucine and hydrophobic amino acids in the family 2 [87-891). The presence of Co’+ for BSAP Pl-position [82]. Generally speaking, broad spectrum and Ca2+ for the leucine aminopeptidase modulates aminopeptidases hydrolyse few or no peptides con- both the catalytic activity and the specificity of these taining an acid residue (Asp, Glu, pGlu) in the enzymes. In addition, for both these enzymes, the PI-position, or a proline in positions Pl and P’l. regulatory cation possesses its own fixation site, This characteristic helps explain the existence of distinct from that of the catalytic cations. This type several aminopeptidase families displaying strict of regulation, using metal cations, may be used by specificity (aspartate aminopeptidases, Peps, proline bacteria to regulate the in vivo activity and speci- iminopeptidases, X-proline dipeptidyl aminopepti- ficity of metallo-aminopeptidases [86]. dases) and capable of cleaving peptide links which are resistant to broad spectrum aminopeptidases. However, the substrate specificity of an amino- 3. Cellular location and regulation of bacterial peptidase is not only determined by the amino acid aminopeptidases in the N-terminal position. Residues in the P’ l-posi- 3.1. Cellular location tion and downstream of the peptide sequence may also modulate this specificity. For example, the me- It has been demonstrated that aminopeptidase ac- thionine aminopeptidase (Table 1, family 71, which tivity is present in different cellular compartments displays strict specificity for the methionine in the (Table 1). Nevertheless, the majority of these en- Pl-position, only cleaves this amino acid if the zymes (97%) are found in soluble fractions, either in P’ l-position is occupied by either an Ala, Gly, Pro, the cytoplasm (65%), in the periplasm of Gram-nega- Ser or Thr. In fact, the presence of certain residues tive bacteria or embedded in the Gram-positive cell (Arg, Asn, Be, Leu, Lys, Phe) at the P’-1 position wall (16%), or secreted into the external environment abolishes the catalytic activity of this enzyme [83]. (16%). The A. proteoZytica leucine aminopeptidase Likewise, studies of specificity of the E. coli P is the only extracellular aminopeptidase for which aminopeptidase showed that the nature of the residues the nucleotide sequence of the corresponding situated in positions Pl, P’ 1, P’2, P’3 and P’4 influ- has been determined. The gene contains a signal ence the kinetic constants of the reaction (Table 1, sequence at the N-terminal end, which is a character- family 11 [84]). istic of exported proteins [41,42]. In addition, the specificity of metallo-amino- To date only two aminopeptidases, the E. coli Iap peptidases is equally a function of the type of diva- enzyme (Table 1, family 10 [90]> and the APMy of lent cation associated with the enzyme. This phe- M. salicarium (Table 1, family 2 [ 161) have been nomenon was studied for the A. proteolytica leucine shown to be true membrane proteins. The former aminopeptidase (Table 1, family 2 [SS]), a monomeric enzyme contains a classic signal sequence and its enzyme naturally possessing two Zn*+ ions. When activity is directed towards the periplasmic space. these two ions have been removed by extensive The latter is a membrane-bound enzyme which re- dialysis against an EDTA-containing solution, it is quires treatment with Triton-X-100 as well as papain possible to restore the catalytic activity of the apoen- in order to extract and solubilise it from zyme with different metal cations, namely Zn’+, the membrane. Several other aminopeptidases have co2+, cu’+ and Ni*+. Several enzyme types, asso- some interaction with the cytoplasmic membrane, 330 T. Gonzales, J. Robert-Baudouy/ FEMS Microbiology Reviews 18 (1996) 319-344 especially on its internal side, without being consid- (Table 1). Nucleotide sequence information for genes ered as membrane proteins. These enzymes normally that code for bacterial aminopeptidases show that, in display part of their activity as a soluble fraction and general, these genes are monocistronic and display part associated with membranes. Since solubilisation promotor consensus sequences characteristic of genes of the membrane fraction does not always require the transcribed by an RNA polymerase associated with use of detergents, it is possible that the enzyme- gT,,. Two genes coding for aminopeptidases have membrane association is due to weak interactions. been shown to be part of an operon. The B. subtilis

Included amongst this type of aminopeptidases are: methionine aminopeptidase (Table 1, family 7) is Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 the E. coli and P. aeruginosa N-aminopeptidases transcribed as a polycistronic message which con- [91,92]; the aminopeptidase from A. calcoaceticus tains four other transcripts coding for the ribosomal [5 11; C-aminopeptidase, X-prolyl dipeptidyl amino- L 15 and L30 proteins, the SecY membrane protein peptidase and in general the proline-specific pepti- involved in extracellular protein export and adeny- dases from Lc. lactis ssp. cremoris [93-951; the late cyclase [ 1041. The reason why the methionine Klebsiella cloacae Pep [96]; the S. sanguis arginine aminopeptidase gene belongs to this operon is not aminopeptidase [97]; the I-aminopeptidase from B. known. This contrasts with the gene coding for the stearothermophilus [ 17,98,99]; the L.C. lactis ssp. same enzyme in E. coli which is monocistronic [83]. cremoris PepT tripeptidase 156,991 and the dipep- However, the gene encoding the PepC aminopepti- tidy1 peptidase IV from Flavobacterium dase from L. helueticus also belongs to an operon meningosepticum [ 1001. The reason why these en- consisting of two genes [105]. The nature and func- zymes require contact with the cytoplasmic mem- tion of the protein coded for by the second gene is brane is unknown. However, since certain amongst unknown [83]. It has also been proposed that the them are involved in bacterial nutrition, it is possible gene coding for the Lc. lactis ssp. cremoris PepT that these peptidases are located in close proximity tripeptidase (Table 1, family 141, belongs to an to cellular membrane systems involved in the trans- operon since it lacks a typical nucleotide promotor port of exogenous peptides in order to be able to sequence [56]. react with their substrates as soon as they enter the To date only one aminopeptidase-coding gene has cell. been identified that is not transcribed by an RNA Initially, the cellular location of several polymerase associated with u,~ factor. The gene, aminopeptidases, in particular those of E. coli and from Neisseria gonorrhoeae and coding for a pro- the lactic acid bacteria, was controversial. However, line iminopeptidase (Table 1, family 121, displays a with the advent of DNA sequencing techniques it has promoter consensus sequence upstream of the start been demonstrated that these enzymes do not possess codon which is characteristic of genes transcribed by a signal sequence at the N-terminal end and it is now an RNA polymerase associated with a,, [ 1061. Since accepted that they are essentially cytoplasmic this type of promotor is characteristic of genes whose [3,8,9,101,102]. It is not however excluded that some expression is induced by deficiency, it is fraction of aminopeptidase activity may be associ- probable that the enzyme is involved in bacterial ated with the cell envelope. In addition, some au- nitrogen nutrition by degrading certain intracellular thors have suggested an unknown mechanism of proteins. The gene encoding the aminopeptidase from translocation or a ‘specific leak’ of some cytoplas- A. calcoaceticus, an enzyme whose synthesis is mic aminopeptidases [8,102]. strongly increased by a deficiency in nitrogen (Table 1, family 3). may also possess a promotor of this 3.2. Regulation of enzyme synthesis type [5 1I. The regulation of genes coding for aminopepti- 3.2. I. Transcriptional control dases has been best studied in Enterobacteria, in Even though studies on bacterial aminopeptidases particular in E. coli and S. typhimurium. The most have been carried out for over several decades now, well characterised of these enzymes is the E. coli little information is available either on the regulation N-aminopeptidase (Table 1, family 1). This enzyme of synthesis or the expression of these enzymes is synthesised throughout the E. co/i life cycle, but T. Gonzales,J. Robert-BaudouF/ FEMS Microbiology Reviews 18 (1996) 319-344 331 the expression of the corresponding gene is increased aminopeptidases have been carried out for a number by a factor of 4 under both phosphate limitation and of these enzymes. However, such studies involved anaerobis [107- 1091. By contrast, conditions of ei- measurements of enzyme activity only and therefore ther nitrogen or carbon limitation do not affect its could not be used to specify the level of regulation, level of synthesis. Although the molecular basis of i.e. transcriptional, translational or post-translational. regulation is not known, it appears to differ from the Both constitutive and induced expression at different regulation of alkaline phosphatase, an enzyme whose phases or temperatures of growth, have been demon-

production is also induced by phosphate limitation strated (Table 1). It has also been shown that the Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 [ 110. I 1 I]. The physiological importance of amino- expression of certain aminopeptidases from lactic peptidase regulation is still not understood. It has acid bacteria is induced when the culture medium is been suggested that N-aminopeptidases fulfil addi- enriched with peptides. This occurs for both dipepti- tional functions in the cell once a change occurs in dases from Lb. casei [ 1151 and from Lb. bulgaricus the extracellular environment [ 101,112]. Two other [116] and one of the Lc. Zuctis [ 1171. genes that code for Enterobacterial aminopeptidases Such observations agree with the presumptive role of display similarities of regulation with the gene en- these enzymes in nitrogen nutrition in these bacteria. coding the E. coli N-aminopeptidase. These are the The only cited example of regulation at the post- E. coli pepD (Table I, family 14), whose expression transcriptional level is the leucine aminopeptidase is induced under conditions of phosphate limitation from A. proteolytica (Table 1, family 2). This extra- [ 1031, and the S. tvphimurium pepT (Table 1, family cellular enzyme is synthesised as a 43-kDa precursor 14) which is characterised by an increase in expres- [41]. After cleavage of the signal sequence. a double sion under anaerobic conditions [55,113]. In the lat- maturation at the N- and C-terminals occurs which ter case. anaerobic induction is due to the attachment frees the mature active 32-kDa enzyme. This matura- of the product of fnr gene onto the promotor region tion process only occurs when the culture medium is of pepT, but this seems unlikely to be the case for held for 1 h at 70°C and confers remarkable ther- the E. coli pepN gene [55]. Indeed regulation of mostability on the 32-kDa protein. The 43-kDa pre- N-aminopeptidase production by phosphate level is cursor also displays catalytic activity, but in contrast not confined to E. coli but has also been found for this is rapidly inactivated at 70°C [41,42]. N-aminopeptidases from several other Gram-nega- Finally, an endogenous inhibitor of the E. coli tive bacteria [ 1141. N-aminopeptidase has been isolated. The inhibitor, Recently, the gene coding for aminopeptidase E which is found in crude cell extracts, is responsible from S. Qphimurium (Table 1, family 14), an en- for a competitive-type inhibition. Even though the zyme which specifically cleaves Asp-dipeptides, was compound has not as yet been identified, it known to characterised. Expression of this gene is controlled have a molecular weight of less than 1000 Da and is by catabolite repression [22] and is the first reported resistant to incubation for 10 min at pH 2 or 100°C example of this type of regulation for an aminopepti- [11&l 191. dase-coding gene. The physiological reason for this regulation is still unclear, as may not be used as a sole carbon source by this bacterium. 4. Physiological role of bacterial aminopeptidases However, as this amino acid is the origin of the synthesis of a number of other amino acids (Asn, 4. I. Utilisation of exogenous peptides as nutrients Lys, Met, Thr, Be), it is possible that under condi- tions of carbon limitation, the bacterium uses this Luctococcus lactis is one of the most well-char- dipeptidase to free aspartic acid residues from dipep- acterised bacteria with respect to peptidase activity. tides in order to synthesise other amino acids [22]. This organism is usually found as a constituent of lactic acid starter cultures used in the production of 3.2.2. Other mechanisms of regulation milk products such as Dutch-type pressed Studies on the influence of environmental condi- and certain fermented milks [120-1221. Like other tions on the regulation of the production of lactic acid bacteria, Lc. luctis is nutritionally fastidi- 332 T. Gonzales. J. Roben-Baudouy/ FEMS Microbiology Reviews 18 (1996) 319-344

ous and requires amino acids and peptides for growth. Endopeptidase However, these growth factors are not found in sufficient quantities in milk [3,8,9,123]. To achieve optimum growth, Lc. Zuctis hydrolyses milk proteins

() in order to acquire essential amino acids. ProdHis-Phc Imimpeptidase Thus, by liberating small peptides and free amino acids from milk caseins, the proteolytic system of Glx-kiiy-P&u-Leu-Leu Aminopcpddase A

LL. Zactis plays a predominant role in the nitrogen Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 nutrition of this bacterium. The importance of this Gly-PmYx”-La-Leu X-Prolyle-dipeptidyle system to the dairy industry in the maturation of aminopcptidase milk products has stimulated much research in this field over the last 15 years [3,8]. Leu’iLe”-Le” Tnpeptidase The Lc. Zuctis proteolytic system is composed of (caseinases) and peptidases (especially en- dopeptidases) associated with the cell wall, as well HisJPhe Ammopepddase c as intracellular peptidases [3,7- 10,122]. Genes cod- ing for four distinct proteolytic activities are nor- LeUJLe” Dipeptidase mally found on plasmids [8]. These corresponding enzymes display strong similarities at the peptide Gly+ro Pmlidase sequence level [3], but differ with respect to their specificity for different caseins (cQ,-, Q-, k- and lactis p-caseins). In general LL contain one ca- Lys+Alaffilxffily+Pm+ku+Leu+Leu+Pro+His+Phe seinolytic activity [123]. The presence of at least Fig. 2. Example of the degradation of a synthetic into three amino acid transport systems in Lc. lactis has amino acids by the combined action of several peptidases from been demonstrated. The first of these is specific for LX. lads (after [8]). free amino acids, the second for di- and tri-peptides and the third for of at least three residues [8]. capable of cleaving peptide bonds involving a pro- Peptide degradation is carried out within the cell line residue. Some of these enzymes have been with the aid of a variety of peptidases of different purified (X-proline dipeptidyl aminopeptidase and specificity. Many of these enzymes, e.g. aminopepti- prolidase; Table 1, families 13 and 14) while others dases, dipeptidyl aminopeptidases, di- or tri-pepti- have only been observed (prolinase, proline dases, have been purified and their biochemical iminopeptidase [ 1231). Since caseins are proteins rich properties studied (Table 1). Some display overlap- in proline (10% for as2- up to 35% for P-casein ping specificities and the contribution of each en- [ 12411, it is evident that Lc. lactis has adapted its zyme to casein degradation remains unclear [3,8,9]. proteolytic system to suit the conditions of its pre- In addition, the regulation of expression of amino- ferred environment [8]. An example of the degrada- peptidase activities is unknown. However, the pre- tion of an oligopeptide by the concerted action of sent exhaustive characterisation of the Lc. lactis several peptidases is given in Fig. 2. proteolytic system at the biochemical and molecular levels, as well as the isolation of mutants lacking 4.2. Degradation qf intracellular proteins and pep- certain activities [56], should soon allow the determ- tides nation of the importance of each enzyme to the casein degradative process [3,6]. Since carboxypepti- 4.2.1. Protein turnover dase activity has never been detected in LL lactis Since the intracellular protein content of bacteria [8,122], it seems that peptide degradation from ca- is constantly renewed during the growth cycle, a sein occurs at the N-terminal extremity only. Finally, dynamic equilibrium is established between the syn- L.C. Zactis appears to contain several aminopeptidases thesis of new proteins and the catabolism of proteins T. Gonzales, J. Robert-Baudoug/ FEM.? Microbiology Reviews 18 (19%) 319-344 333 previously synthesised (protein turnover). The rate of newly synthesised proteins. As a consequence, the protein degradation depends on the physiological viability of strains mutated for several aminopepti- status of the cell [125,126]: bacteria in exponential dase coding genes is reduced with time, which clearly phase growth degrade l-2% of their intracellular demonstrates the importance of these enzymes in the protein per hour whereas 5- 12% of such proteins are adaptative process of a bacterium to its environment degraded in the same period of time during station- [129]. ary phase [loll. These variations may be explained It was subsequently shown that these same

by the necessity to adapt the protein content of the aminopeptidases are responsible for the final stages Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 cell to modifications in the external environment. of degradation of abnormal proteins, the latter having Under conditions of limitation (carbon, ni- been produced either as a result of treatment of cells trogen, amino acids), the degradation of certain nor- with puromycin (an antibiotic causing incomplete mally stable proteins is essential in order to supply protein synthesis) or by the incorporation of canavine, the amino acids required for new protein syntheses an analogue of arginine [ 1351. Since these peptidases [ 10 1,127,128]. Under certain conditions, these amino are also involved in the degradation of exogenous acids may also be used as a source of energy by the peptides [ 136,137], it can be concluded that different cell [ 127,129]. In addition to protein turnover, the classes of aminopeptidases do not exist and that the catabolism of certain proteins and particular peptides same enzymes are responsible for the breakdown of is also necessary since the cell needs to eliminate both exogenous peptides as well as the degradation abnormal proteins and signal sequence peptides de- of peptides resulting from the catabolism of endoge- rived from exported proteins [ 101,127,128]. nous proteins [ 1351. The role of the aminopeptidases PepP and PepQ in the degradation of intracellular 4.2.2. The role of peptidases in the degradation of peptides was also demonstrated [138]. endogenous proteins It is clear that the ability of a bacterium to 4.2.3. Recognition of proteins to be catabolised degrade intracellular proteins and peptides is a Under conditions of limiting carbon supply, strains physiological necessity. Aminopeptidases were of S. typhimurium mutated in one or several amino- thought to free amino acids from the peptides result- peptidase-coding genes are also characterised by a ing from the catabolism of proteins by proteases significant reduction in radioactivity present in free [ 130-1321. This hypothesis was confirmed in the amino acids and small peptides with respect to the early 1980’s by work carried out on S. typhimurium. parental strain [ 134,135]. If the predominance of In particular, studies were performed on the fate of small partially degraded radioactive peptides was proteins labelled with radioactive [ ‘“Clleucine in both predictable for mutated strains, the general reduction wild-type and mutant strains for several genes cod- in the level of radioactivity (free amino acids and ing for aminopeptidases ( pepA, pepB, pepD, pepN). small peptides) in the same strains is difficult to Strains mutated for one or several aminopeptidase explain. One hypothesis proposed that these genes, display, at the intracellular level, proportion- aminopeptidases, involved in the final degradation of ally lower levels of free radioactive leucine and peptides to amino acids, could also be involved in higher levels of small radioactive peptides containing earlier stages of catabolism [ 134,135]. The absence leucine as compared to the parent non-mutated strain. of aminopeptidases in mutant strains also results in a These results clearly show the involvement of general slowing down in the degradation of proteins aminopeptidases A, B, D and N in the final stages of into small peptides. In support of this, some amino- the degradation of intracellular proteins into amino peptidase enzymes, e.g. PepA, are active mainly on acids. This phenonemon is observed under different large peptides and are thus capable of degrading physiological conditions. i.e. for bacteria in exponen- proteins 11391. It was subsequently proposed that tial- [ 1331 as well as in stationary-phase growth in a certain aminopeptidases could be involved in the medium under limiting carbon conditions [134]. In very first stage of catabolism by cleaving the N- the latter case, the deficit in free amino acids in the terminal amino acid of certain proteins [140]. Once cell also involves a reduction in the quantity of deleted these proteins would be unstable, in accor- 334 T. Gonzales, J. Robert-Baudouy/ FEMS Microbiology Rehws 18 (1996) 319-344

Table 2 mechanisms used to label proteins for degradation, ‘N-end rule’ in E. coli and (after the role of aminopeptidases in these mechanisms is t 1401) still hypothetical. A better understanding of gene X X-pgalactosidases half-life expression for aminopeptidase-coding genes as well E. coli wt E. coli aat- S. cerevisiae wt as the conditions for in vivo activity of these en- Arg 2 min > 10h 2 min zymes would allow the clarification of their role in LYS 2 min > 10h 3 min the process of intracellular protein degradation. Phe 2min 2min 3 min 2min 2 min 3 min Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 LeU 4.3. Protein maturation Trp 2 min 2 min 3 min Tyr 2min 2 min 10 min His >lOh > IOh 3 min Bacterial aminopeptidases have often been pro- Ile > 10h > IOh 30 min posed to be involved in the maturation of protein Asp > 10h > 10h 3 min precursors [101,132], even though few examples of Glu > 10h > 10h 30 min such processes have been reported. The methionine Asn > 10h > 10h 3 min Gln > 10h > 10h 10 min aminopeptidase (MAP or PepM) is responsible for Cys > 10 h > 10h > 20 h the cleavage of the N-terminal methionine of newly Ala > 10h >lOh > 20 h produced polypeptide chains. Many of these en- Ser > 10h > 10h > 20 h zymes, of diverse bacterial origins, have been stud- Thr >lOh > 10h > 20 h ied at the biochemical or the molecular level (cf. GlY > 10h > 10h > 20 h Val > IOh > 10h > 20 h references Table 1, family 7). These aminopeptidases pro ? ? > 20 h display a strict specificity for the methionine at the Met > 10h > 10h > 20 h Pl-position, but at the same time cleavage of the protein is also a function of the amino acid adjacent The half-lives of different @galactosidase enzymes, modified at amino acid ‘X’ at the end-terminal position, depends upon the to the methionine residue (Table 3). As a result, nature of the modified residue. In E. coli, arginine and lysine are certain mature proteins possess a methionine residue ‘secondary’ destabilising amino acids. In fact, the ‘amino acid at their extremity whereas others, e.g. for E. coli ’ (our gene products) catalyse the addition of ‘primary’ about 50% of proteins [143], would have lost this destabilising residues (Leu or Phe) at the N-terminal end. In a residue. It is therefore evident that methionine strain mutated in the aat genes, Arg- and Lys-P-galactosidases are more stable. aminopeptidase is involved in the determination of the half-life of a protein as a function of the N-end rule. Strains mutated for the corresponding gene dance with the N-end rule according to which the in (map or pepM) could not be obtained either for E. vivo half-life of a protein is a function of the nature of the amino acid situated at the N-terminal position (Table 2). An ATP-dependant Ti would Table 3 therefore recognise and degrade these proteins [141]. Cleavage of the N-terminal methionine by the bacterial methion- ine aminopeptidase as a function of the amino acid in the P’l- If this theory is correct, then aminopeptidases, or at position (after [83]) least certain amongst them, are involved in both the Cleaved Variable Non-cleaved recognition of proteins to be degraded and the final cleavage stages of degradation. Ala cys Meanwhile, it was shown that certain mechanisms Lys GlY His Ar g of degradation are based on principles other than the Pro Met Leu N-end rule and therefore do not require aminopepti- Ser Trp Ile dase enzymes to signal proteins for degradation. In Thr Tyr Asn particular the E. coli ATP-dependant La protease, Asp Phe Glu coded for by ion, is involved, at least in part, in the Gln degradation of abnormal proteins [101,141,142]. Val Also, given the lack of information regarding the T. Gon:ales. J. Robert-Baudoq/ FEMS Microbiokqq Reviews IX f 1996)31 Y-344 335 co/i or for S. t?iphimurium [143,144]. It therefore role in this process which is independent of its appears that mutations in this gene are lethal for the aminopeptidase activity [ 1471. bacterium and that certain essential proteins, nor- The 0. anthropi D-aminopeptidase provides a mally mature, remain inactive most probably as me- second example of an aminopeptidase which may thionyl precursors [ 1441. It is interesting to note that possess an additional function. This enzyme specifi- mutation of the equivalent gene (mupl) in Saccha- cally cleaves D-form amino acids and displays strong mmyces crre~~isiae, is not lethal and it is possible similarities at the peptide sequence level with DD-

that an alternative mechanism of N-terminal methio- carboxypeptidases, enzymes involved in peptidogly- Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 nine cleavage exists in this yeast [ 1451. can biosynthesis. It has been suggested that this Another example of post-translational maturation D-aminopeptidase could be involved in the synthesis due to the action of an aminopeptidase is the E. coli and/or degradation of peptidoglycan. The fact that alkaline phosphatase. This periplasmic enzyme con- this cell wall constituent contains D-Ala-Giy-Gly sists of two sub-units coded for by the same gene peptide motifs, which are excellent substrates for and exists as three possible isoforms. The N-terminal D-aminopeptidase. supports the above hypothesis extremity of each sub-unit is made-up of an arginine m. which may be cleaved by Iap-aminopeptidase, the Some bacterial aminopeptidases also seem to be latter being a membrane enzyme whose catalytic involved either in the mechanism of activation or the domain extends into the periplasm. The various sub- transport of antibiotics into the cell. Both the E. co/i unit combinations may or may not display an argi- PepN as well as the S. Qphimurium PepN and PepA nine at the N-terminal position which results in the aminopeptidases are responsible for the activation of three isoforms [90]. albomycin in the cytoplasm, since strains mutated for these aminopeptidase activities are insensitive to this 4.4. Other known ,functions antibiotic 11481. The Xc aminopeptidase of Xun- thornonus citri is involved in transport of the antiobi- As well as their role in the maturation of proteins otic ascamycin across the cytoplasmic membrane. and in the degradation of peptide and proteins of This antibiotic naturally exists as a alanylated form both endogenous and exogenous origin, certain which cannot cross the cell membrane. The Xc aminopeptidases may fulfil additional functions in aminopeptidase, which is located in the cell enve- the cell. For example, the E. co/i PepA peptidase is lope, cleaves the alanine residue and allows the a necessary accessory factor for the stability of high transport of the antibiotic into the cell [149]. Thus, copy number ColEl plasmids [40]. These plasmids the presence of aminopeptidase activities results in have the tendency to form multimers in vivo as a an increased sensitivity of bacteria to certain antibi- result of homologous recombination. In E. cofi a otics. It is possible that antibiotic-secreting organ- specific recombination system exists which allows isms exploit the presence of certain aminopeptidase the formation of stable monomers from multimeric in target bacteria in order to suppress their develop- plasmids. This system requires the presence of the ment. Finally, other potential functions of bacterial 250- (bp) cer on the ColEl plasmid aminopeptidases, such as the degradation of toxic as well as several proteins coded for by the chromo- peptides or the inactivation of physiologically impor- somally located genes. xerA, xerB, xerC and xerD tant proteins or peptides, have been proposed [147]. The xerC and xerD genes code for two [101,132]. recombinases while xerA codes for a repressor of the arginine biosynthetic pathway whose role in the recombination process is not very clear. The xerB 5. Conclusions gene codes for PepA aminopeptidase [40]. Recently it has been shown that a mutation in pepA which Bacterial cells contain several aminopeptidases of results in abolishing enzyme activity does not alter broad specificity, displaying overlapping activities, aminopeptidase function in the recombination pro- and several aminopeptidases with narrow specificity. cess and it is probable that PepA plays a structural In E. coli eleven different aminopeptidase activities have been detected [loll. They comprise three logical substrates are required. The considerable in- broad-spectrum enzymes (PepA, PepB, PepN), three terest in bacterial aminopeptidases, whether basic or dipeptidases (pepD, PepE, PepQ), one tripeptidase applied, should help advance our knowledge on their (PepT) and four aminopeptidases of narrow speci- functioning and physiological role. ficity (PepM, PepP, Iap, iminopeptidase). Certain enzymes, such as PepN-type, seem to be universally present amongst bacteria, whereas others, e.g. Peps, Acknowledgements

X-proline dipeptidyl aminopeptidases, PepC, are Downloaded from https://academic.oup.com/femsre/article/18/4/319/529845 by guest on 24 September 2021 found in certain bacterial species only. It is interest- ing to note that the bacterial content in aminopepti- We thank M.J. Storrs for help with the translation dases, even though considerable, is much less than of this publication and G. Luthaud for typing parts of that of the yeast S. cerellisiue. In fact, for this latter the manuscript. organism about thirty distinct activities, either aminopeptidase, dipeptidyl aminopeptidase or dipep- tidase, have been reported [ 146,150- 1541. References The biochemical and enzymatic characterisation of bacteria1 aminopeptidases, which began almost [II El Soda, M. (1993) The role of lactic acid bacteria in three decades ago, has revealed the existence of three accelerated ripening. FEMS Microbial. Rev. 12. major catalytic types. The largest family is composed 239-252. of metallo-aminopeptidases. Catalytic types which 121Gasson, M.J. (1993) Progress and potential in the biotech- nology of lactic acid bacteria. FEMS Microbial. Rev. 12. require a highly reactive serine or cysteine residue 3-20. are less common and are found mainly amongst 131 Kok. J. (1990) Genetics of the proteolytic system of lactic aminopeptidases that display narrow substrate speci- acid bacteria. FEMS Microbial. Rev. 87, 15-42. ficity. Although the catalytic mechanisms which al- [41 MacKay, L.L. and Baldwin. K.A. (1990) Applications for low substrate hydrolysis are more or less understood, biotechnology: present and future improvements in lactic acid bacteria. FEMS Microbial. Rev. 87, 3-14. the molecular basis of substrate specificity remains 151 Mulholland, F. (1991) Flavour peptides: the potential role to be elucidated. However, recently initiated crystal- of Ltrctococctrl peptidaaes in their production. Biochem. lographic studies should help advance our under- Sot. Trans. 19. 685-690. standing of this. [61 Olson. N.F. (1990) The impact of lactic acid bacteria on To date forty genes coding for bacterial cheese flavor. FEMS Microbial. Rev. 87. 13 I - 148. I71 Pritchard, G.G. and Coolbear, T. (1993) The physiology aminopeptidases have been characterised. This has and biochemistry of the proteolytic system in lactic acid allowed the demonstration of phylogenetic links be- bacteria. FEMS Microbial. Rev. 12. 179-206. tween enzymes that often possess similar biochemi- [81 Tan, P.S.T.. Poolman. B. and Konings. W.N. (1993) Prote- cal or enzymatic properties. The available data shows olytic enzymes of Lnctococ~cus hcris. J. Dairy Re\. 60. the existence of distinct classes of aminopeptidases 269-286. 191 Thomas, T.D. and Pritchard. G.G. (1987) Proteolytic en- within a given catalytic type. In addition, peptide zymes of dairy starter cultures. FEMS Microhiol. Rev. 46. sequence similarities have been detected with hydro- 245-268. lases not belonging to the aminopeptidase family as [lOI Visser, S. (1981) Proteolytic enzymes and their action on well as with eucaryotic aminopeptidases. milk proteins. A review. Neth. Milk Dairy J. 35. 65-88. Aminopeptidases are involved in the [l II Sweeney. P.J. and Walker, J.M. (1993) Aminopeptidases. In: Methods in Molecular Biology; Enzymes of Molecular of exogenous peptides and in the turnover of intra- Biology (Burrel, M.M. and Totowa, N.J., Eds.). Vol. 16, pp. cellular proteins as well as the elimination of abnor- 319-329. Humana Press Inc. mal proteins. In addition, methionine aminopeptidase iI21 Manafi, M.. Kneifel. W. and Bascomb, S. (1991) Fluoro- has been shown to play a role in the maturation of genie and chromogenic substrates used in bacterial diapnoa- newly synthesised polypeptide chains. However, de- tic.\. Microbial. Rev. 55, 335-348. [I31 Taylor. A. 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