Báo cáo khoa học: In silico analysis of the adenylation domains of the freestanding enzymes belonging to the eucaryotic nonribosomal peptide synthetase-like family pot

Rossi Fanelli’, Universita` di Roma ‘La Sapienza’, 00185 Roma, Italy Fax: +39 06 49917566 Tel: +39 06 49917574 E-mail: Stefano.Pascarella@uniroma1.it Website: http://w3.uniroma1.it/bio_c

freestanding enzymes belonging to the eucaryotic

nonribosomal peptide synthetase-like family

Leonardo Di Vincenzo1, Ingeborg Grgurina1and Stefano Pascarella1,2

1 Dipartimento di Scienze Biochimiche ‘A Rossi Fanelli’, Universita` di Roma ‘La Sapienza’, Roma, Italy

2 Centro Interdipartimentale di Ricerca per l¢ Analisi dei Modelli e dell’Informazione nei Sistemi Biomedici (CISB), Universita` di Roma

‘La Sapienza’, Roma, Italy

Nonribosomal peptide synthetases (NRPSs) are

multi-domain, multifunctional enzymes involved in the

bio-synthesis of many bioactive microbial peptides [1,2]

This class of natural products includes a variety of

compounds with interesting biological activities

(phyto-toxins, siderophores, biosurfactants, and antiviral

agents), as well as several clinically valuable drugs

[3,4] NRPSs are organized in iterative modules, one

for each amino acid to be built into the peptide

pro-duct The minimal module required for a single

mono-mer addition consists of a condensation domain (C),

an adenylation domain (A) and a peptidyl carrier

pro-tein (PCP) also denoted as thiolation (T) domain The

A domain is involved in the selection and activation of the amino acid substrate, which is then covalently attached to the enzyme via a thioester bond with the phosphopantetheine residue of the T domain C domains are localized between every consecutive pair

of A domains and PCPs and catalyze the formation of the peptide bond between the upstream amino acyl or peptidyl moiety tethered to the phosphopantetheinyl group and the free amino group of the downstream aminoacyl moiety, thus facilitating the translocation

of the growing chain onto the next module The structural diversity of NRPS products is enriched through the occasional presence of epimerization (E),

Keywords

nonribosomal peptide synthetase; homology

modelling; docking; specifity conferring

code; freestanding NRPSs

Correspondence

S Pascarella, Dipartimento di Scienze

Biochimiche ‘A Rossi Fanelli’, Universita` di

Roma ‘La Sapienza’, 00185 Roma, Italy

Fax: +39 06 49917566

Tel: +39 06 49917574

E-mail: Stefano.Pascarella@uniroma1.it

Website: http://w3.uniroma1.it/bio_chem/

homein.html

(Received 8 August 2004, revised 30

November 2004, accepted 9 December

2004)

doi:10.1111/j.1742-4658.2004.04522.x

This work presents a computational analysis of the molecular characteris-tics shared by the adenylation domains from traditional nonribosomal pep-tide synthetases (NRPSs) and the group of the freestanding homologous enzymes: a-aminoadipate semialdehyde dehydrogenase, a-aminoadipate reductase and the protein Ebony The results of systematic sequence com-parisons allow us to conclude that a specificity-conferring code, similar to that described for the NRPSs, can be recognized in such enzymes The structural and functional roles of the residues involved in the substrate selection and binding are proposed through the analysis of the predicted interactions of the model active sites and their respective substrates The indications deriving from this study can be useful for the programming of experiments aimed at a better characterization and at the engineering of this emerging group of single NRPS modules that are responsible for amino acid selection, activation and modification in the absence of other NRPS assembly line components

Abbreviations

A domain, adenylation domain; AASDH, a-aminoadipate semialdehyde dehydrogenase; ACV, synthetase [ L -d-(a-aminoadipoyl)- L -cysteine- D -valine] synthetase; AS, putative amine-selecting domain; GrsA, gramicidin S synthetase A; HMM, hidden Markov models; NRPS,

nonribosomal peptide synthetase; PQQ, pyrroloquinoline quinone; T domain, thiolation domain; RMSD, root mean square deviation.

cyclization (Cy), N-Methylation (N-Met) and

oxida-tion (Ox) domains [1]

Intense research work carried out in the last decade

led to the characterization of a number of new gene

clusters and to the discovery of nonclassical NRPS

sys-tems [2,5] The crystallographic structure of three

members of the adenylate-forming enzyme family,

fire-fly luciferase of Photinus pyralis [6], the A domain of

the gramicidin S synthetase A (GrsA) from Bacillus

brevis [7] and, recently, DhbE (2,3-dihydroxy-benzoate

activating module) [8], have been solved Likewise, the

structure of VibH, representative of C domains, is now

available [9] The wealth of sequence and structure

information pertaining to the A domains has been

exploited to understand the molecular bases of their

substrate specificity [10,11] Systematic comparative

analyses identified 10 sequence positions lining the

act-ive site pocket that are responsible for substrate

recog-nition and selection The nature of the residues at such

positions was correlated with the known substrates

and a specificity-conferring code was proposed also

with predictive potential [10]

Recently, it was pointed out that modules composed

of an adenylation and a thiolation domain, followed

by a domain having a redox function and not inserted

in the context of a typical NRPS cluster, can be found

in eucaryotes [12] Indeed, a-aminoadipate

semialde-hyde dehydrogenase (AASDH) and a-aminoadipate

reductase (Lys2), enzymes involved in lysine

meta-bolism in eucaryotes, display a 3-domain architecture

where the two N-terminal domains are homologous

to the A and T domains from NRPS systems and the

C-terminal part contains a redox cofactor binding

site for either pyrroloquinoline quinone (PQQ) or

NADPH In particular, AASDH, containing a PQQ

binding domain, is supposed to be involved in lysine

degradation and to convert the a-aminoadipate

semi-aldehyde to a-aminoadipate [12] Lys2, possessing a

NADPH-binding domain, is involved in lysine

biosyn-thesis; it converts the a-aminoadipate to

a-aminoadi-pate semialdehyde [13,14] Furthermore, the protein

Ebony, an enzyme from Drosophila melanogaster

involved in conjugation of b-alanine to histamine and

sharing homology to NRPS domains A and T, was

recently characterized [15]

The occurrence of gene assets, typically encountered

in the microbial world, in evolutionarily higher

organ-isms is intriguing It appears worthwhile to carry out a

deeper investigation on the extent of similarity between

the A domains of the aminoacyl adenylate-forming

enzymes of the freestanding enzymes and those of the

traditional NRPS systems In particular, how many

sequences of freestanding A domains are known and

which are the evolutionary relationships to the NRPSs? Can the nonribosomal code of the traditional NRPS systems be applied in the freestanding A domains and, if so, what is the potential role of the residues involved? To address these issues, systematic sequence comparisons, homology modelling and dock-ing simulations were employed to predict the structure

of the active site of such enzymes and to propose func-tional roles for the conserved residues

Results and Discussion

Databank searches and sequence comparison The available sequences of the freestanding NRPS modules from eucaryotic organisms were collected by means of exhaustive databank searches The psi-blast [16] suite was applied over the NR and UniProt data-banks Query sequences were Ebony from Drosophila melanogaster, AASDH from Mus musculus and Lys2 from yeast Each sequence is representative of a domain pattern: A-T-AS (AS stands for puta-tive amine-selecting domain [15]), A-T-PQQ and A-T-NADPH, respectively Only the A and T domains were included in the query sequence Table 1 reports the homologous sequences collected by these databank searches including 10 sequences from genes coding for putative A domains not yet annotated in the protein databanks which were predicted through genome scans Overall, 39 sequences were identified from dif-ferent eucaryotic species and the domain assignments were confirmed by CDD [17] and Pfam [18] queries The sequence subset formed by the A-T domains was aligned utilizing the hmmer package [19] A set of 62 sequences, corresponding to the domain A of micro-bial NRPSs, were extracted from the seed alignment of the Pfam AMP-binding family (Pfam code: PF00501) The adjacent T domain was subsequently added to each sequence The extended sequences were aligned with clustalw [20] and the final alignment was manu-ally refined to match functionmanu-ally important residues such as the Asp235 that binds the a-amino group of the substrate [11] and Ser573 site of the phosphopant-etheine attachment The resulting alignment was finally utilized to train the HMM The resulting HMM was used to align a subset of the A-T domains listed in Table 1 The alignment was manually refined and used

in turn to train the final HMM, now specific for the eucaryotic A-T domains, to carry out the alignment of all the 39 sequences (Fig 1)

On the basis of the structural equivalencies con-tained in this multiple alignment, the occurrence of a specificity-conferring code similar to that described for

the NRPS systems [10] was tested Substrate

specifici-ties were assigned either on the basis of literature

data or by use of the NRPS prediction server [11] The

sequence positions equivalent, in the multiple sequ-ence alignment, to those involved in the described nonribosomal specificity code [10] are reported in

Table 1 List of freestanding and NRPS-like enzymes retrieved from databanks All accession numbers refer to UniProt database except where noted Boldface names denote in silico predicted proteins not included in databanks A stands for adenylation domain, T for thiolation,

C for condensation, AS for amine-selecting, PQQ for PQQ-binding domain, NADPH for NADPH-binding domain, X for other domains not commonly present in NRPSs Numeric subscript to parentheses indicate the repetition of those modules MNRPS stands for monomodular NRPS Question marks denote unassigned function Every PSI-BLAST search was performed with three iterations using as a probe the sequence of GrsA (P14687).

Protein length

Putative function

BLAST

E-value

a EMBLCDS entry name and b EnsEMBL peptide databank Boldface names denote in silico predicted proteins not included in databanks.

c,d,e denote that the TBLASTN searches against genomes used as input query sequences, P07702, Q8L5Z8, Q80WC9, respectively The genes were predicted from the nucleotide sequences:fEMBL accession number AABZ01000259 (positions coding for the protein: 3300–7700),

g EMBL AACF01000123 (15700–20800), h EMBL AAAA01021459 (854–2040), i EMBL AAAA01023971 (610–2070), j EMBL AAAA01000789 (18780–31200), k EnsEMBL ctg11952 (800001–1000000), l EnsEMBL Chr_scaffold_632 (38782–48782), m EMBL AABS01000029, n EMBL AACT01000010,oEnsEMBL scaffold_37623 (4535897–4735897).

Fig 1 Multiple sequence alignment of adenylation domains Only conserved portions from the multiple sequence alignment obtained as described in Results are shown Dashes represent insertion and deletion Numbers above the sequences refer to the sequence numbering

of the gramicidin synthetase;  is used as block separator The sequence positions equivalent to those involved in the nonribosomal speci-ficity-conferring code described for the A domain of the gramicidin synthetase are marked with blue triangles The positions of the core motifs are marked underneath with grey bars labelled according to [1] Secondary structure assignments are shown for GrsA: a-helices and b-strands are rendered as squiggles and arrows, respectively; T stands for turn; blank for coil and irregular conformations; dots represent gaps introduced in the alignment Identically conserved residues are displayed as white characters on red background Conserved regions are denoted by boxed red characters.

Fig 1 (Continued).

Table 2 The residues equivalent to those which in the

GrsA were observed to interact with the a-amino and

a-carboxyl groups of the amino acid substrate, Asp235

and Lys517, respectively [7], are conserved, the only

exceptions being the freestanding NRPS from Leptos-phaeria maculans (UniProt accession no Q873Z1) (lacks the Asp235) and AASDH from Acremonium chrysogenum (UniProt accession no Q9HDP9) (lacks

Table 2 Nonribosomal specifity-conferring code in the freestanding enzymes All accession numbers refer to UniProt database except those noted Boldface codes denote in silico predicted protein not included in databanks MNRPS is monomodular NRPS; ACV stands for ( L -d-(a-aminoadipoyl)-cysteine- D -valine) tripeptide synthetase Question marks denote unassigned function or substrate L -a-Aa stands for

L -a-aminoadipate, L -a-Aas stands for L -a-aminoadipate semialdehyde, b-Ala for b-alanine, Hty for hydroxyl tyrosine, the other three letters code stand for standard amino acid abbreviations.

Activated substrate

Residue position according to GrsA A domain numbering

a EMBLCDS entry name and b EnsEMBL peptide databank c Module 1 of ACV synthetase is included for comparison with the code of Lys2 and AASDH d Predicted using the NRPS prediction BLAST server [11].

the Lys517), and the sequences of Ebony from

Dro-sophila melanogaster and from Anopheles gambiae

(UniProt accession no Q7QKF0) where the Asp235 is

missing In this latter case, the absence of the Asp235

can be explained in the light of the model of the

inter-action of the substrate with the active site (vide infra)

It should be noted that the specificity code for the A

domains recognizing the substrate b-alanine (Table 2)

is similar to that already predicted for the A module

of exochelin synthetase from Mycobacterium smegmatis

(UniProt accession no O87313) [11], the only

differ-ences being in Ebony, at the positions 239 (Ser vs

Thr), 278 (Val vs Leu), 299 (Val vs Ile) and 322 (Phe

vs Ser) The specificity codes of Lys2 and AASDH

share the residues Asp235 and Pro236 The residue

Pro236 seems to be specific for the aminoadipate

sub-strates Indeed, the only other system in which it is

present in the same position is the module 1 of the

chloroeremomycin synthetase (UniProt accession no

O52821) from Amycolatopsis orientalis specific for

3,5-hydroxy-l-phenylglycine [11] The specificity code of

the module 1 of ACV

[l-d-(a-aminoadipoyl)-l-cys-teine-d-valine] synthetase from Penicillium

chrysoge-num that activates the l-a-aminoadipate, displays

strong similarities to the Lys2 code (Table 2) with the

remarkable difference at position 235 where a Glu

resi-due replaces the conserved Asp, and at position 330,

where a Phe residue replaces the conserved Arg⁄ His

The marginal resemblance of the AASDH code to that

of Lys2 and ACV module 1 provides a structural basis

for the current view that the physiological substrate of

the dehydrogenase is l-a-aminoadipate semialdehyde

rather than l-a-aminoadipate

Traditional and freestanding A and T domains share

also some conserved core motifs In particular, the

core motifs A3 to A10 [1] are conserved in the

eucary-otic NRPS-like domains while the motifs A1 and A2

are positioned in a nonconservative section of the

alignment (not shown in the figure) However, A1 and

A2 are away from the active site and probably only

conserved in the NRPSs for structural reasons [1]

Phylogenetic analysis

To visualize evolutionary relationships among the

freestanding NRPS A domains and the

correspond-ing domains of the traditional NRPS in a

phylo-genetic tree, the A domains of 25 bacterial NRPS

and the A domain of the ACV synthetase from

Peni-cillium chrysogenum were added to the multiple

sequence alignment shown in Fig 1 The 25 bacterial

NRPS sequences were selected taking one

representa-tive from each of the different substrate specificity

groups defined by Challis et al [11] to have a view

of the substrate range utilized by these enzymes The phylogenetic tree shown in Fig 2A, was built from the portion of the multiple sequence alignment shown in Fig 1 comprised between the positions 190–331, that contain the specificity code residues and the core motifs A3 to A5, using the neighbor-joining method as implemented in the module neigh-bor of the phylip package [21] The tree accuracy was tested with 1000 bootstrap replicates On the basis of the assumption that the nine amino acids lining the binding pocket determine substrate specific-ity [11], we used maximum parsimony method imple-mented in the program protpars of the phylip package [21] to establish a relationship between these important residues and substrate specificities in the

65 A-domains considered, i.e 39 freestanding plus 26 NRPS A domains Therefore, the tree in Fig 2B was derived considering only nine sequence positions cor-responding to the eight involved in the nonribosomal specificity code [11] and the Asp235 which was inclu-ded because not always conserved On the contrary, Lys517 was not included because it was conserved in all cases considered The resulting tree obviously has

no phylogenetic meaning The phylogenetic tree based on the positions 190–331 of the complete alignment revealed two clusters containing the a-ami-noadipate reductase from fungi and the a-aminoadi-pate semialdehyde dehydrogenase from metazoa, with independent segregation from the other bacterial sequences This pattern parallels that observed in the specificity code tree reported in Fig 2B and confirms that Lys2 and AASDH recognize different substrates Another independent cluster in both trees is made by the two Ebony proteins (UniProt accession nos Q7QKF0 and O76858), domain A of exochelin syn-thetase from Mycobacterium smegmatis module 2 (UniProt accession no O87313) and the two plant hypothetical NRPS-like proteins (UniProt accession

no Q8L5Z8 and in silico predicted protein O_SATI-VA3 in Table 1) This segregation could suggest that b-alanine or a very similar compound might be the substrate of the two plant proteins ACV synthetase module 1 from Penicillium chrysogenum (UniProt accession no P26046) displays a substrate specificity identical to fungal Lys2 although its sequence is more similar to that of the metazoa AASDH Finally, it is interesting to observe the unexpected position in the trees of the protein sequences from Caenorhabditis elegans and Caenorhabditis briggsae (UniProt accession nos Q95Q02 and Q17301) These proteins, containing 2870 and 4767 residues, respect-ively, display a typical NRPS modular structure and,

in the phylogenetic tree, are grouped with

bacter-ial NRPSs Two sequences in the same species

homologous to AASDH are observed to cluster, as

expected, in the AASDH group (UniProt accession

no Q9XUJ4 and EnsEMBL accession no ENS-CBRP00000001007)

Evolutionary trace analysis [22] (results not shown) was also applied to confirm the presence of functionally

Fig 2 Phylogenetic trees based on the multiple alignment of A domain sequences Metazoa, plants, fungi and bacteria are represented with red, green, brown and black colours, respectively All names and numbers used in the phylogenetic trees are defined in Table 1 except for the following UniProt accession numbers: P35854, D -alanine activating enzyme, Lactobacillus casei; Q50857, saframycin Mx1 synthetase B., Myxococcus xanthus; O87313, FxbB, Mycobacterium smegmatis; O30409, tyrocidine synthetase 3, Brevibacillus brevis; Q9Z4X5, CDA pep-tide synthetase II, Streptomyces coelicolor; P19828, AngR protein, Listonella anguillarum; Q45295, LchAA protein, Bacillus licheniformis; P39845, putative fengycin synthetase, Bacillus subtilis; P45745, dhbF, Bacillus subtilis; O68008, bacitracin synthetase 3, Bacillus lichenifor-mis; O68006, bacitracin synthetase 1, Bacillus licheniforlichenifor-mis; O52819, PCZA363.3, Amycolatopsis orientalis; O68007, bacitracin synthetase 2, Bacillus licheniformis; O87606, peptide synthetase, Bacillus subtilis; Q9ZGA6, FK506 peptide synthetase, Streptomyces sp.; O07944, Pristi-namycin I synthetase 3 and 4, Streptomyces pristinaespiralis; P11454, enterobactin, Escherichia coli; O52820, PCZA363.4, Amycolatopsis orientalis; O52821, PCZA363.5, Amycolatopsis orientalis; P71717, phenyloxazoline synthetase MBTB, Mycobacterium tuberculosis; Q9Z4 · 6, CDA peptide synthetase I, Streptomyces coelicolor; Q50858, saframycin Mx1 synthetase A, Myxococcus xanthus; O69246, LchAB protein, Bacillus licheniformis; P26046, N-(5-amino-5-carboxypentanoyl)-L-cysteinyl- D -valine synthetase, Penicillium chrysogenum The

‘M’ followed by a number in bacterial NRPS refers to the module A used for building the trees Enzyme substrates are indicated at the end

of the databank code with the standard one-letter code for amino acids or with the following abbreviations Aa: L -a-aminoadipate; Orn:

L -ornithine; DHPG: 3,5-hydroxy-L-phenylglycine; PGly: L -phenylglycine; b-A: b-alanine; Aas: L -a-aminoadipate semialdehyde; 3hTyr: 3-hydroxy-L-tyrosine; HPG: 4-hydroxy-L-phenylglycine; 3h4mF: 3-hydroxy-4-methyl-phenylalanine (A) Neighbor-joining phylogenetic tree based on the comparison of alignment positions 190–331 The numbers on the branches indicate the number of times the partition of the species into the two sets which are separated by that branch occurred among the 1000 bootstrap trees; (B) maximum parsimony tree calculated with the nine amino acid lining the substrate binding pocket of adenylation domains.

important residues conserved at different levels of

parti-tion of the freestanding NRPS family This method

exploits the information inherent in a family of

homo-logous proteins by dividing it to maximize functional

similarity within the groups and functional variation

between the single groups The analysis was conducted

using the TraceSuite II server (http://www.cryst

bioc.cam.ac.uk/jiye/evoltrace/evoltrace.html) with the

same multiple sequence alignment used for building

the tree shown in Fig 2A The results showed that the

core motifs A3 to A5 are conserved in almost all

parti-tions and are characteristic of the NRPS A domains

Furthermore, the variability of the residues of the

spe-cificity code confirms that they are group-specific

except for the residues Asp235 and Pro236 that are

shared by the two groups, AASDH and Lys2, which

bind similar substrates (Table 2)

Modelling of active sites and docking studies

Molecular modeling, manual and automated docking

have been utilized to map the conserved residues

onto a hypothetical active site structure, to

under-stand the role of their conserved residues and predict

their interaction with the substrates Figures 3, 4 and

5 report the model active sites of Ebony from

Drosophila melanogaster, Lys2 from Saccharomyces

cerevisiae and the AASDH from Homo sapiens,

respectively

The reliability of docking experiments using homo-logy models built at a sequence identity to the tem-plate of 25–30%, as in the reported case, can be

Fig 3 Model structure of Ebony from Drosophila melanogaster.

Ebony model is represented in teal blue cartoons AMP molecule is

rendered as a stick model The specifity code residues are shown

as stick models with superimposed slate blue CPK models Carbon,

oxygen, nitrogen and phosphorous atoms are displayed with green,

red, blue, purple colors, respectively b-Alanine is represented as a

stick model with grey carbon atoms Dashes indicate hydrogen

bonds This figure was rendered using PYMOL [31].

Fig 4 Model structure of the active site of Lys2 from Saccharo-myces cerevisiae AMP molecule is shown as a stick model Car-bon, oxygen, nitrogen and phosphorous atoms are displayed with green, red, blue, purple colors, respectively The two possible assets of the substrate L -a-aminoadipate ( L -a-Aa) are superimposed and represented as sticks Carbon atoms are colored in two differ-ent way: cyan for L -a-Aa in which the d-carboxyl group forms an hydrogen bond with Lys517; green for L -a-Aa in which a-carboxyl forms a hydrogen bond with Lys517 The other atoms are colored

as in AMP All the residues in the active site are rendered as CPK and colored in slate blue This figure was rendered using PYMOL [31].

Fig 5 Model structure of AASDH from Homo sapiens AASDH main chain is represented in teal blue cartoons; AMP is shown as stick model Carbon, oxygen, nitrogen and phosphorous atoms are displayed with violet, red, blue, purple colours, respectively.

L -a-Aminoadipate semialdehyde ( L -a-Aas) is represented as stick and carbon atoms are green The specifity code residues are shown as sticks and CPK Sticks are colored as in AMP except for carbon atoms which are in grey, and CPK which are colored in blue marine This figure was rendered using PYMOL [31].

questionable Indeed, the superposition of the three

structures related to the freestanding A domains,

namely GrsA, firefly luciferase and DhbE that share

16% sequence identity on average, shows that the

average RMSD over the Ca of the entire structures is

2.6 A˚ On the contrary, the average RMSD calculated

over the Caenclosed in a sphere of radius 9 A˚ centered

at the GrsA residue Asp235 in the active site, is

0.95 A˚ Indeed, the active sites of the enzymes tend to

be structurally more conserved during evolution [23]

Therefore the error affecting the active site is expected

to be lower than that regarding the rest of the protein

Consequently, the docking studies can still provide

useful and testable indications

In the active site of Ebony (Fig 3), two residues

of the traditional nonribosomal code Asp235 and

Pro236, are replaced by Val and Asp, respectively

The aspartate in position 236 can form a hydrogen

bond to the b-amino group of the b-alanine substrate,

which interacts also via hydrogen bonds with Ser301

and Asp331 The other residues line the active site

pocket A bulky aromatic residue (Phe322) serves as

the floor of the active site pockets Apparently, the

rearrangement of the side chains at the active site

enabled the enzyme to recognize a substrate with a

b-amino instead of a a-amino group Interestingly,

substitution of Asp235 is indicative of the substrate

structure For example, in the case of DhbE position

235 is occupied by Asn and the relative susbstrate

lacks a a-amino group [8]

It has been proposed, for Lys2 from S cerevisiae,

that the l-a-aminoadipate substrate could be

adenyl-ated at the d-carboxylate rather than the

a-carboxy-late and that the a-amino and a-carboxyl groups of

the substrate bind at the bottom of the pocket

inter-acting with the Arg239 and Glu322 [14] Analogous

arrangement was proposed also for the binding of

l-a-aminoadipate to the adenylation domain of the

ACV synthetase from Penicillium chrysogenum [7]

The results of the docking experiments indicated

(Fig 4) that the possible binding modes cluster into

two solutions According to the first possibility, the

substrate a-aminoadipate is bound to the active site

with a salt bridge between the a-amino group and

the a-carboxyl group of Asp235 and a hydrogen

bond to the carbonyl group of Arg330 In yeast

Lys2, the d-carboxylate group of the substrate forms

a salt bridge with Arg239 Finally, the substrate

a-carboxylate interacts via hydrogen bonds with the

e-amino group of Lys517 The other residues of the

putative specificity code line the walls of the active

site In particular, the conserved Pro236 shapes the

pocket to host the substrate An alternative

inter-action way of binding of the substrate to the active site involves the formation of a salt bridge between the d-carboxylate group and the e-amino group of Lys517 and between the a-carboxylate and Arg239 The a-amino group interacts via hydrogen bonds with the carbonyl oxygens of Met322, Gly324 and Arg330 The first binding mode of the substrate (the a-carb-oxylate interacting with Asp235) is supported by the invariancy of Asp235 that usually stabilizes the a-amino group of the amino acid substrate The importance of Asp235 in Lys2 is evidenced also by mutational analysis which showed a complete loss of catalytic activity for the mutant Asp235fiAsn, while the mutant Asp235fiGlu retained only 4% of cata-lytic activity [24] Also, this binding mode is in line with the absence of a negatively charged side chain in the position 322 of the putative a-aminoadipate spe-cificity code (Table 2) whose role is to stabilize the a-amino group of the substrate Such a residue (Glu322) is present in ACV synthetase However, most importantly, the same binding mode does not account for the experimental evidence of the existence

of the a-aminoadipoyl-C6-AMP [13], which can be explained by the binding mode with the d-carboxylate

in proximity of Asp235

The results of the docking studies of a-aminoadipate semialdehyde, assumed to be the substrate of AASDH [12] (Fig 5), show that the substrate can interact with the active site in only one orientation It involves the formation of a salt bridge between the a-amino group

of the substrate and the carboxylic group of Asp235 and a hydrogen bond to the carbonyl atom of Ser330 The d-aldehyde group of the substrate interacts with Gln278 Finally, the substrate a-carboxylate, as expec-ted, interacts via hydrogen bonds with the e-amino group of Lys517 in both enzymes Once again, this binding mode can explain the invariancy of Asp235 and this model can account for the lack of a negatively charged side chain at position 322 of the putative spe-cificity code (Table 2) able to stabilize the a-amino group of the substrate which is instead present in ACV synthetase (Glu322)

The results reported in this work demonstrate that a specificity-conferring code can be recognized also in the freestanding eucaryotic NRPS-like enzymes A role for some of the specificity residues could be predicted

on the basis of in silico studies These indications can

be useful for programming experiments aimed at a bet-ter characbet-terization and at the engineering of this emerging group of single NRPS modules responsible for amino acid selection, activation and modification

in the absence of other NRPS assembly line compo-nents