Functional sites and evolutionary connections of acylhomoserine lactone synthases

Saikat Chakrabarti and R. Sowdhamini1,

National Centre for Biological Sciences, Tata Institute of Fundamental Research, UAS-GKVK Campus, Bangalore 560 065, India

1 To whom correspondence should be addressed.E-mail: mini{at}ncbs.res.in


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Acylhomoserine lactone (AHL) synthases act as chemical communication signals or pheromones in Gram-negative bacteria and regulate diverse physiological events in a cell density-dependent manner. The recent crystal structure determination of EsaI, a key enzyme in this pathway, shows that the AHL synthase superfamily members adopt the fold of the N-acetyltransferase superfamily. We suggest, by the identification of intermediate sequences, that the two superfamilies are evolutionarily related. Evolutionary trace analyses of aligned sequences and docking studies have been used to discuss functionally important residues of EsaI homologues.

Keywords: distant similarity/intermediate sequences/N-acetyl transferase/OHHL synthase/protein structure prediction/quorum sensing


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Many Gram-negative pathogenic bacteria sense their growth environment and elicit an adaptive response. They synthesize low molecular weight pheromones or autoinducers such as acylhomoserine lactone (AHL) that serve in a cell-to-cell communication system, termed ‘quorum sensing’ [reviews (Salmond et al., 1995Go; Fuqua and Greenberg, 1998Go)]. Probably the best-characterized quorum-sensing system is the LuxI–LuxR of Vibrio fischeri. LuxI protein produces V.fischeri autoinducer [VAI, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL)], which binds to the transcriptional activator protein LuxR [review (Fuqua et al., 1996Go)]. Complexes of LuxR–VAI activate transcription of the lux operon, resulting in bioluminescence. S-Adenosyl methionine (SAM) and an acylated acyl carrier protein (acyl-ACP) are the potent substrates for the quorum sensing mechanism in bacteria and the substrate specificity depends mainly on the length of the acyl chain of acyl-ACP (Parsek et al., 1999Go).

AHL synthase superfamily members were identified in five other species by means of a lux-plasmid bioluminescent sensor for OHHL and by gene complementation studies (Swift et al., 1993Go). Some of these proteins differ in their substrate specificity and activate diverse biological events but in a similar cell density-dependent manner. The plant pathogen Agrobacterium tumefaciens requires the conjugal transfer factor of OOHL (N-3-oxooctanoyl-L-homoserinelactone) produced by the traI gene product (Fuqua and Winans, 1994Go). In Pseudomonas aeruginosa, the production of exoproducts such as elastase is regulated via quorum sensing and two pairs of LuxRI homologues have been identified, i.e. RhlRI and LasRI (Jones et al., 1993Go; Latifi et al., 1995Go). The major signal molecule produced via RhlI and LasI, respectively, are N-butanoyl-L-homoserine lactone (BHL) and N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL). Some strains of the Gram-negative bacteria such as Erwinia carotovora make the simple carbapenem antibiotic via the CarRI system. CarI and EagI of Enterobacter agglomerans produce the freely diffusible molecule OHHL (Swift et al., 1993Go). Subsequently, 35–40 AHL synthase proteins have been identified [review (Swift et al., 1996Go)]. Our earlier structure prediction studies, like fold recognition and three-dimensional modelling on EagI, suggested that the AHL synthase superfamily members are compatible with the N-acetyltransferase fold (NAT) (S.Chakrabarti and R.Sowdhamini, unpublished results).

Recently, the crystal structure of EsaI (one of the AHL synthase superfamily) has shown that these proteins adopt the fold observed in N-acetyltransferases (Watson et al., 2002Go). The structures of the NAT superfamily (e.g. PDB codes, 1qst, 1b87 and 1cjw) reveal an alpha- and beta-fold for this superfamily and a central V-shaped cavity which forms the acetyl-CoA (AcCoA) and coenzyme binding site. Despite the similarity in their AcCoA-binding regions, the three structures differ significantly in other regions presumed to be involved in binding the substrate to be acetylated.

The present study involved the sequence and structural analyses of AHL synthase superfamily members. We show, by recursive PSI-BLAST searches (Altschul et al., 1997Go) and by the identification of intermediate sequences (ISS), that the structural similarity observed between AHL synthase and NAT superfamilies may have evolutionary meaning. We further propose that the intermediate sequences could effectively be a connecting link between the two superfamilies. The crystal structure of EsaI (Watson et al., 2002Go) has enabled us to obtain three-dimensional models for the other AHL synthase superfamily members and to examine the spatial positions of putative functionally important residues identified by evolutionary trace methods. Evolutionary class-specific clusters point to several interesting residue substitutions in the hydrophobic region of the substrate-binding site that might account for their substrate specificity.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Members of the AHL synthase superfamily were identified by performing PSI-BLAST runs (Altschul et al., 1997Go) using EsaI, CarI, LuxI, RhlI, TraI, LasI and EagI sequences as the initial query against the non-redundant sequence database. Three NAT structural representatives (PDB codes, 1cjw, 1b87 and 1qst) and the three intermediate sequences (ISS, see Results and discussion for details) were used to obtain further NAT members and homologues of ISS, respectively. The secondary structural positions of AHL synthase members were predicted using PHD (Rost and Sander, 1993Go), PSI-PRED (Jones, 1999aGo), J-PRED (Cuff and Barton, 1999Go) and NN-predict (Kneller et al., 1990Go). Homologous sequences were multiply aligned using CLUSTALW (Thompson et al., 1994Go) followed by a manual annotation. Three structural members of NAT superfamily and the sequence of EsaI (pdb code, 1kzf) were aligned using COMPARER (Sali and Blundell, 1990Go) and the alignment template was elaborated to include other AHL synthase superfamily members and related sequences using CLUSTALW (Thompson et al., 1994Go) and manual intervention.

For the recursive PSI-BLAST runs, sequences from NAT (1cjw, 1qst, 1b87) and AHL synthase (EagI, EsaI, CarI, LuxI, RhlI, TraI and LasI) superfamilies were used as queries to search against the non-redundant sequence database. Subsequently, each hit identified was passed through a further round of PSI-BLAST. The intention behind such searches was to identify intermediate search sequences (ISS) that are similar to both superfamily members.

Hierarchical clustering and dendrogram construction were performed for AHL synthases, NAT representatives and ISS using sequence dissimilarity measures and PHYLIP3.5 (Felsenstein, 1985Go). Non-redundant homologues of the three sets of proteins (at 90% identity cut-off), obtained by PSI-BLAST, were included in an expanded multiple alignment using MALIGN (Johnson et al., 1993Go). From the multiple alignment, distances based on sequence similarity were extracted and used as an input to the principal component analysis (PCA) (M.Johnson, unpublished results) program. Inter-sequence similarity profiles are represented in a three-dimensional projection where each point in the three-dimensional plot represents a particular sequence (Figure 1aGo). PCA involves a mathematical procedure that transforms a number of (possibly) correlated variables into a smaller number of uncorrelated variables called principal components. The first principal component accounts for as much of the variability in the data as possible and each succeeding component accounts for as much of the remaining variability as possible.



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Fig. 1. Similarities between NATs, AHL synthase superfamilies and the intermediate sequences (ISS) to denote evolutionary distances (see Methods for definition). (a) Principal component analysis using PCA (M.Johnson, unpublished results) of 45 distinct sequences. Three major clusters, corresponding to the three sets (AHL synthase, NATs and ISS), with ISS as an intermediate cluster in the evolutionary sequence space are evident. (b) Hierarchical clustering using PHYLIP (Felsenstein, 1985Go). Ten vertical lines show equal partitions in the evolutionary dendrogram. Key members of AHL synthase superfamily, the structural representatives of NAT superfamily and the three ISS are clustered. NAT structural representatives are denoted by their PDB codes; coordinates corresponding to the ‘A’ chain identifier have been used. Three major clusters are observed each roughly corresponding to the three sets of protein sequences. The ISS cluster includes one of the NAT members (1cjw) suggesting higher similarity between them.

 
The evolutionary trace method (Litcharge et al., 1996Go; Innis et al., 2000Go) maps the evolutionary path in different partitions of the dissimilarity-based dendrograms and categorizes alignment positions into invariant, cluster (class)-specific and variable types. Residues that are invariant may be important for the structure of the fold or crucial for the function. Residues that are class-specific could account for the substrate specificity: in this case, for binding to substrates with acyl chains of different lengths. Variable residues are unlikely to be important and could correspond to non-functional loop regions.

Three-dimensional modelling was performed using MODELLER (Sali and Blundell, 1993Go) and subsequently loop re-modelling was performed using COMPOSER (Sutcliffe et al., 1987aGo,bGo). The crystal structure of EsaI (Watson et al., 2002Go) served as the structural template for EagI, CarI, LuxI, RhlI and LasI. Models were energy minimized using standard TRIPOS parameters and validated using VERIFY3D (Eisenberg et al., 1997Go). The crystal structure of serotonin acetyltransferase [1cjw (Hickman et al., 1999Go)] when used as the structural template gave rise to the best ISS models (for C82060 and BAB06701.1) and 1b87 was similarly used as the template for T34942.

The different substrates for AHL synthases were modelled starting from a phosphopantetheine (PNT) backbone using CHNGEN (C.Ramakrishnan, unpublished results). The docking of S-adenosylmethionine (SAM) and the corresponding substrates was performed using GRAMM (Katchalski-Katzir et al., 1992Go; Vakser, 1995Go). Acyl-ACP was first docked to the protein model followed by modelling the interaction with SAM. Subsequently ligands of the template NAT structures were docked to the ISS models. Selected docked models were further refined using an energy-driven docking procedure in the SYBYL package (Tripos Associates). The final docked structures were examined for non-bonded energies, possible short contacts and hydrophobic interactions at the binding site. Residues in contact with SAM and acyl-PNT were identified by a liberal distance cut-off of 8 Å to account for possible structural alterations at the protein backbone that can occur during ligand binding.


    Results and discussion
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Homologous sequences

Thirty-five members of AHL synthase superfamily identified were closely related to each other (25–57% sequence identity). More than 50 NAT members (13–18%) and 25 homologues of ISS (28–50%) were also identified. Both NAT and AHL synthase superfamilies catalyse similar biochemical reactions although they differ in the nature of the substrates. Moreover, the two superfamilies have similar folds (Watson et al., 2002Go), very similar active sites and ligand-binding clefts. The r.m.s.d. value between serotonin acetyltransferase (PDB code, 1cjw) and EsaI (PDB code, 1kzf) after the best superimposition is 1.8 Å in spite of poor sequence identity (11%).

Search for intermediate sequences

We investigated the possibility that the similarities observed would imply common ancestry or evolutionary origin. Through recursive PSI-BLAST searches, three sequences (C82060, BAB06701.1 and T34942) were identified that share distant similarity with both NAT and AHL synthase superfamilies. These intermediate search sequences (ISS), although of bacterial origin, are not functionally annotated as belonging to either NAT or AHL synthase superfamily. C82060 is an elaA protein in one of the chromosomes of the O1 strain of Vibrio cholerae. BAB06701.1 is from Bacillus halodurans and is a conserved unknown protein; T34942 is a hypothetical protein from Streptococcus coelicolor. Table IGo provides the cut-off parameters and the PSI-BLAST iteration number at which these similarities could be recognized and provides pairwise sequence identities. The mean sequence identity between the three ISS is 37%. Twenty-five sequence homologues of ISS were obtained starting from the ISS sequences where the sequence identity with the three ISS ranges from 28 to 50%. Figure 1aGo shows the principal component analysis of the three sets of proteins (NATs, AHL synthase and ISS) along with their respective homologues. Table IIGo summarizes the fold prediction results of ISS using three different methods [3DPSSM (Kelley et al., 2000Go), GenThreader (Jones, 1999bGo) and BIOINBGU (Fischer and Eisenberg, 1996Go)]. The top-ranking scores by three independent methods relate ISS to NAT fold ensuring that these sequence connections are true positives. The absence of AHL superfamily members in these predictive exercises is due to the recent determination of structural information for this superfamily and therefore not being considered in the fold library. Furthermore, fold prediction exercises obtained by using other ISS homologues as query sequences are likely to yield similar results. This was confirmed by performing fold prediction for five most distantly related ISS homologues. In each instance, the predicted folds were NAT-fold members.


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Table I. Intermediate sequence searches using recursive, PSI-BLAST (Altschul et al., 1997Go).
 

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Table II. Summary of the fold prediction resultsa of intermediate sequences
 
Secondary structure prediction and multiple-sequence alignment

Secondary structural equivalences between the two superfamilies and the intermediate sequences are fairly high. Pairwise alignments of the two superfamilies and the intermediate sequences annotated for predicted secondary structural positions show above 75% secondary structural conservation (data not shown). Figure 2Go shows the multiple sequence alignment of EagI and its homologues (AHL synthases), three structural representatives from the NAT superfamily (PDB codes 1cjw, 1b87 and 1qst) and three ISS. The observed secondary structures in NAT members and in EsaI have been projected on this alignment



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Fig. 2. Structure-annotated multiple sequence alignment of representatives of NAT members and ISS aligned with representative AHL synthase sequences. Where possible, structure-based sequence alignment has been performed using COMPARER (Sali and Blundell, 1990Go). The observed secondary structural positions in NAT structural members and EsaI are indicated: filled arrows for strands and filled cylinders for helices. ET-identified stretches of invariant or class-specific residues are underlined and regions marked as I–VI (evolutionary trace was performed by including close homologues). Single point mutations that lead to loss of function in LuxI (Hanzelka et al., 1997Go) appear as grey shades. Residues predicted to be within 8 Å of bisubstrate (AcCoA, SAM and the acyl-PNT), by homology modelling and GRAMM docking (Katchalski-Katzir et al., 1992Go; Vakser, 1995Go) are marked in bold.

 
Evolutionary trace analysis

Sequence similarity scores between AHL synthase, NATs and three ISS show a more conserved N-terminal part compared with their C-terminal half (data not shown), suggesting that the binding of SAM and AcCoA is common between these proteins and acyl-PNT-binding region is variable and class-specific. Evolutionary trace analyses (Litcharge et al., 1996Go; Innis et al., 2000Go) were performed on NAT and AHL synthase superfamilies and ISS along with their respective homologues to identify invariant and class-specific residues. This analysis was not applied to a joint alignment across superfamilies since the method is only effective in classifying amino acid replacements amongst protein sequences where the sequence identity is reasonably high (30–70%).

Invariant and class-specific ET-identified residue patches are marked in the alignment (see Figure 2Go). These residues occur in six equivalent regions when compared between the three superfamilies. ISS are similar to AHL synthase superfamily members with a conserved Arg and Phe in Region I, LFGI/W motif in Region II and RLL in Region III. This suggests that there are additional players in ISS associated with a NAT-like function. Perhaps ISS is involved in protein–protein interactions, in region II, analogous to ACP binding of AHL synthases. Region VI shows high general conservation across all the members in the alignment (e.g. RXG{phi}, where {phi} corresponds to a hydrophobic residue). The last 20–25 residues in the C-terminal half of the AHL synthase superfamily members may not be critical for their enzymatic activity as suggested by deletion mutation studies (Swift et al., 1993Go). Interestingly, residues in this region are identified as neither invariant nor class-specific within the AHL superfamily by the ET method.

Amongst the AHL synthase superfamily members, ET-identified invariant residues such as Arg in alignment positions 30, 84 and 118 and negatively charged residues in Region I (alignment positions, 53, 55 and 58) could be critical for function. Interestingly, single mutations in all these corresponding positions in LuxI have rendered the protein inactive (Hanzelka et al., 1997Go). Invariant ET-residues in other regions (including Arg at alignment position 38 in Region I, positions 115, 117 and 119 in Region IV, positions 201 and 202 following Region V) could also be important for binding to the other substrate. RhlI, which is involved in the synthesis of BHL, shows anomalous behavior in the invariant residues. Residues which remain invariant in the other AHL synthase superfamily members, such as Ser82, Ser116, Phe119, Ser/Thr140 and Thr161 (alignment position number), are replaced by Cys, Leu, Tyr, Ala and Ala in RhlI, respectively.

Our evolutionary trace analysis identifies sets of class-specific residues within AHL synthases (Table IIIGo). As seen by independent docking studies of the ligands to the models (see later), these residues are within ligand-interacting distances. The class-specific amino residues in Region III are perhaps serving as a secondary interaction site for substrate (acyl-PNT) binding. In the final docked models, Region III is within interacting distance of the variable substrate (indicated in bold in the alignment in Figure 2Go). Bulky hydrophobic residues such as Phe85, Tyr113, Phe120, Ile138, Leu142 and Ile162 (alignment position) in AHL synthase superfamily members are replaced by smaller hydrophobic residues such as Leu/Ile, Met/Ile, Cys/Ala, Ala/Cys, Ala and Ala/Val in 8/12-acyl AHL synthases, respectively. On the other hand, the class-specific amino acid exchanges in RhlI (that binds to a smaller substrate) relate to the accommodation of bulkier hydrophobic residues at the putative acyl-binding site (e.g. Trp114, Tyr198 and Phe199 at alignment position). RhlI recognizes a non-oxidized butyl chain as its substrate (Winson et al., 1995Go), whereas the other members bind to oxidized acyl chains. Interestingly, one class-specific residue identified at alignment position 161, that is a conserved Thr in other AHL synthases but an alanine in RhlI, interacts with the 3-oxo state of the acyl chain in other members. Even amongst the ET-marked class-specific residues, RhlI exhibits anomalous residue substitutions in several alignment positions (in Table IIIGo, for example, Ala at alignment position 120, Val at position 162). Certain class-specific mutations involve polar/charged residue substitutions in longer-acyl AHL synthases at the corresponding hydrophobic putative substrate-binding site (e.g. residues Glu and Arg in LasI at alignment positions 141 and 144).


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Table III. List of class-specific residues within the AHL synthase superfamily
 
Three-dimensional modelling and ligand docking

Three-dimensional models were obtained for the AHL synthases such as EagI, LuxI, CarI, RhlI, TraI, LasI and the ISS using the template structures as described in Methods. The models, especially of the AHL synthases, were reasonable as reflected by high validation scores (Table IVGo). EagI, LuxI, CarI, RhlI, TraI and LasI are very similar in their backbone positions (average r.m.s.d. 0.80 Å). Facile docking was possible in all the cases where the acyl-PNTs and SAMs are embedded inside the V-shaped cleft such that the amine N-atom is oriented close to first carbonyl carbon of PNT acyl chain. This follows the mechanism where cyclization of SAM is driven by the nucleophile N being proximate to the C1 carbon of the phosphopantetheine moiety. A nucleophilic attack initiates the lactonization and cyclization of SAM.


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Table IV. Summary of results obtained for three-dimensional modelling and structure validation of AHL synthases (AS) and intermediate sequences (ISS)
 
The docking studies were performed with all interacting members for which we have reliable information. Interactions could not be considered since the loop that binds to ACP is ill-defined in the template. The N-terminal segment of AHL synthases, comprising Regions I and II, may be involved in ACP binding. The crystal structure of EsaI shows conformational heterogeneity and movements in Region I exhibited as weak electron density. In fact, the lack of coordinates of Region I prevented us (and indeed Watson and co-workers) from modelling the binding of ACP. We generated a homology model where the loop comprising Region I was built using a general loop search and was therefore not very reliable. However, it is interesting that in our docking studies using this model and ACP, the interacting site involves residues 20–50 of EsaI with residues 40–50 as the primary interacting region (data not shown).

The accesssible surface area buried as a result of docking the two small molecules, acyl-PNT and SAM, is of the order of 350 Å2 (380 for EagI and 304 for LasI). The final docked structures provide around 25 residues within interacting distance to the ligands (25 and 28 for EagI and LasI, respectively). A majority of the interacting residues are non-polar/hydrophobic (80–85%) in nature. In all the homologous structures except RhlI, the bisubstrate binding modes were very similar. In RhlI, the orientation of SAM is such that the cyclic group of SAM is docked opposite to the binding pocket but the amine N-atom points to the PNT acyl chain. This difference could be due to the small size of butanoyl-PNT leading to tighter binding or due to limitations of the docking. The acyl-PNT binding mode is similar in all AHL synthase members: in the longer acyl-PNTs, the acyl chain is extended towards the open face of the groove (facing side in Figure 3Go) surrounded by hydrophobic residues. The greater extent of acidic residues at the acyl-PNT and substrate binding site in LasI (also picked up by the ET method) is unclear; perhaps this allows the longer-chain products to be released with lesser affinities to the protein.



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Fig. 3. GRASP (Nicholls et al., 1991Go) representation of the model structure of EagI (a) compared with LasI model (b). SAM and the corresponding acyl-PNT substrate are docked using GRAMM (Katchalski-Katzir et al., 1992Go; Vakser, 1995Go) and shown in stick form. Hydrophobic residues are shown in white, acidic residues in red and basic residues in blue. The acyl-PNT-binding site of LasI is broader and is acidic. (c), (d) Close-up of the binding site for (a) and (b), respectively. Interacting residues are shown in pink, SAM in green and acyl-PNT in purple.

 
Conformational changes and hydrophobic content

In the form where serotonin acetyltransferase is complexed with the bisubstrate analogue, helix 1 in loop 1 (residues 40–80) is longer and more rigid. A similar conformation of ‘loop 1’ is also observed in the other structural members of this superfamily bound only to AcCoA implying that this is the binding site for the acetyl donor. This region is mobile and ill-defined in the uncomplexed form of EsaI structure (Watson et al., 2002Go). However, these conformational changes in helix-1 upon binding could be secondary and may not correspond to the actual region of contact with SAM. Our examination of the final bisubstrate-docked models shows that residues in Regions I–IV (see Figure 2Go) are within interacting distance of SAM and Regions V–VI are involved in interaction with acyl-PNT. SAM and acyl-PNT binding are in predominantly hydrophobic regions similar to the crystal structures of NAT members. In addition, hydrophobic patches of an amphipathic helix prior to ET-marked Region IV (77–87 in EsaI; alignment position 100) are conserved amongst the AHL synthase superfamily; an equivalent helix is not present in the NAT superfamily except 1cjw. This helix could provide additional hydrophobic stabilization to the substrates, especially for the molecules with a larger number of acyl groups.

This paper reports the presence of intermediate sequences connecting two superfamilies that have similar folds. Such intermediate sequence searches (Park et al., 1997Go), are powerful tools in recognizing distant similarities and discovering new evolutionary pathways. There is a pull-down of ISSs using PSI-BLAST with AHL synthase sequence as a query and vice versa. This distant but clear relationship between ISSs and AHL synthases despite the closer similarity to NAT members demonstrates that ISSs are not just homologues of NATs but are connecting links between the two superfamilies. ISS also retains local similarity to AHL synthases at the putative binding site responsible for the interaction of AHL synthases with acyl-carrier proteins. The evolutionary bridge between NAT and AHL synthases could therefore suggest that the intermediate sequences perform N-acetyltransferase activity but are involved in protein–protein interactions. These additional proteins could have a direct role (such as in the transport of the substrate or the product) or a facilitatory role in the acetylation reaction.

This paper also reports a search for sequence determinants of dramatic differences in specificity amongst homologous proteins. These include the class-specific residue exchanges observed by ET and the observation of an acidic patch in our LasI model and the anomalous behaviors of RhlI in residue substitutions. Several interesting ET-identified sites, such as invariant sites at alignment positions 38 and 115 and class-specific residues at alignment positions 85, 99, 120, 123, 137, 141, 142, 144 and 162, have not been discussed earlier. The identification of class-specific residues are especially hard to identify in the absence of an objective algorithm. A total complementarity in amino acid exchanges is not expected since the binding of these homologous proteins for their respective substrate, although high, is not extremely specific; RhlI and LasI are shown also to promote the synthesis of OHHL in small amounts. In addition, conformational changes at the backbone of homologous AHL synthases could doubtless contribute to substrate specificity amongst closely related members. The current modelling strategies are, however, insufficient to depict huge conformational differences. In spite of this limitation, several interesting residue replacements between EsaI, LasI and TraI have been identified. The class-specific residue exchanges can be starting points to design targets for mutagenesis and to develop drugs to inhibit quorum sensing.


    Acknowledgments
 
R.S. is a Wellcome Trust Senior Research Fellow. We thank the National Centre for Biological Sciences for financial support. We thank Dr Simon Swift (University of Nottingham, UK) and Dr Axel Innis for useful discussions and Dr N.Srinivasan for his critical reading of the manuscript. The evolutionary trace method was kindly provided by Professor Tom Blundell’s laboratory (University of Cambridge, UK). The principal component analysis (PCA) program was kindly provided by Professor Mark Johnson (University of Turku, Finland).


    References
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
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Received October 11, 2002; revised January 20, 2003; accepted February 5, 2003.





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