La noción temporal y el referente curricular para la educación inicial

lanB genes are characteristic of class I lantibiotic clusters. They encode a ~120 kDa protein responsible for the dehydration of serine and threonine amino acids to form 2,3-didehydroalanine (Dha) and (Z)-2,3-didehydrobutyrine (Dhb), respectively (Figure 1.11).

The first evidence for a role in the formation of these characteristic non-proteogenic amino acids came through in vivo work. Targeted mutations in producing strains indicated a role for LanB in the dehydration of serine and threonine residues in Pep5 and nisin (Meyer et al. 1995; Koponen et al. 2002). Further work demonstrated that LanB is necessary for dehydration in a non-producing L. lactis strain. Expression of the nisABTC genes efficiently produced fully post-translationally modified prenisin, while expression of just nisBT could produce dehydrated prenisin without thioether rings and a dehydrated form of a non-lantibiotic peptide (Kuipers et al. 2004). Interestingly, the dehydrations of Ser/Thr yielding Dha/Dhb groups in thiopeptides are mediated by enzymesresembling LanB-type lantibiotic dehydratases (Li and Kelly 2010).

48 Subsequently, focus has moved to the expression and purification of an active LanB in

order to study the enzymatic reaction mechanism. Although this has so far proved elusive, a substantial amount of information has been accrued along the way. LanB enzymes are overall hydrophilic proteins, but contain some hydrophobic domains which imply it may be membrane associated. This is supported by the observed co-sedimentation of NisB and SpaB with membrane vesicles (Engelke et al. 1992). Yeast-two-hybrid and immunoprecipitation studies have suggested that LanBCT enzymes are only active in a multimeric lanthionine synthetase complex. For example in nisin biosynthesis, yeast-two-hybrid revealed an interaction between NisB and NisC as well as an interaction between NisC and the NisT, and implied that at least two molecules of NisC and two molecules of NisT are part of the complex (Siegers et al. 1996). Likewise a complex of at least two each of SpaT, SpaB, and SpaC were demonstrated to be associated with the substrate subtilin through yeast-two-hybrid and coimmunoprecipitation experiments (Kiesau et al.

1997). Yet in vivo data indicates a lack of mutual dependence for activity, as deletions of lanC do not prevent the dehydration by LanB, and deletions of lanB do not prevent cyclisation by LanC (Meyer et al. 1995; Koponen et al. 2002).

In the absence of active purified LanB, heterologous expression systems have been established to demonstrate the substrate promiscuity of LanB through the expression of chimeras and non-lantibiotic peptides. The production of non-naturally occurring peptides containing a series of Dhb residues from L. lactis was achieved through expressing both nisBT and a plasmid encoding a specific leader peptide fusion construct (Rink et al.

2007c). Novel hexapeptides fused to the nisin leader and expressed in a L. lactis strain Figure 1.11 : Dehydration and cyclisation of lantibiotic prepropeptides.

The dehydration of Ser/Thr to form Dha/Dhb is catalysed by LanB or LanM. The intramolecular addition of Cys thiols onto Dha/Dhb to form Lan/MeLan is catalysed by LanC or LanM.

49 containing the nisin modifying and export enzymes resulted in the production of correctly

modified fusion peptides (Rink et al. 2005). A L. lactis strain containing nisBTC was found to effectively dehydrate and secrete a wide range of non-lantibiotic peptides, many of which also demonstrated ring formation (Kluskens et al. 2005). The non-lantibiotic peptides included a number of animal hormones such as vasopressin, angiotensin and erythropoietin. Post-translational modifications by NisB and NisC still occur when the nisin leader is preceded by a 27 amino acid Sec signal peptide, enabling exploitation of the Sec pathway of L. lactis for the secretion of dehydrated variants of therapeutic peptides (Kuipers et al. 2006).

1.3.4.1.2 LanC

lanC genes encode 400-450 amino acid enzymes responsible for cyclisation reactions yielding Lan and MeLan bridges. Dha and Dhb are subject to nucleophilic attack by the thiol group from a cysteine residue located elsewhere in the peptide chain. This intramolecular cyclisation reaction forms characteristic thioether crosslinks. Those formed from Dha are Lan bridges, while those formed from Dhb are MeLan bridges (Figure 1.11).

Section 1.3.4.1.1 referred to studies in which LanBCT enzymes were found only to be active in a multimeric lanthionine synthetase complex (Siegers et al. 1996; Kiesau et al.

1997). Evidence for the role of NisC was initially provided through in vivo work. His-tagged nisin precursors expressed in a nisC mutant were dehydrated but did not contain (Me)Lan bridges, implying NisC is required for nisin maturation (Koponen et al. 2002).

Subsequently, purified NisC was able to perform all five cyclisation reactions on a dehydrated NisA substrate, confirming the activity of NisC in vitro. (Li et al. 2006). Further in vivo work has demonstrated the relaxed substrate specificity of LanC enzymes. NisC is able to cyclise a variety of peptides unrelated to nisin, as long as they are fused to the C-terminus of the NisA leader peptide (Rink et al. 2007a).

Overall, sequence identity between LanC enzymes is low, only 20-30 %, but there are a few strictly conserved histidine and cysteine residues. These were proposed to act as ligands to coordinate zinc binding which likely has a role in the activation of Dha and Dhb residues (Okeley et al. 2003). This has been verified through the X-ray-structure of NisC (Li et al. 2006). Subsequent mutagenesis identified residues essential for catalysis in NisC and SpaC (Helfrich et al. 2007; Li and van der Donk 2007).

In a regioselective reaction, bond formation occurs in one direction. In type A lantibiotics, cyclisation occurs in the C-to-N terminal direction to form an endocyclic enolate, whereas some substrates of type B lantibiotics undergo cyclisation in the opposite direction to form

50 an exocyclic enolate (Zhou and van der Donk 2002). There is evidence the leader peptide

controls the regioselectivity of (Me)Lan formation (Section 1.3.4.1.3). In the cyclisation reaction, the thiol functional group of the cysteine residue undergoes a 1,4-nucleophilic attack on the Michael acceptor system of the Dha/Dhb. It has been observed that the cyclisation of short peptide analogues of type A lantibiotic ring systems occurs spontaneously to form the naturally occurring Lan or MeLan diastereomer (Zhou and van der Donk 2002). However in type B lantibiotics, some cyclisations occur in the N-to-C terminal direction and there is evidence to suggest that in the absence of LanC, a mixture of stereoisomers is created (Zhou and van der Donk 2002). Likewise there is an indication of a similar tendancy towards the natural stereoisomer in nonenzymatic cyclisations of precursors. Although a mixture of stereoisomers is created, the major product has the stereochemistry of the natural lathionine. However these results are only clear-cut when the substrate only has the capacity to form one ring. In more complex substrates chemoselectivity is apparent, as cysteine residues will react preferentially with Dha rather than Dhb to form Lans over MeLans. (Zhou and van der Donk 2002). Therefore the main challenge for LanC enzymes is not the activation of the reaction or the control of stereochemistry and regioselectivity of this cyclisation but is instead to overcome the chemoselectivity that would otherwise result in Lan formation over MeLan formation.

1.3.4.1.3 LanM

As an alternative to the individual LanB and LanC genes, type AII lantibiotic gene clusters encode a single bifunctional LanM responsible for both the dehydration and cyclisation reactions (Figure 1.11). While the N-termini of LanM enzymes share no similarity to LanB enzymes, the C-termini share ~20 % identity to LanC enzymes (Okeley et al. 2003).

Subsequent to the successful in vitro reconstitution of LanC enzymes, three LanM enzymes were purified for use in assays. LctM, HalM1 and HalM2, responsible for the modification of lacticin 481, haloduracin α and haloduracin β, respectively, all demonstrated dehydratase and cyclase activity in vitro (Xie et al. 2004; McClerren et al.

2006). Studies on LctM and HalM2 suggested that substrate binding via an N-terminal leader results in dehydration and cyclisation with a strong preference for N to C directionality, providing evidence on the role of the leader peptide in controlling the regioseletivity of (Me)Lan formation (Levengood et al. 2007).

The two functions performed by LanM enzymes are likely achieved through different active sites. The detection of completely dehydrated substrates with no or little cyclisation supports the hypothesis that all the dehydration reactions are catalysed before ring cyclisation begins (Miller et al. 2006). Conserved residues in the cyclase domain on LanM

51 include three zinc ligands and the equivalent of His212 in NisC, the residue proposed to be

involved in the activation of Dha/Dhb (Li and van der Donk 2007). In accordance with this, site-directed mutagenesis of these residues in LctM decreased or abolished cyclisation without affecting the dehydration of lacticin 481 (Paul et al. 2007). These results imply separate dehydratase and cyclase domains perform the two reactions independently.

In general, the biosynthetic gene clusters of two component lantibiotics encode an individual LanM for each peptide. Exceptions occur when the two substrates are highly similar, such as with cytolysin (Gilmore et al. 1994). This would tend to imply substrate specificity, but as with LanC enzymes, LanM have remarkable versatility in cyclisation of a variety of peptides. Purified LctM was able to process nonlantibiotics and even non-proteinogenic residues such as homocysteine and β-homocysteine to generate novel thioether linkages (Chatterjee et al. 2006). This promiscuity has subsequently been exploited in the preparation of analogues of both type AI (e.g. subtilin (Liu and Hansen 1992)) and type B (e.g. mersacidin (Szekat et al. 2003)) lantibiotics. Whilst most studies have focused on model antibiotic-producing bacteria, the smaller genomes of cyanobacteria have made use of the promiscuous nature of LanM enzymes. The planktonic marine bacterium Prochlorococcus MIT9313 encodes a single LanM enzyme able to recognise up to 29 different linear ribosomally synthesised peptides as substrates to generate an array of polycyclic, conformationally constrained products with highly diverse ring topologies (Li et al. 2010).

1.3.4.1.4 RamC/LanL

RamC is the cyclase responsible for the formation of Lan bridges in the lantipeptide SapB introduced in Section 1.3.2 (Kodani et al. 2004). The structure of this cyclase is unlike that of the LanB-like or LanM-like enzymes. The N-terminus of RamC exhibits similarity to catalytic domains of Ser/Thr kinases while the C-terminus has some sequence similarity to the C-terminal domain of LanM (Hudson and Nodwell 2004). The kinase domain is thought to be involved in phosphorylation of the Ser residues to facilitate dehydration, with the C-terminal domain catalysing subsequent Lan formation (despite lacking the characteristic zinc-binding and catalytic residues of LanC-like domains).

More recently it has become apparent that RamC is part of a larger group of lantipeptide synthetases, sometimes referred to as class III (whereas LanB/C are class I and LanM are class II). This class includes enzymes with the generic name LanL, of which VenL was the first to be characterised (Goto et al. 2010). Individual expression of each VenL domain revealed that Ser and Thr residues are first phosporylated by the kinase domain, followed by an elimination catalysed by the lyase domain, which results in Dha and Dhb residues

52 (Goto et al. 2010). Subsequent addition of the Cys thiols onto these dehydrated residues

is performed by the cyclase domain, which contains a LanC-like zinc-binding site and active-site residues, yielding (Me)Lan bridges (Goto et al. 2010). Significantly, the expression of the VenL N-terminus is the first example of an in vitro reconstituted peptide dehydratase, as previously only bifunctional lantipeptide synthetases had been reconstituted in vitro.

1.3.4.1.5 Linaridins

Recently, a new class of post-translationally modified peptides have been identified in which modifications previously described in lantibiotics are carried out by unusual enzymes or via an alternative modification pathway to those described in lantibiotic biosynthesis. These clusters contain cypL homologs and are proposed to be involved in the biosynthesis of non-cyclised peptides containing dehydrated amino acids, referred to as linaridins (Claesen and Bibb 2010). The type member of the linaridin family is cypemycin (described in Section 1.3.2). The cyp cluster does not contain a conventional LanB enzyme, instead CypH and CypL are essential for cypemycin biosynthesis. It is proposed that CypH and/or CypL are responsible for dehydration of the Thr residues of cypemycin to Dhb, however neither CypH nor CypL activity could be reconstituted in vitro (Claesen and Bibb 2010).