Identification of the Catalytic Residues in the Cyclase Domain of the Class IV Lanthipeptide Synthetase SgbL [original]

Identification of the Catalytic Residues in the Cyclase

Domain of the Class IV Lanthipeptide Synthetase SgbL

Julian D. Hegemann*[a] and Roderich D. Süssmuth*[a]

Lanthipeptides belong to the family of ribosomally synthesized

and post-translationally modified peptides (RiPPs) and are

subdivided into different classes based on their processing

enzymes. The three-domain class IV lanthipeptide synthetases

(LanL enzymes) consist of N-terminal lyase, central kinase, and

C-terminal cyclase domains. While the catalytic residues of the

kinase domains (mediating ATP-dependent Ser/Thr phosphor-

ylations) and the lyase domains (carrying out subsequent

phosphoserine/phosphothreonine (pSer/pThr) eliminations to

yield dehydroalanine/dehydrobutyrine (Dha/Dhb) residues)

have been characterized previously, such studies are missing for

LanL cyclase domains. To close this gap of knowledge, this

study reports on the identification and validation of the

catalytic residues in the cyclase domain of the class IV

lanthipeptide synthetase SgbL, which facilitate the nucleophilic

attacks by Cys thiols on Dha/Dhb residues for the formation of

β-thioether crosslinks.

Representatives of the RiPP natural product family are defined

through a shared biosynthetic logic, where genetically encoded

precursor peptides are matured into the final natural products

by the activity of processing enzymes.[1]

Lanthipeptides are members of the RiPP family that contain

characteristic β-thioether crosslinks; so-called (methyl)

lanthionine ((Me)Lan) residues.[1] These crosslinks are installed

by: 1) activation/elimination of Ser/Thr hydroxy groups to yield

Dha/Dhb residues, and 2) nucleophilic attacks on these

unsaturated double bonds by Cys thiolates to yield, after an

additional protonation step, the (Me)Lan moieties.

The lanthipeptide precursors contain N-terminal leader and

C-terminal core peptide regions.[1] The leader regions feature

conserved motifs needed for substrate recognition by the

processing enzymes.[1–2] The core peptide regions are those

where the dehydroalanine/dehydrobutyrine (Dha/Dhb) residues

are intermediately formed and (sometimes only partially)

converted into the (Me)Lan crosslinks (Figure 1). After full

modification of the core peptide, the mature lanthipeptide is

released via proteolytic removal of the leader and subsequently

often exported into the extracellular space.[1–3]

Lanthipeptides can be further subclassified on the basis of

their corresponding biosynthetic enzymes and there are

currently five different classes known.[1,4] Class IV lanthipeptide

synthetases, so-called LanL enzymes, feature a three-domain

architecture consisting of a lyase, a kinase, and a cyclase

domain (Figure 1A).[1,3a,4e,5] LanLs accomplish the generation of

the Dha/Dhb residues through the concerted action of the lyase

and kinase domains. First, the central kinase domain binds the

precursor and facilitates the ATP-dependent Ser/Thr

phosphorylation.[1,2,6] Then, the N-terminal lyase domain cata-

lyzes the phosphate elimination from the pSer/pThr residues,

yielding the Dha/Dhb residues[1,7] (Figure 1B). Finally, the C-

terminal cyclase domain mediates the nucleophilic attack of the

Dha/Dhb residues by Cys thiolates and facilitates the subse-

[a] Dr. J. D. Hegemann, Prof. Dr. R. D. Süssmuth

Institute of Chemistry, Technische Universität Berlin

Strasse des 17. Juni 124, 10623, Berlin (Germany)

E-mail: [email protected]

[email protected]

Supporting information for this article is available on the WWW under

https://doi.org/10.1002/cbic.202100391

an open access article under the terms of the Creative Commons Attribution

Non-Commercial NoDerivs License, which permits use and distribution in

any medium, provided the original work is properly cited, the use is non-

commercial and no modifications or adaptations are made.

Figure 1. (A) Schematic representation of the domain organization in class IV

lanthipeptide synthetases. (B) (Me)Lan formation by class IV lanthipeptide

synthetases. The domain active during a specific step is shown in color,

while the domains not participating in this step of catalysis are shown in

grey. (C) Schematic representation of the class IV lanthipeptide globisporin;

Abu=aminobutyric acid.

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quent protonation of the resulting enolates to yield the (Me)Lan

crosslinks (Figure 1B).[1,2b,4e,6]

In our recent review[1b] on class III and IV lanthipeptide

synthetases, we noted that whereas the catalytic residues of the

lyase[7] and kinase[6] domains of class IV lanthipeptide synthe-

tases have been reported, the catalytic residues of the LanL

cyclase domains still remained uncharacterized. Due to the

homology between the cyclases of class I, II, and IV lanthipep-

tide synthetases, we further hypothesized that class IV cyclase

domains would feature the same conserved catalytic residues

that were previously reported[1b,c,8] for class I and II cyclases.

Indeed, an alignment of well-studied class I[1c,8a–d] and II

cyclases[1c,8b,e] with the cyclase domain of the previously

characterized class IV lanthipeptide synthetase SgbL[2b,6] as well

as with the cyclase domains of a selection of other reported

class IV enzymes[2b,3a,4e,5] (Figure 2A) shows the full conservation

of the putative catalytic residues. To experimentally validate the

role of these residues in (Me)Lan formation, we performed a

mutational analysis of the SgbL enzyme that catalyzes the core

peptide modification during the biosynthesis of the class IV

lanthipeptide globisporin (Figure 1C).[2b,6]

By mutation of the his6-sgbL gene in an expression vector,

heterologous expression in E. coli, and in vitro reconstitution,

the five predicted catalytic residues were exchanged to Ala.

According to bioinformatic analysis, three of these residues

(Cys769, Cys814, His815) would be coordinating a zinc ion that

would act as a Lewis acid to increase the nucleophilicity of the

Cys thiols in the SgbA core region (Figure 2B).[1b,c,8a–d] In addition,

His710 would fulfill the role of the catalytic acid that protonates

the enolate intermediates and that is further activated by

interaction with Asp642 (Figure 2B).[1b,c,8a–d]

To assess if the generated SgbL variants (SgbL(D642A), SgbL

(H710A), SgbL(C769A), SgbL(C814A), SgbL(H815A)) are indeed

unable to catalyze the (Me)Lan formation, a series of in vitro

assays were performed using the SgbL WT enzyme as a positive

control. However, whereas Ser/Thr dehydrations are easily

observable by mass spectrometry (MS) due to the loss of a

water molecule (18 Da), the (Me)Lan formation is mass

neutral. Hence, the lack of (Me)Lan formation can only be

detected indirectly by labeling of the free Cys residues present.

This labeling can be accomplished by addition of N-ethyl-

maleimide (NEM), a thiol-selective electrophile.[2b,6,9] Whenever a

free thiol adds to an NEM molecule, the mass of the compound

increases by 125 Da, which can be readily tracked by MS.

Another important aspect to consider for these assays is

that the steric hindrance imposed by the methyl groups in Thr-

derived Dhb side chains efficiently suppresses non-enzymatic

cyclizations to occur, while the less sterically hindered unsatu-

rated double bonds in Dha residues can be more readily

attacked by Cys thiol nucleophiles in the absence of an

enzyme.[2b] Thus, a variant of the precursor peptide SgbA was

heterologously produced, where the only ring-forming Ser in

the core region was replaced with a Thr (His6-SgbA(S20T),

Figure 3A) to suppress the background of non-enzymatic Lan

formation in our assays.

Accordingly, the His6-SgbA(S20T) precursor variant was

incubated overnight under assay conditions either by itself

(negative control), with SgbL (positive control), or with one of

the five SbgL variants (Figure 3B).

On the next day, the reaction mixtures were digested with

trypsin to facilitate the MS analysis of the modified SgbA(S20T)

core peptides.[2b] The samples were then split to enable the

comparison of the mass spectra before and after NEM treat-

ment. Hence, one part of each sample was directly desalted and

applied to MS, while the other part was first treated with NEM

before desalting and MS analysis (Figure 3B).

Indeed, all SgbL variants introduced up to four dehydrations

into the His6-SgbA(S20T) precursor peptide as did the WT

enzyme (Figure 4), which was expected as it was

previously[2b,4e,6] shown that the LanL lyase/kinase domains can

incorporate the dehydrations independently from the cyclase

domain.

However, when comparing the results of the NEM assays, it

became apparent how the Ala exchanges affected the ability of

SgbL to introduce the MeLan crosslinks into His6-SgbA(S20T).

The assay with WT SgbL yielded a significant amount of fully-

cyclized core peptide that did not add any NEM. In contrast,

none of the SgbL variants were able to install all four MeLan

crosslinks into the precursor peptide and the major products of

all the SgbL variant assays always had four NEM molecules

added. Thus, these experiments clearly demonstrate that the

Ala exchange of any of the five catalytic residues predicted

through homology analysis (almost) completely abrogates the

ability of SgbL to introduce β-thioether crosslinks into His6-

SgbA(S20T).

Figure 2. (A) Excerpt of the alignment of a selection of known functional

LanL enzymes[2b,4e,5] with the representative cyclases of class I (NisC[8a]) and

class II (CylM[8e]) lanthipeptide synthetases. The red labels above the

alignment refer to the corresponding predicted catalytic residues in SgbL.

(B) Putative catalytic mechanism of the SgbL cyclase-mediated (Me)Lan

formation.

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In conclusion, this study provides the first experimental

validation of the function of the predicted[1b] conserved catalytic

residues in the cyclase domain of a class IV lanthipeptide

synthetase. Adding to previous studies that were focused on

identifying and characterizing the catalytic residues situated in

the kinase[6] and lyase[7] domains of LanL enzymes, these

experiments therefore lead to a more complete understanding

of the underlying principles of class IV lanthipeptide biosyn-

thesis.

Figure 3. (A) An S20T exchange in the SgbA precursor peptide yields a core

peptide that can only form MeLan crosslinks. (B) Schematic representation of

the general workflow of the in vitro modification assays.

Figure 4. Results of the in vitro modification assays of His6-SgbA(S20T) with

SgbL and variants thereof. Traces in teal and orange show the MS data

before and after NEM labeling, respectively. For the negative control shown

on top, the precursor was incubated overnight under assay conditions in the

absence of any modification enzyme. The MS signals shown were obtained

by MALDI-TOF-MS analysis of the samples after desalting and using sinapic

acid as MALDI matrix; u=unmodified peptide.

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Acknowledgements

We would like to thank Dr. Chris Weise (Freie Universität Berlin)

for assistance with the MALDI-TOF-MS measurements. Open

Access funding enabled and organized by Projekt DEAL.

Conflict of Interest

The authors declare no conflict of interest.

Keywords: biocatalysis ·biosynthesis ·lanthipeptides ·natural

products ·RiPPs

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Manuscript received: August 3, 2021

Revised manuscript received: September 6, 2021

Accepted manuscript online: September 7, 2021

Version of record online: September 12, 2021

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