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\begin{document}

\title{Mechanistic Investigations into the Catalytic Mode of a Dehydratase
Complex Involved in the Biosynthesis of Lantibiotic Cacaoidin}



\author[1]{Yanqing Xue}%
\author[1]{Miao Li}%
\author[1]{Ling Hu}%
\author[1]{Jianing Liu}%
\author[2]{Lifeng Pan}%
\author[1]{Wen Liu}%
\affil[1]{Shanghai Institute of Organic Chemistry Chinese Academy of Sciences}%
\affil[2]{Shanghai Institute of Organic Chemistry}%


\vspace{-1em}



  
  \date{June 15, 2024}


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\selectlanguage{english}
\begin{abstract}
Dehydration of serine/threonine residues necessitates the activity of a
dehydratase enzyme (domain) during the biosynthesis of ribosomally
synthesized and post-translationally modified peptide (RiPP). Recently,
it was reported that the dehydration process in thioviridamide relies on
a distinct dehydratase complex which showcases the activities of a
phosphotransferase TvaC for serine/threonine phosphorylation and a lyase
TvaD for subsequent phosphate elimination. Herein, we report that the
dehydration process of lantibiotic cacaoidin involves a similar
dehydratase complex, CaoK/CaoY. Remarkably, this dehydratase complex
exhibits flexible enzymatic activity and tolerates significant
variations in its substrate peptide sequence. By binding with the leader
peptide (LP) sequence of precursor peptide CaoA, the dehydration
reactions proceed directionality from the C-terminus of the core peptide
(CP) to its N-terminus, and C-terminally truncated variants of CP are
acceptable. We show that fusing CaoK to CaoY in a 1:1 molar ratio
enables the resulting enzyme CaoYK to exert enhanced dehydration
activity. CaoK binds with the LP to improve its own solubility and to
ensure the phosphate transfer activity, while CaoY functions
independently of the LP. This work advances our understanding of the
dehydration process of cacaoidin, and provides valuable enzymes and
methods for the studies of the rapidly emerging RiPPs.%
\end{abstract}%



\sloppy


\textbf{Cite this paper:}\emph{Chin. J. Chem.} \textbf{2023} , \emph{41}
, XXX---XXX. \textbf{DOI: 10.1002/cjoc.202300XXX}

Mechanistic Investigations into the Catalytic Mode of a Dehydratase
Complex Involved in the Biosynthesis of Lantibiotic Cacaoidin

Yanqing Xue,\textsuperscript{++,a}Miao Li,\textsuperscript{++,a,\emph{b}}
Ling Hu,\textsuperscript{a}Jianing Liu,\textsuperscript{a} Lifeng
Pan,*\textsuperscript{,a,\emph{b}} and Wen Liu*\textsuperscript{,a}

\textsuperscript{a} State Key Laboratory of Bioorganic and Natural
Products Chemistry, Shanghai Institute of Organic Chemistry, University
of Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032,
China\textsuperscript{b} School of Chemistry and Materials Science,
Hangzhou Institute for Advanced Study, University of Chinese Academy of
Sciences, 1 Sublane Xiangshan, Hangzhou 310024, China\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
\textbf{Keywords}\tabularnewline
\midrule
\endhead
Dehydratase complex \textbar{} Cacaoidin \textbar{} Dehydration reaction
\textbar{} Ribosomally synthesized and post-translationally modified
peptide\tabularnewline
\textbf{Comprehensive Summary}\tabularnewline
Dehydration of serine/threonine residues necessitates the activity of a
dehydratase enzyme (domain) during the biosynthesis of ribosomally
synthesized and post-translationally modified peptide (RiPP). Recently,
it was reported that the dehydration process in thioviridamide relies on
a distinct dehydratase complex which showcases the activities of a
phosphotransferase TvaC for serine/threonine phosphorylation and a lyase
TvaD for subsequent phosphate elimination. Herein, we report that the
dehydration process of lantibiotic cacaoidin involves a similar
dehydratase complex, CaoK/CaoY. Remarkably, this dehydratase complex
exhibits flexible enzymatic activity and tolerates significant
variations in its substrate peptide sequence. By binding with the leader
peptide (LP) sequence of precursor peptide CaoA, the dehydration
reactions proceed directionality from the C-terminus of the core peptide
(CP) to its N-terminus, and C-terminally truncated variants of CP are
acceptable. We show that fusing CaoK to CaoY in a 1:1 molar ratio
enables the resulting enzyme CaoYK to exert enhanced dehydration
activity. CaoK binds with the LP to improve its own solubility and to
ensure the phosphate transfer activity, while CaoY functions
independently of the LP. This work advances our understanding of the
dehydration process of cacaoidin, and provides valuable enzymes and
methods for the studies of the rapidly emerging RiPPs.\tabularnewline
\bottomrule
\end{longtable}

Background and Originality Content

Dehydroalanine (Dha) and dehydrobutyrine (Dhb) are nonproteinogenic
residues commonly arising from the dehydration of Ser/Thr residues in
bacterial ribosomally synthesized and post-translationally modified
peptides (RiPPs) (Figure 1A). \textsuperscript{{[}1{]}} We and others
recently reported that the dethiolation of cysteine can serve as a
supplementary method for the generation of Dha residue, as observed in
linaridins and several novel class I
lanthipeptides.\textsuperscript{{[}2{]}} Dha/Dhb residues contain polar
double bonds, serving as reactive functional groups for a variety of
enzymatic post-translational modifications (PTMs) that contribute to the
formation of elaborate functionalities in RiPPs.
\textsuperscript{{[}3{]}}

The formation of Dha/Dhb residues has been well established in several
subfamilies of RiPPs, such as the four known classes of
lanthipeptides.\textsuperscript{{[}4{]}} They utilize either
glutamination (class I) or phosphorylation (classes II-IV) to activate
the \selectlanguage{greek}β-\selectlanguage{english}hydroxy groups of Ser/Thr for subsequent elimination reactions
(Figure 1A). In contrast to class II lanthipeptides (LanM) where a
single dehydration domain is responsible for catalysis, both class III
(LanKC) and class IV (LanL) lanthipeptides employ a coordinated lyase
domain and phosphotransferase domain for efficient formation of Dha/Dhb
residues (Figure S1). The lyase domain found in LanKC and LanL exhibits
a limited similarity to the HopA1 effector family proteins.
\textsuperscript{{[}5{]}} This family protein functions as a
phosphothreonine lyase through targeting and irreversibly eliminating
the phosphate group of several cognate phosphorylated kinases with the
purpose of disrupting host signal transduction pathways.
\textsuperscript{{[}6{]}} Recent structural studies have revealed that
in both LanKC and LanL, the phosphotransferase domain couples with the
lyase domain and leader peptide (LP), facilitating the efficient
transfer of the phosphorylated core peptide (CP) to the cavity of the
lyase domain.\textsuperscript{{[}7{]}}

We and others evidenced \emph{in vivo} and \emph{in vitro} that the
Dha/Dhb formation in thioviridamide (TVA) involves a dehydratase complex

\textbf{Figure 1} Dha/Dhb formation (A), the structure of cacaoidin (C),
and the sequence of precursor peptide CaoA (C). Residues that undergo
dehydration reactions are highlighted in blue.

comprising of a phosphotransferase protein TvaC and a lyase protein TvaD
(Figure S1). \textsuperscript{{[}8{]}} A similar dehydratase complex has
also been identified in cacaoidin (CAO), a potent lantibiotic against
MRSA (Figure 1B). \textsuperscript{{[}9{]}} In the putative dehydratase
complex, CaoK exhibits 39.2 \% identity with TvaC, while CaoY shows 33.5
\% identity with TvaD. The dehydration process of CAO can differ from
that of TVA, as the former lacks the thioamidation modification that
occurs prior to the dehydration reaction in the latter. The number of
residues undergoing dehydration in the precursor peptide of CAO amounts
to eight (Figure 1C), significantly more than in TVA, which contributes
to the increased complexity of the dehydration process.

Results and Discussion

\textbf{The activities of CaoK and CaoY in the dehydration of
CaoA\emph{in vivo}} \emph{.}

We bioinformatically identified homologue genes of \emph{caoK}
and\emph{caoY} in a commercially available strain \emph{Streptomyces
cacaoi} subsp. cacaoi ATCC 19732 encoding a putative BGC of cacaoidin
and accordingly designated their gene product as CaoK (WP\_086817888.1)
and CaoY (WP\_086817890.1), respectively (Figure S2). A genes
coexpression strategy was adopted to examine dehydrated intermediates of
precursor peptide CaoA (\textbf{1} , WP\_169729690.1) by heterologous
expression of precursor encoding gene \emph{caoA} with related PTMs
genes \emph{caoK} and \emph{caoY} in \emph{Escherichia coli}
.\textsuperscript{{[}8b{]}} The major product is an eight-dehydrated
peptide \textbf{1-1} (- 144 Da, - 8 x H\textsubscript{2}O compared with
CaoA), accompanied by several minor intermediates which are partially
phosphorylated or dehydrated, i.e., \textbf{1-2} to \textbf{1-9} (Figure
S3). \textbf{1-1} was characterized by high-performance liquid
chromatography (HPLC) with high-resolution mass spectrometry (HR-MS) and
tandem MS. The observed m/z of \textbf{1-1} shows good agreement with
the calculation value (Figure S3). However, the MS/MS fragmentation
of\textbf{1-1} was seldom observed in the CP region, which is highly
hydrophobic and does not ionize effectively (Figure S4). We were unable
to fully determine the structures of the aforementioned intermediates,
which would benefit providing insights into the directionality of
dehydration. Nevertheless, the limited fragments obtained
from\textbf{1-2} and \textbf{1-3} supported the proposal that
dehydration reaction occurs from the C-terminus of CP to the N-terminus
(Figures S5-S6).

To assess the individual activities of CaoK and CaoY in the dehydration
process, we engineered a substrate peptide
gene,\emph{caoA\textsubscript{2}} which encodes substrate peptide
\textbf{2}(Table 1), to coexpress with either the gene \emph{caoK} or
both the\emph{caoK} and \emph{caoY} genes in \emph{E. coli} . Only one
dehydratable residue (Thr6) is retained in \textbf{2} , which minimizes
the formation of byproducts caused by incomplete transformation and thus
facilitates the tracking and differentiation of intermediate(s). A
dominated mono-dehydrated product \textbf{2-1} (- 18 Da, - 1 x
H\textsubscript{2}O compared with \textbf{2} ) was observed in the
coexpression of genes \emph{caoA\textsubscript{2}KY} (Figure 2). Tandem
MS data supported that the dehydration reaction takes place on the Thr6
residue as expected (Figure S7). Omitting \emph{caoY} from the
above\emph{E. coli} coexpression system failed to produce \textbf{2-1}
and led to the accumulation of a new mono-phosphorated variant
\textbf{2-2}(+ 80 Da, + 1 x HPO\textsubscript{3} compared with
\textbf{2} ), resulting from the CaoK-catalyzed phosphorylation of the
Thr6 residue (Figures 2 and S8). These data clearly show that the
phosphotransferase CaoK initially phosphorylates the Ser/Thr residues,
which are subsequently subjected to phosphate elimination catalyzed by
the lyase CaoY in the dehydration process. These observations agree well
with a recent study on the dehydration of cacaoidin by H. Liang \emph{et
al} .\textsuperscript{{[}9c{]}}

\textbf{The Dehydration Reaction Allows for Significant Truncation in
the CP Sequence\emph{.}}

The PTMs of \textbf{2} conducted by the CaoK/CaoY complex, indicate that
there is a tolerance for variations in the CP sequence during the
dehydration process. To further explore the substrate scope,

\textbf{Figure 2} Examination of CaoK and CaoY activities in the
dehydration of CaoA \emph{in vivo} . (A) Structures of the substrate
peptide \textbf{2} and its derivatives. (B) Examination of the product
profiles of the \emph{E. coli} strains by HR-MS. The genes expressed
in\emph{E. coli} include the precursor peptide
gene\emph{caoA\textsubscript{2}} alone (i), and the combinations of
gene\emph{caoA\textsubscript{2}} with gene \emph{caoK} (ii); or
genes\emph{caoYK} (iii). For {[}M + 5H{]}\textsuperscript{5+}
ions:\textbf{2} , calc. 983.2496, obs. 983.2507, and [?]ppm 1.12;
\textbf{2-1} , calc. 979.6475, obs. 979.6484, and [?]ppm 0.92; and
\textbf{2-2} , calc. 999.2427, obs. 999.2423, and [?]ppm 0.40.

we performed additional genetic engineering by variating the amino acid
sequence composition of the core portion in \textbf{2} . Specifically,
we obtained substrate gene \emph{caoA3} \textasciitilde{}
\emph{caoA5}encoding peptide \textbf{3} \textasciitilde{} \textbf{5} ,
respectively (Table 1). In each instance, the engineered substrate gene
was coexpressed with genes \emph{caoK} and \emph{caoY} , the resulting
product was purified and subsequently monitored using HPLC-MS and tandem
MS. \textbf{3} was completely consumed by the CaoK/CaoY complex \emph{in
vivo} , mainly resulting in the formation of two dehydrated variants,
i.e., \textbf{3-1} (- 54 Da, - 3 x H\textsubscript{2}O compared
with\textbf{3} ) and \textbf{3-2} (- 36 Da, - 2 x H\textsubscript{2}O
compared with \textbf{3} ), with a ratio of \textasciitilde{}2:1
(Figures 3A and S9-S10). Trace amount of monophosphorylated \textbf{3-3}
(+ 80 Da, + 1 x HPO\textsubscript{3} compared with \textbf{3-2} ) was
also observed as the precursor for \textbf{3-1} (Figure 3A). This
evidence, combined with the PTMs observed in \emph{E. coli} for
substrate peptide\textbf{2} , supports that the dehydration reaction
allows for C-terminal truncation of the CP sequence.

Peptides \textbf{4} and \textbf{5} were further evaluated to determine
whether non-native peptides could be accepted as substrates for
dehydration. \textsuperscript{{[}10{]}} A small quantity of two variants
of \textbf{4} arise from the CaoK/CaoY-catalyzed dehydration,
i.e.,\textbf{4-1} (- 18 Da, - 1 x H\textsubscript{2}O compared
with\textbf{4} ) and \textbf{4-2} (+ 80 Da, +1 x
HPO\textsubscript{3}compared with \textbf{4} ), while a significant
portion of \textbf{4}remains unmodified (Figures 3B and S11-S12).
Similarly, only a small amount of \textbf{5} was dehydrated to form
\textbf{5-1} (- 18 Da, - 1 x H\textsubscript{2}O) while a relatively
large ratio of mono-phosphorylated variant \textbf{5-2} (+ 80 Da, + 1 x
HPO\textsubscript{3}) was accumulated (Figures 3C and S13-S14). We did
not observe any other dehydrated or phosphorylated variants. It appears
that the CaoK/CaoY complex might not be well-coordinated in their
actions on these two tested non-native peptides, leading to the presence
of residual \textbf{4} and \textbf{5} in each case. The accumulation of
phosphorylated intermediates, \textbf{4-2} and \textbf{5-2} , implies
the impaired elimination activity during the catalysis.

\textbf{The Fused Protein CaoYK Exerts Enhanced Dehydration
Activity\emph{in vitro}.}

Both TvaC/TvaD and CaoK/CaoY were previously coexpressed and copurified
as soluble protein pairs. \textsuperscript{{[}8c, 9c{]}} It was
indicated that CaoK and CaoY form a heterodimer by their elution profile
in size exclusion chromatography. \textsuperscript{{[}9c{]}} We
envisioned here that a fused

\textbf{Figure 3} Substrate tolerance of the dehydration reaction
in\emph{E. coli} . (A) Structures of the products derivatized from the
designed substrate peptides. For the sequences of substrate peptides,
see Table 1. (B) Examination of the product profiles of the
recombinant\emph{E. coli} strains by HR-MS. The genes coexpressed in
\emph{E. coli}include the genes \emph{caoYK} with gene
\emph{caoA\textsubscript{3}}(i), \emph{caoA\textsubscript{4}} (ii), or
\emph{caoA\textsubscript{5}}(iii). For {[}M + 5H{]}\textsuperscript{5+}
ions: \textbf{3-1} , calc. 978.8412, obs. 978.8493, and [?]ppm 8.28;
\textbf{3-2} , calc. 982.4433, obs. 982.4492, and [?]ppm 6.01;
\textbf{3-3} , calc. 998.4365, obs. 998.4343, and [?]ppm 2.20; \textbf{4}
, calc. 979.2478, obs. 979.2447, and [?]ppm 3.17; \textbf{4-1} , calc.
975.6458, obs. 975.6425, and [?]ppm 3.38;\textbf{4-2} , calc. 995.2410,
obs. 995.2374, and [?]ppm 3.62; \textbf{5} , calc. 997.4751, obs.
997.4695, and [?]ppm 5.61; \textbf{5-1} , calc. 1013.4683, obs. 1013.4642,
and [?]ppm 4.05; \textbf{5-2} , calc. 993.8730, obs. 993.8698, and [?]ppm
3.22.

protein CaoYK, in which the phosphotransferase CaoK is covalently linked
to the C-terminus of the lyase CaoY, can exert enhanced activity due to
spatial adjacency. A flexible sequence (\textbf{L1} , Table 1) that
contains the recognition and cleavage site for Human Rhinovirus (HRV) 3C
protease, was employed as the internal linker between CaoY and CaoK
(Figure 4A). The design protein CaoYK was characterized to be a monomer
in solution by Multi-Angle Light Scattering (MALS) (Figure 4B). In the
presence of ATP and Mg\textsuperscript{2+}, CaoYK can effectively
transform \textbf{2} into the -18 Da mono-dehydrated product\textbf{2-1}
, compared to the purified CaoK/CaoY complex \emph{in vitro}(Figure 4C),
supporting the notion that the phosphotransferase CaoK and the lyase
CaoY work together as a heterodimer during the dehydration progress.

\textbf{The Design and Characterization of LP/Substrate Fused Proteins.}

The fusion of LP sequence to its corresponding PTM enzyme succeeded in
creating a variety of constitutively active RiPP maturases. This
strategy enables the utilization of CPs as substrates, eliminating the
need for larger full-length precursor peptides \emph{in vitro} and has
been developed for the biosynthesis of
lanthipeptides,\textsuperscript{{[}11{]}} cyanobactins,
\textsuperscript{{[}12{]}} and microviridins. \textsuperscript{{[}13{]}}
We obtained one such fused protein CaoYK\textsubscript{LP} by linking
the leader sequence to the C-terminus of protein CaoYK via another
flexible sequence (\textbf{L2} , Table 1), which contains the
recognition and cleavage site for Tobacco Etch Virus (TEV) protease
(Figure 4A). For the sake of convenient nomenclature, peptides fused to
the PTM enzymes are subscripted in the final protein.
CaoYK\textsubscript{LP} is also an active monomer, as observed through
MALS analysis and the \emph{in vitro} transformation from \textbf{2} to
\textbf{2-1} (Figure 4B, C). However, its dehydration activity is not as
potent as that of CaoYK. Interestingly, CaoYK\textsubscript{LP} has a
more compact size compared to CaoYK, indicating structural changes in
enzymes triggered by LP binding (Figure 4B). Surprisingly, it was
observed that CaoK\textsubscript{LP} exists in a soluble form,
indicating that the binding of the LP contributes to the improved
solubility of CaoK (Figure S15). However, neither of these two enzymes
(CaoK\textsubscript{LP} and CaoYK\textsubscript{LP}) could phosphate or
dehydrate octapeptide \textbf{6} \emph{in vitro} (Table 1), using
standard reaction conditions described above. Possibly, the covalent
linkage of CP with LP is essential for the phosphorylation step which
initiates the complete dehydration reaction.

To better depict the catalytic mode of the LP fused proteins, we further
fused DNA codons encoding \textbf{6} to
gene\emph{caoYK\textsubscript{LP}} , resulting in the recombinant
gene\emph{caoYK\textsubscript{LP-6}} encoding the new design
CaoYK\textsubscript{LP-6} (Figure 4A, Table 1).
CaoYK\textsubscript{LP-6} is also a monomer revealed by the MALS
analysis (Figure 4B). The C terminus portion of
CaoYK\textsubscript{LP-6}, \textbf{LP-6} (or its derivatives), was then
released proteolytically using TEV protease. Only the mono-dehydrated
product, \textbf{LP-6-1} , was observed and confirmed by HR-MS and
tandem MS analysis, indicating that the dehydration reaction
specifically targets the Thr6 residue in \textbf{LP-6} (Figures 4D and
S16). No substrate or intermediate \emph{en route} to product
\textbf{LP-6-1} was detected in the digestion mixture by TEV protease.
These data suggest that the substrate peptide \textbf{LP-6} , provided
\emph{in cis} to CaoYK, can access the cavities of both CaoK and CaoY in
CaoYK\textsubscript{LP-6}. This phenomenon is reminiscent of
N-methyltransferase in fungi and BURP-domain cyclase in plant, where the
enzymes are naturally fused to their respective substrate peptides for
efficient catalysis. \textsuperscript{{[}14{]}}

\textbf{Figure 4} The design and characterization of fused proteins. (A)
Diagram of the fused enzymes CaoYK, CaoYK\textsubscript{LP}, and
CaoYK\textsubscript{LP-6}. (B) MALS analysis. For theoretical monomer
molecular weight, CaoYK, 80.4 kDa; CaoYK\textsubscript{LP}, 85.0 kDa;
CaoYK\textsubscript{LP-6}, 85.8 kDa. (C) \emph{In vitro} assays of the
fused enzymes activity. HPLC-MS examination of the conversion of
peptide\textbf{2} to \textbf{2-1} . i, peptide \textbf{2} (control); and
ii, iii, iv, in the presence of CaoY/CaoK complex, CaoYK, or
CaoYK\textsubscript{LP}, respectively. For extracted ion chromatograms
(EICs), the {[}M+5H{]}\textsuperscript{5+} for \textbf{2}
and\textbf{2-1} are 983.65 (black trace) and 980.05 (blue trace),
respectively. (D) HR-MS data of \textbf{LP-6-1} . The core portion
of\textbf{LP-6-1} are indicated. {[}M + 4H{]}\textsuperscript{4+} m/z:
calc. 1060.2458, obs. 1060.2430, and [?]ppm 2.64; {[}M + 3H +
K{]}\textsuperscript{4+} m/z: calc. 1069.7348, obs. 1069.7295, and [?]ppm
4.95; {[}M + 2H + 2K{]}\textsuperscript{4+} m/z: calc. 1079.2238, obs.
1079.2178, and [?]ppm 5.56.

\textbf{Mechanistic Insights into the Catalytic Mode of CaoK and CaoY.}

Despite significant efforts, including the utilization of intact monomer
protein or complex formed by HRV 3C and/or TEV protease(s) digestion, we
were unable to crystallize our two designs, CaoYK\textsubscript{LP} and
CaoYK\textsubscript{LP-6}. To further investigate the molecular
catalytic mode, AlphaFold was used to generate a predicted structure
model for the monomer CaoYK\textsubscript{LP-6} (Figure
S17A).\textsuperscript{{[}15{]}} The prediction accuracy of \textbf{L1}
and\textbf{L2} sequences are not as high as the other regions (Figure
S17B-C), consistent with their flexible nature. Interactions between
CaoK and CaoY (Figure S17D), as well as those between CaoK and the LP
(Figure S17E), were identified in the structural model which are akin to
the model of the CaoK/CaoY/CaoA complex. \textsuperscript{{[}9c{]}} The
well-ordered interfaces between CaoK/CaoY and CaoK/LP, indicate that the
designed fused protein CaoYK\textsubscript{LP-6} are primarily
stabilized by the interfaces instead of the linkers. Based on the
structural model (Figure S17E), we suggested that the \emph{in
cis}provided LP in CaoYK\textsubscript{LP} may compete with the leader
portion of substrate \textbf{2} for the same LP-binding site, resulting
in impaired activity compared to CaoYK.

To determine the LP dependence of CaoY, both His\textsubscript{6} and
thioredoxin tags were fused to its N-terminus for protein purification
(Figure S15). The purified recombinant CaoY was used for elimination of
phosphorylated analog \textbf{7} \emph{in vitro} (Table 1). However, the
transformation of \textbf{7} was not detected by HR-MS (Figure 5).
Parallelly, a new peak of the dehydrated product \textbf{7-1} emerged
when substrate \textbf{7} was incubated with CaoK/CaoY complex, the
fused design enzymes CaoYK, or CaoYK\textsubscript{LP} (Figure 5). The
structure of \textbf{7-1} was further determined by tandem MS (Figure
S18). The phosphate elimination activity of CaoYK competes with that of
CaoYK\textsubscript{LP} in the conversion of \textbf{7} to
\textbf{7-1}(Figure 5). These results suggest that CaoY functions
independently of LP, but its activity is turned off in the absence of
CaoK.

\textbf{Figure 5} Phosphate elimination on a phosphorylated
octapeptide\textbf{7} . (A) Structures of the substrate peptide
\textbf{7} and its dehydrated product \textbf{7-1} . (B) HR-MS profiles
of \textbf{7} alone (i) and incubated with CaoY (ii), CaoY/CaoK complex
(iii), CaoYK (iv), or CaoYK\textsubscript{LP} (v). For {[}M + H{]}
\textsuperscript{+}ions: \textbf{7} , cal. 889.3520, obs. 889.3563, and
[?]ppm 4.83;\textbf{7-1} , cal. 791.3756, obs. 791.3770, and [?]ppm 1.77.

\textbf{Table 1. Amino Acid Sequences of Substrate Peptide in This
Study.}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Name & Amino acid sequence\textsuperscript{a}\tabularnewline
\midrule
\endhead
\emph{In vivo} & \emph{In vivo}\tabularnewline
\textbf{1} & LP-SSAPCTIYASVSASISATASWGC\tabularnewline
\textbf{2} & LP-AAAPCTIY\tabularnewline
\textbf{3} & LP-SSAPCTIY\tabularnewline
\textbf{4} & LP-KFSSSF\tabularnewline
\textbf{5} & LP-KLVFSIS\tabularnewline
\emph{In vitro} & \emph{In vitro}\tabularnewline
\textbf{6} & AAAPCTIY\tabularnewline
\textbf{7} & AAAPC\emph{pT}IY\tabularnewline
In fusion & In fusion\tabularnewline
\textbf{L1\textsuperscript{b}} & GSGSLEVLFQ|GPGSGS\tabularnewline
\textbf{L2\textsuperscript{b}} & GSGSENLYFQ|GSGS\tabularnewline
\textbf{LP-6} & LP-AAAPCTIK\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a} For the sequence of LP, see Figure
1.\textsuperscript{b} The cleavage sites of HRV 3C protease and TEV
protease in \textbf{L1} and \textbf{L2} are indicated, respectively.

Conclusions

In this study, we investigated the dehydration process which initiates
the biosynthesis of lantibiotic cacaoidin in detail. The
phosphotransferase CaoK and the lyase CaoY naturally tends to form a
heterodimer to function as a dehydratase. CaoK binds with the LP
sequence to phosphorylate the Ser/Thr residues in CP. The subsequent
phosphate elimination reaction is independent of the LP sequence. In
particular, we developed methods for creating fused RiPP maturases with
the purpose of increasing activity and detailed mechanism studies. The
designed enzyme CaoYK has the potential to be a valuable tool for
peptide dehydration, given its potent dehydration activity and wide
substrate scope. As genome mining has revealed hundreds of BGCs
containing the CaoK/CaoY-related protein pairs, \textsuperscript{{[}8a,
16{]}} this research will accelerate the discovery, biosynthetic
investigation, and synthetic biology study of the rapidly expanding
RiPPs.

Experimental

\textbf{Materials, bacterial strains and plasmids}

Chemical, biochemicals and media were purchased from Sinopharm Chemical
Reagent Co., Ltd. (China), Oxoid Ltd. (U.K.) or Sigma-Aldrich
Corporation (USA) unless otherwise stated. Enzymes and kits for DNA
manipulations were purchased from Takara Bio (Japan), Vazyme Biotech
(China) or Thermo Fisher Scientific (USA). The bacterial strains,
plasmids and primers used in this study are summarized in Tables S1 and
S2.

\textbf{DNA isolation, manipulation, and sequencing}

DNA isolation and manipulation in \emph{Escherichia coli}
or\emph{Streptomyces} strains were carried out according to standard
methods. \textsuperscript{{[}17{]}} PCR amplifications were carried out
on an Applied Biosystems Veriti Thermal Cycler either using Taq DNA
polymerase (Vazyme Biotech) for routine genotype verification or KOD DNA
polymerase (Takara Bio) for high fidelity amplification. The plasmid
subcloning was performed using In-Fusion method (Takara Bio). Primer
synthesis was performed at Shanghai Biosune Biotech Co., Ltd. (China).
DNA sequencing was performed at Shanghai Biosune Biotech Co., Ltd.
(China).

\textbf{Chemical analysis}

Analysis by HPLC-coupled high resolution mass spectrometry (HR-MS) and
high-resolution tandem MS (HR-MS/MS) were performed on a Thermo Fisher
LTQ XL ESI-MS spectromer and a Q ExactiveTM Plus Mass Spectrometer
(Thermo Fisher Scientific Inc., USA). Related data were processed using
Thermo Xcalibur software.

\textbf{Constructs for the production of precursor peptide and its
variants}

\textbf{Constructs for the production of precursor peptide (1).} For the
sake of enhancing expression and purification, the \emph{caoA} gene was
fused to the gene encoding small ubiquitin-related modifier protein
(SUMO), resulting in a recombinant gene encoding N-terminally
SUMO-His\textsubscript{6}-tagged CaoA. This recombinant gene was then
cloned into the plasmid pETDuet-1, generating the recombinant
pETDuet-1-SUMO-His\textsubscript{6}-CaoA.

\textbf{Constructs for the production of precursor peptide variants 2,
3, 4, and 5.} The partial gene encoding core peptide of CaoA in the
recombinant plasmid pETDuet-1-SUMO-His6-CaoA was replaced for the DNA
sequence of \textbf{2} , \textbf{3} , \textbf{4} , or \textbf{5} by
rolling cycle PCR amplification. Long primer pairs listed in Table S2
were utilized, followed by DpnI digestion according to the standard
procedure of the Mut Express II Mutagenesis Kit (Vazyme Biotech Co. Ltd,
China). The corresponding constructs were listed in Table S1.

\textbf{Constructs for the production of CaoK, CaoY and the CaoK/CaoY
complex.}

For the sake of better expression and purification, the recombinant
genes encoding N-terminally His\textsubscript{6}-SUMO-tagged CaoK and
His\textsubscript{6}-Thioredoxin A (Trx)-tagged CaoY were individually
cloned into the plasmid pRSFDuet-1 used for overexpression. The two
recombinant genes, encoding N-terminally His\textsubscript{6}-Trx-tagged
CaoY and CaoK, were cloned into the two respective multiple cloning
sites of pRSFDuet-1 to facilitate the purification of the CaoK/CaoY
complex.

\textbf{Constructs for coexpression of gene encoding precursor peptide
or its variant with genes encoding CaoK and/or CaoY.}

The PCR products containing \emph{caoK} , and/or \emph{caoY} were cloned
into pRSFDuet-1, yielding the constructs used for coexpression with the
recombinant pETDuet-1 derivatives encoding precursor peptide or its
variants (see Table S1).

\textbf{Constructs for the production of fused proteins.}

\textbf{Constructs for the production of CaoYK.} The 48-bp fragment
containing the DNA sequence of \textbf{L1} were attached to
\emph{caoK}and \emph{caoY} by PCR using primer pairs, i.e., SUMO-CaoY-F
and L1-CaoY-R, L1-CaoK-F and HindIII-CaoK-R, respectively. The two PCR
products containing \emph{caoK} and \emph{caoY} , along with the PCR
product of the SUMO gene using primer pair, i.e., EcoRI-SUMO-F and
CaoY-SUMO-R, were cloned into the plasmid pRSFDuet-1, resulting in the
recombinant pRSFDuet-1-SUMO-CaoYK.

\textbf{Constructs for the production of CaoYKLP.} The 42-bp fragment
containing the DNA sequence of \textbf{L2} were attached to the partial
gene encoding for the CaoA leader peptide and gene encoding for the
fused CaoKY protein using primer pair, i.e., SUMO-CaoY-F and LP-L2-R.
The two PCR products along with the PCR product of the SUMO gene using
primer pair, i.e., EcoRI-SUMO-F and CaoY-SUMO-R, were cloned into the
plasmid pRSFDuet-1, resulting in the recombinant
pRSFDuet-1-SUMO-CaoYKLP.

\textbf{Constructs for the production of CaoYKLP-6.} The 24-bp DNA codon
encoding \textbf{6} were fused to the C-terminus of gene encoding for
CaoYKLP by rolling cycle PCR amplification. Long primer pair LP-6-F and
LP-6-R were utilized, followed by DpnI digestion to produce the
recombinant pRSFDuet-1-SUMO-CaoYKLP-6.

\textbf{Constructs for the production of CaoKLP.} The PCR product
containing CaoKLP was amplified from the template
pRSFDuet-1-SUMO-CaoYKLP using primer pair SUMO-CaoK-F and HindIII-LP-R,
and then cloned into the plasmid pRSFDuet-1 together with the PCR
product of the SUMO gene using primer pair, i.e., EcoRI-SUMO-F and
CaoK-SUMO-R. This resulted in the recombinant plasmid
pRSFDuet-1-SUMO-CaoKLP.

\textbf{Protein expression and purification}

\emph{E. coli} BL21(DE3) served as a general host for heterologous
expression. The culture of each recombinant \emph{E. coli} strain was
incubated in Luria-Bertani (LB) medium (5 g/L yeast extract, 10 g/L
tryptone and 10 g/L NaCl) containing 50 \selectlanguage{greek}μ\selectlanguage{english}g/mL kanamycin, and/or 100
\selectlanguage{greek}μ\selectlanguage{english}g/mL ampicillin, at 37 \selectlanguage{ngerman}°C and 220 rpm until the cell density reached
0.6-0.8 at OD600. and the induction was carried out overnight
(\textasciitilde{} 16 hours) at 16 °C.The cells were harvested by
centrifugation at 5000 × g for 10 min, flash-frozen and then stored at
-- 80 °C.

\emph{E. coli} cells were re-suspended in lysis buffer (1 x PBS, 10 \%
glycerol and 5 mM imidazole, pH 8.0). Recombinant proteins that contain
a His\textsubscript{6}-tag alone or coupled with SUMO or Trx tags were
purified on a HisTrap HP column (GE Healthcare, USA), which was
pre-treated with 10 column volumes (CVs) of lysis buffer followed by 10
CVs of wash buffer (1 x PBS, 10 \% glycerol and 40 mM imidazole, pH
8.0), using elution buffer (1 x PBS, 10 \% glycerol and 250 mM
imidazole, pH 8.0). Desired protein fractions were concentrated
(\textasciitilde{} 100 \selectlanguage{greek}μ\selectlanguage{english}M) using Amicon\selectlanguage{ngerman}® Ultra-15 Centrifugal Filter
Devices (MILLIPORE, USA) and desalted using a PD-10 Desalting Column (GE
Healthcare, USA) according to the manufacturer's protocols, and then
quantified in concentration by Bradford assay using bovine serum albumin
as the standard. The purity of recombinant proteins was determined by
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),
see Figure S16.

\textbf{Measurement of protein molecular weight.}

Before the measurement of molecular weight of the designs, the SUMO tag
was removed by proteolysis scaled to a 1 mL reaction mixture described
above. Size exclusion chromatography coupled with Multi-Angle Light
Scattering (MALS) was performed on a dynamic light scattering detector
(DynaPro NanoStar, Wyatt), a static light scattering detector (DAWN
HELLOS-II, Wyatt) and a differential refractive index detector (Optilab
TrEX, Wyatt) coupled with an AKTA pure system (GE Healthcare) at room
temperature. All the protein samples (with the concentrations \selectlanguage{english}> 3 mg/mL)
were filtered and loaded onto a Superdex 200 Increase 10/300 GL column
pre-equilibrated by the buffer containing 20 mM Tris-HCl (pH 7.5), 100
mM NaCl, and 1 mM DTT overnight. The data were analyzed by ASTRA
software (version 7.1) and were further output to the Origin 9.0
software and aligned with each other.

\textbf{Examination of precursor peptide and its related peptides}

To examine precursor peptide CaoA and its variants that resulted from
the heterologous (co)expression of related genes in \emph{E. coli} ,
purified peptide products were treated with SUMO protease (i.e., Ulp-1)
to remove the N-terminal SUMO tag. Each proteolysis was conducted at 30
\selectlanguage{ngerman}°C for 30 min in a 50 \selectlanguage{greek}μ\selectlanguage{english}L reaction mixture containing 5 \selectlanguage{greek}μ\selectlanguage{english}M Ulp-1, 100 \selectlanguage{greek}μ\selectlanguage{english}M
SUMO-tagged CaoA or its derivative, and 50 mM Tris-HCl (pH 8.0). The
products were then subjected to HPLC-MS and MS/MS analyses on an Agilent
SB-C18 Zorbax column (3.5 \selectlanguage{greek}μ\selectlanguage{english}m, 2.1 mm \selectlanguage{ngerman}× 100 mm) by gradient elution of
solvent A (H\textsubscript{2}O containing 0.1 \% formic acid) and
solvent B (CH\textsubscript{3}CN containing 0.1 \% formic acid) at a
flow rate of 0.3 mL/min over a 25 min period as follows: T = 0 min, 5 \%
B; T = 5 min, 35 \% B; T = 20 min, 60 \% B; and T = 25 min, 90 \% B.

\emph{In vitro} \textbf{biochemical assays}

\emph{In vitro} \textbf{assays of dehydration activity on substrate
peptide 2.} For in vitro assays of dehydration activity, reactions were
conducted at 30 °C for 2 h in a 50 \selectlanguage{greek}μ\selectlanguage{english}L reaction mixture containing 50 mM
Tris-HCl (pH 8.0), 10 mM MgCl\textsubscript{2}, \textasciitilde{} 50 \selectlanguage{greek}μ\selectlanguage{english}M
peptide \textbf{2} , and 5 \selectlanguage{greek}μ\selectlanguage{english}M enzymes. The enzymes tested are CaoK/CaoY
complex, CaoYK, and CaoYKLP. Each reaction was quenched with 50 \selectlanguage{greek}μ\selectlanguage{english}L of
CH\textsubscript{3}CN and then analyzed by HPLC-MS as described above.

\emph{In vitro} \textbf{assays of phosphorylation activity on oligo
peptide 6.} For \emph{in vitro} assays of phosphorylation activity,
reactions were conducted at 30 \selectlanguage{ngerman}°C for 2 h in a 50 \selectlanguage{greek}μ\selectlanguage{english}L reaction mixture
containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl\textsubscript{2}, 100 \selectlanguage{greek}μ\selectlanguage{english}M
peptide \textbf{6} , and 10 \selectlanguage{greek}μ\selectlanguage{english}M enzymes. The enzymes tested are CaoKLP
and CaoYKLP. Each reaction was quenched with 50 \selectlanguage{greek}μ\selectlanguage{english}L of
CH\textsubscript{3}CN and the products were then subjected to HPLC-MS
and MS/MS analyses on an Agilent SB-C18 Zorbax column (3.5 \selectlanguage{greek}μ\selectlanguage{english}m, 2.1 mm \selectlanguage{ngerman}×
100 mm) by gradient elution of solvent A (H\textsubscript{2}O containing
0.1 \% formic acid) and solvent B (CH\textsubscript{3}CN containing 0.1
\% formic acid) at a flow rate of 0.3 mL/min over a 25 min period as
follows: T = 0-3 min, 5 \% B; T = 20 min, 65 \% B; and T = 25 min, 90 \%
B.

\emph{In vitro} \textbf{assays of phosphate elimination activity on
oligo peptide 7.} For \emph{in vitro} assays of phosphate elimination
activity, reactions were conducted at 30 °C for 2 h in a 50 \selectlanguage{greek}μ\selectlanguage{english}L reaction
mixture containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl\textsubscript{2},
100 \selectlanguage{greek}μ\selectlanguage{english}M peptide \textbf{7} , and 10 \selectlanguage{greek}μ\selectlanguage{english}M enzymes. The enzymes tested are
CaoY, CaoK/CaoY complex, CaoYK and CaoYK\textsubscript{LP}. Each
reaction was quenched with 50 \selectlanguage{greek}μ\selectlanguage{english}L of CH\textsubscript{3}CN and the
products were then subjected to HPLC-MS and MS/MS as described above.

\textbf{Bioinformatic analysis}

Protein function analysis was carried out using available BLAST methods
(http://www.ncbi.nlm.nih.gov/blast/). The alignment of gene cluster was
performed by Easyfig. \textsuperscript{{[}18{]}} The AlphaFold Model of
CaoYKLP-6 was conducted by ColabFold and visualized using
ChimeraX.\textsuperscript{{[}19{]}}

Supporting Information

The supporting information for this article is available on the WWW
under https://doi.org/10.1002/cjoc.2023xxxxx.

Acknowledgement

This work was supported in part by grants from the National Key Research
and Development Program of China (2022YFC2303100 for L.P and W. L), the
National Natural Science Foundation of China (32030002 and 22193070 for
W.L) and the Science and Technology Commission of Shanghai Municipality
(20XD1425200 for L.P), and the CAS Youth Interdisciplinary Team
(JCTD-2022-10 for L.P).

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\begin{longtable}[]{@{}l@{}}
\toprule
Manuscript received: XXXX, 2023 Manuscript revised: XXXX, 2023
Manuscript accepted: XXXX, 2023 Accepted manuscript online: XXXX, 2023
Version of record online: XXXX, 2023\tabularnewline
\bottomrule
\end{longtable}

The Authors\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
& After acceptance, please insert a group photo of the authors taken
recently. &\tabularnewline
& \textbf{Left to Right:} Authors Names &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Entry for the Table of Contents}\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
\textbf{Mechanistic Investigations into the Catalytic Mode of a
Dehydratase Complex Involved in the Biosynthesis of Lantibiotic
Cacaoidin} Yanqing Xue,\textsuperscript{++,a} Miao
Li,\textsuperscript{++,a,b} Ling Hu,\textsuperscript{a} Jianing
Liu,\textsuperscript{a} Lifeng Pan,\textsuperscript{*,a,b} and Wen
Liu\textsuperscript{*,a} \emph{Chin. J. Chem.} \textbf{2023}, \emph{41},
XXX---XXX. \textbf{DOI: 10.1002/cjoc.202300XXX}\tabularnewline
Herein, we report that the dehydration process of lantibiotic cacaoidin
involves a dehydratase complex, which is composed of a
phosphotransferase CaoK and a lyase CaoY in a 1:1 molar ratio. CaoK
interacts with leader peptide for its own solubility and activity, while
CaoY functions independently of the leader portion.\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{english}
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