Promiscuous Enzymes Cooperate at the Substrate Level en Route to Lactazole A

Article abstract of DOI:10.1021/jacs.0c05541

Enzymes involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) often have relaxed specificity profiles and are able to modify diverse substrates. When several such enzymes act together during precursor peptide maturation, a multitude of products can form, yet usually the biosynthesis converges on a single natural product. For the most part, the mechanisms controlling the integrity of RiPP assembly remain elusive. Here, we investigate the biosynthesis of lactazole A, a model thiopeptide produced by five promiscuous enzymes from a ribosomal precursor peptide. Using our in vitro thiopeptide production (FIT-Laz) system, we determine the order of biosynthetic events at the individual modification level and supplement this study with substrate scope analysis for participating enzymes. Our results reveal an unusual but well-defined assembly process where cyclodehydration, dehydroalanine formation, and azoline dehydrogenation events are intertwined due to minimal substrate recognition requirements characteristic of every lactazole enzyme. Additionally, each enzyme plays a role in directing LazBF-mediated dehydroalanine formation, which emerges as the central theme of the assembly process. Cyclodehydratase LazDE discriminates a single serine residue for azoline formation, leaving the remaining five as potential dehydratase substrates. Pyridine synthase LazC exerts kinetic control over LazBF to prevent the formation of overdehydrated thiopeptides, whereas the coupling of dehydrogenation to dehydroalanine installation impedes generation of underdehydrated products. Altogether, our results indicate that substrate-level cooperation between the biosynthetic enzymes maintains the integrity of lactazole assembly. This work advances our understanding of RiPP biosynthesis processes and facilitates thiopeptide bioengineering.

Full text of DOI:10.1021/jacs.0c05541

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Article
Promiscuous enzymes cooperate at the substrate level en route to lactazole A
Alexander A. Vinogradov, morito shimomura, Naokazu Kano, Yuki Goto, Hiroyasu Onaka, and Hiroaki Suga
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c05541 • Publication Date (Web): 14 Jul 2020
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1 Promiscuous enzymes cooperate at the substrate level en
2 route to lactazole A
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Alexander A. Vinogradov1, Morito Shimomura2, Naokazu Kano3, Yuki Goto1, Hiroyasu
Onaka2,4 and Hiroaki Suga1,*
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1Department of Chemistry, Graduate School of Science, The University of Tokyo,
Bunkyo-ku, Tokyo 113-0033, Japan
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2Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The
University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
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3 Department of Chemistry, Faculty of Science, Gakushuin University, 1-5-1 Mejiro,
Toshima-ku, Tokyo 171-8588, Japan
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4Collaborative Research Institute for Innovative Microbiology, The University of Tokyo,
Bunkyo-ku, Tokyo 113-8657, Japan
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*Correspondence to Hiroaki Suga (Department of Chemistry, Graduate School of Science,
The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan, +81-3-5841-8372,
hsuga@chem.s.u-tokyo.ac.jp)
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The authors declare no competing interests.
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18 Abstract
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Enzymes involved in biosynthesis of ribosomally synthesized and post-translationally
modified peptides (RiPPs) often have relaxed specificity profiles and are able to modify
diverse substrates. When several such enzymes act together during precursor peptide
maturation, a multitude of products can form, and yet usually, the biosynthesis converges
on a single natural product. For the most part, the mechanisms controlling the integrity of
RiPP assembly remain elusive. Here, we investigate biosynthesis of lactazole A, a model
thiopeptide produced by five promiscuous enzymes from a ribosomal precursor peptide.
Using our in vitro thiopeptide production (FIT-Laz) system, we determine the order of
biosynthetic events at the individual modification level, and supplement this study with
substrate scope analysis for participating enzymes. Our results reveal an unusual, but well-
defined assembly process, where cyclodehydration, dehydroalanine formation, and azoline
dehydrogenation events are intertwined due to minimal substrate recognition requirements
characteristic of every lactazole enzyme. Additionally, each enzyme plays a role in directing
LazBF-mediated dehydroalanine formation, which emerges as the central theme of the
assembly process. Cyclodehydratase LazDE discriminates a single serine residue for
azoline formation, leaving the remaining five as potential dehydratase substrates. Pyridine
synthase LazC exerts kinetic control over LazBF to prevent formation of overdehydrated
thiopeptides, whereas coupling of dehydrogenation to dehydroalanine installation impedes
generation of underdehydrated products. Altogether, our results indicate that substrate level
cooperation between the biosynthetic enzymes maintains the integrity of lactazole assembly.
This work advances our understanding of RiPP biosynthesis processes and facilitates
thiopeptide bioengineering.
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41 Main text
42 Introduction
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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are
structurally and functionally diverse natural products united by a common biosynthetic logic.1
Usually during RiPP maturation, biosynthetic enzymes utilize the N-terminal sequence of a
ribosomally produced precursor peptide as a recognition motif (leader peptide; LP) and
install post-translational modifications (PTMs) in the C-terminal section of the same
substrate (core peptide; CP). This mode of action leads to relaxed substrate requirements
around the modification sites, often enabling one RiPP enzyme to introduce multiple PTMs
in
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single substrate, as exemplified by lanthipeptide synthetases,2 YcaO
cyclodehydratases3 and methyltransferases from polytheonamide biosynthesis.4,5 Unique
enzymology of RiPP biosynthetic enzymes has come under intense scrutiny in the recent
years, which explained observed substrate specificities in many cases.6–17 During
biosynthesis of complex RiPPs, when multiple enzymes capable of differentially modifying
their substrate act together, a multitude of products can often form, and yet usually the
biosynthetic pathway manages to produce a single natural product. Molecular mechanisms
controlling the integrity of RiPP biosynthesis are only beginning to be elucidated,4,18–25 and
many details remain unclear, especially in the cases where enzymes can apparently
compete over the substrate. For example, during biosynthesis of some thiopeptides, Ser
and Thr residues in the precursor peptide CP are selectively modified to either
oxazoline/oxazole or dehydroamino acids by cyclodehydratase/dehydrogenase and
dehydratase enzymes, and the basis for such a cooperative action despite the potential for
competition has not yet been firmly established.
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In this study, we aim at investigating the roots of cooperative biosynthesis of lactazole A,
a cryptic thiopeptide from Streptomyces lactacystinaeus (Fig. 1).26 Lactazole biosynthesis
involves 5 dedicated enzymes colocalized with the precursor peptide gene (lazA) into laz
biosynthetic gene cluster (BGC; Fig. 1a–c). During thiopeptide maturation, LazD and LazE
operate as a single cyclodehydratase enzyme,27–29 responsible for the installation of azoline
PTMs (Fig. 1d), which are further dehydrogenated to azoles by the C-terminal domain of
LazF in an FMN-dependent manner.3,13 Dehydroalanines (Dha) are accessed from Ser by
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the combined action of LazB and the N-terminal domain of LazF, which utilize glutamyl-
tRNAGlu for dehydration similarly to class I lanthipeptide synthetases (Fig. 1e).30–33 The
remaining enzyme, LazC, performs macrocyclization and eliminates LP as a C-terminal
amide (LP-NH2) to yield the thiopeptide (Fig. 1f).34,35
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Lactazole A is an example of a RiPP assembled by multiple enzymes that can compete
over the substrate. LazA CP contains 6 Ser residues, 4 of which are converted into Dha, 1
into oxazole (Oxz), and 1 which remains unmodified (Fig. 1b, c). Previously, we reconstituted
biosynthesis of lactazole A in vitro by combining flexible in vitro translation (FIT)36 — utilized
to access LazA precursor peptide — with recombinantly produced Laz enzymes.37 Using
this platform, termed the FIT-Laz system, we showed that selective biosynthesis of
lactazole A occurred only when all enzymes were present in the reaction mixture from the
beginning, whereas stepwise treatments led to either under- or overdehydrated thiopeptides
containing 3 or 5 Dha, respectively. These results suggest that cooperation between Laz
enzymes extends beyond the “azoles form first, Dha second” model observed for
thiopeptides studied to date,22,23,38 and indicate that lactazole may be a good model system
to study concerted action of multiple enzymes in RiPP biosynthesis. Furthermore, our
previous findings37 indicated that Laz enzymes can convert extensively mutated LazA
analogs to corresponding thiopeptides, exemplified by the synthesis of over 90 lactazole-
like thiopeptide containing up to 25 nonnative amino acids. How the enzymes maintain the
integrity of biosynthesis for a diverse set of substrates constitutes the second major question
of this study.
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According to our aims, we intercepted biosynthetic intermediates and reconstructed the
order of events leading up to the final macrocyclization reaction during the lactazole A
assembly process at a single PTM resolution. When supplemented with substrate
preference studies for individual enzymes, these results help in rationalizing the roots of
cooperation between Laz enzymes, and establish the basis for selective lactazole A
production. Bioengineering of RiPPs to harness their potential for human health holds a lot
of promise,39,40 but it requires thorough understanding of the underlying biosynthesis
mechanisms. Our study elucidates how several promiscuous enzymes coordinate the
assembly of a complex RiPP, facilitating thiopeptide bioengineering, and ultimately,
functional reprogramming.
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Figure 1. Biosynthesis of lactazole A. a) The biosynthetic gene cluster from Streptomyces
lactacystinaeus responsible for lactazole A production (laz BGC; GenBank accession:
AB820694.1; MIBIG accession: BGC0000606). Genes encoding the enzymes responsible
for synthesis of azolines are color coded in light blue, azoline dehydrogenase in blue,
dehydroalanine — green, and pyridine — orange. b) Primary amino acid sequence of LazA
precursor peptide. Residues eventually converted to Dha, azoles and pyridine are
highlighted in green, blue, and orange, respectively. c) Chemical structure of lactazole A. d)
LazDE performs a cyclodehydration reaction furnishing azoline heterocycles, which are
further dehydrogenated by FMN-dependent LazF. e) Ser dehydration is catalyzed by LazBF
via a two-step process involving Ser glutamylation by LazB. f) Macrocyclization is achieved
by LazC, which utilizes two Dha residues, Dha1 and Dha12, to form the central
dihydropyridine and concomitantly macrocyclize the peptide. The same enzyme
consequently eliminates LP-NH2 to aromatize the structure.
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117 Results
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Because our previous results indicated that modification of amino acid residues 4–7 in the
CP of LazA (Fig. 1b) is not essential for macrocyclization,37 we hypothesized that maturation
of LazA is modular, i.e. residues 10–13 undergo PTM independent of residues 4–7.
Accordingly, we sought to establish the order of PTM installation for two LazA mutants, LazA
S4-C7A (LazAmin; Fig. 2a) and LazA S10-C13A (LazAaux; Fig. 2b), before proceeding to the
wild type peptide (LazAwt).
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First, we investigated in vitro modification of LazAmin utilizing the FIT-Laz system.37
Synthetic DNA bearing lazAmin gene was in vitro transcribed and translated, generating the
precursor peptide, which was incubated with a mixture of recombinantly produced enzymes
(LazB, LazC, LazD, LazE, LazF, Streptomyces lividans GluRS and synthetic S.
lactacystinaeus tRNAGlu (LazBCDEF/GluRS/tRNAGlu); S.I. 2.3) at 25 °C. Reactions were
stopped by the addition of cold methanol containing iodoacetamide (IAA), and the outcomes
were analyzed by LC-MS. Selective IAA alkylation on unmodified Cys residues and
quantitative acidic hydrolysis of Oxn under HPLC conditions enabled unambiguous
identification of PTMs based on mass shifts (S.I. 3.1), but not their location within the CP.
Accordingly, the captured intermediates were further analyzed by CID tandem mass
spectrometry (MS/MS) in a data-dependent acquisition (DDA) mode (S.I. 2.4-2.5). Our initial
experiments showed that LazAmin maturation is complete in under 3 h (Fig. S6). To intercept
biosynthetic intermediates at a finer temporal resolution, we performed a time course study
and quenched the reactions after 5, 15, 30, 45 and 60 min (Fig. 2a and S7). These
experiments revealed formation and consumption of multiple linear intermediates during
thiopeptide assembly, and enabled assignment of the PTM installation order (Fig. 4a, S.I.
3.3).
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Figure 2. Time course analysis reveals the order of enzymatic action during lactazole A
assembly. a) LazAmin maturation time course. In vitro translated precursor peptide was
incubated with LazBCDEF/GluRS/tRNAGlu for 5, 15, 30, 45 and 60 min, after which reaction
outcomes were analyzed by LC-MS and DDA MS/MS. Displayed are the changes in the
amounts of starting material (S.M.), LP-NH2, thiopeptide (lactazole S4-C7A) and the key
intermediate, Ser10-Oxn11-Dha12-Thn/Thz13, as a function of time. For full product
distribution and characterization of intermediates refer to S.I. 3.3. b) LazAaux maturation time
course; data as panel in a), except LazC was omitted from the enzyme mixture, and the 45-
min time point was skipped. Displayed are the changes in the amounts of S.M. and three
key products as a function of time. See also S.I. 3.4. c) Lactazole A biosynthesis time course;
data as panel in a). Maturation of LazAwt proceeds in a modular fashion: after a 5-min
treatment with Laz enzymes, the key Ser4-Thz5-Dha6-Thz7 intermediate accumulates
similarly to the LazAaux case (compare to panel b). Fast accumulation of this peptide points
to a modular biosynthetic logic. The second key intermediate, Thz5-Dha6-Thz7/Oxn11-
Dha12-Thn13, accumulates analogously to the LazAmin case (compare to panel a). See also
S.I. 3.5. d) The chemical structure of the key Ser4-Thz5-Dha6-Thz7 LazAwt intermediate
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observed after a 5-min treatment with the enzymes. e) MS/MS spectrum supporting
structural assignment of the intermediate from panel d). See also Fig. S37.
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Maturation of LazAmin started with formation of oxazoline at Ser11 (Oxn11), followed by
heterocyclization at Cys13 (Thn13; Fig. S10). After that, Ser12 was dehydrated to Dha12
(Fig. S11), and Thn13 was dehydrogenated to give thiazole13 (Thz13; Fig. S15), arriving at
a prominent intermediate, Ser10-Oxn11-Dha12-Thz13 (Fig. 2a). The following steps, Oxn11
dehydrogenation and Dha10 formation, happened fast relative to the temporal resolution of
our experiments, and we were unable to capture the corresponding intermediates even
when using diluted enzyme mixtures or performing reactions at 4°C (Fig. S16). The next
observed peptide bore the Dha10-Oxz11-Dha12-Thz13 motif required for macrocyclization
(Fig. S12). Dehydration of Ser1 to Dha1 was independent of other modifications, because
most intermediates formed as a mixture of Ser1 and Dha1 throughout the time course.
Treatment of LazAmin with LazBF/GluRS/tRNAGlu for 2 h confirmed that LazDE-independent
dehydration of Ser1 is kinetically competent (Fig. S17). Additionally, the time course study
indicated that LazBF dehydrates Ser adjacent to azolines. We confirmed the azoline-
dependent activity of LazB by treating LazAmin with the enzyme mix lacking dehydrogenase
(LazBDE/GluRS/tRNAGlu; Fig. S18).
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The order of Ser10 dehydration and Oxn11 dehydrogenation could not be determined from
the time course. LazF can convert Oxn11 to Oxz11, but this process is kinetically
incompetent. Although a 17 h treatment of LazAmin with LazDEF yielded the Oxz11/Thz13
product, an analogous 2 h reaction mainly resulted in the formation of Oxn11/Thz13 (Fig.
3a). In contrast, a 2 h incubation of LazAmin with LazBDEF/GluRS/tRNAGlu afforded fully
modified Dha10-Oxz11-Dha12-Thz13, suggesting that Dha formation is required for facile
oxidation of Oxn11. To test which Dha is important, we prepared two LazAmin mutants, S10A
and S12A, and treated them with LazBDEF/GluRS/tRNAGlu for 2 h. The S10A mutant yielded
the Ala10-Oxn11-Dha12-Thz13 peptide (Fig. 3b), and modification of LazAmin S12A led to a
complex mixture of products, none of which bore Dha10 and/or Oxz11 (Fig. 3c). These
results suggest that installation of Dha12 is required for dehydration of Ser10, and in turn,
Dha10 greatly facilitates dehydrogenation of Oxn11. Thus, during LazAmin maturation, the
key intermediate Ser10-Oxn11-Dha12-Thz13 is converted to Dha10-Oxn11-Dha12-Thz13,
which sets the stage for Oxn11 oxidation and the follow-up macrocyclization (Fig. 4a). The
elusive Dha10-Oxn11-Dha12-Thz13 intermediate could be captured for LazAmin S11T
treated with LazBCDEF/GluRS/tRNAGlu for 2 h (Fig. S19).
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To test whether this curious sequence of events has biosynthetic significance, we forced
oxidation of Oxn11 prior to adding LazB to the reaction mixture. A 17 h treatment of LazAmin
with LazDEF furnished the Oxz11/Thz13 product, which upon further incubation with
LazBCF/GluRS/tRNAGlu for 2 h accumulated the Ser10-Oxz11-Dha12-Thz13 intermediate,
and a mixture of thiopeptides bearing either Ser10 or Dha10 (Fig. S20). Similarly, LazAmin
S11C, readily modified by LazDEF to the Thz11/Thz13 intermediate, underwent slow
dehydration at Ser10 and formed a mixture of thiopeptides (Fig. S21). These results indicate
that premature oxidation of Oxn11 hampers Dha10 formation, and highlight the importance
of Dha-dependent Oxn11 dehydrogenation during the biosynthesis. Lastly, we found that
the modification order persisted for three LazAmin variants (although maturation speed and
efficiency varied between the mutants; Fig. S22a-c), and when the enzymes were mixed in
different ratios (Fig. S22d). The Ser10-Ser11-Ser12-Cys13 motif is conserved in over 60
thiopeptides from predicted lactazole-like BGCs (Fig. S23),41 which lends further support to
our findings.
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Figure 3. Both Dha10 and Dha12 are required for facile Oxn11 dehydrogenation. a) Dha-
dependent Oxn11 dehydrogenation in LazAmin. In vitro translated LazAmin was incubated with
LazBDEF/GluRS/tRNAGlu (2 h), LazDEF (2 h or 17 h) or with buffer only, and the outcomes
were evaluated by LC-MS. Displayed are brEIC chromatograms and integrated mass spectra
showing the product distribution. These data suggest that in the absence of LazB, LazF-
mediated dehydrogenation of Oxn11 is kinetically incompetent. b) Kinetically incompetent
Oxn11 dehydrogenation in LazAmin S10A. Analogous to panel a), data for LazAmin S10A. In
the absence of Dha10, Oxn11 oxidation does not happen on a relevant time scale. c)
Kinetically incompetent Oxn11 dehydrogenation in LazAmin S12A. Analogous to panel a),
data for LazAmin S12A. The S12A mutation disrupts azoline installation by LazDE and leads
to formation of multiple products. Nevertheless, when the peptide was treated with
LazBDEF/GluRS/tRNAGlu for 2 h, neither Dha10 formation nor Oxn11 dehydrogenation took
place, suggesting that Dha12 is required for dehydration of Ser10. †: Oxn underwent
quantitative hydrolysis during HPLC (see S.I. 3.1 and 3.2).
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Next, we studied modification of LazAaux. Analogous to the experiments above, LazAaux
precursor peptide was incubated with LazBDEF/GluRS/tRNAGlu for 5, 15, 30 and 60 min,
and reaction outcomes were analyzed by LC-MS and DDA MS/MS (Fig. 2b and S.I. 3.4).
Heterocyclization at Cys5 initiated the chain of PTMs, which then briefly bifurcated and
converged on the key product, Ser4-Thz5-Dha6-Thz7, bearing the wild type modification
pattern (Fig. 4b). This sequence of events was fast — the key intermediate comprised over
50% of total substrate after only 5 min. As before, dehydration of Ser1 to Dha1 was
independent of other PTMs, and was generally slow compared to the modifications at
residues 5–7, taking over 1 h to completion. Even slower was dehydration of Ser4 to Dha4
inside the Ser4-Thz5-Dha6-Thz7 motif, as this product comprised less than 4% of total
substrate after 1 h. These results confirm that Ser inside the Ser-azole-Dha-azole pattern is
a poor substrate for LazBF.
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Finally, we performed time course analysis for LazAwt (Fig. 2c; S.I. 3.5). As anticipated,
biosynthesis proceeded largely in a modular fashion (Fig. 4c). The key intermediate of
LazAaux maturation, Ser4-Thz5-Dha6-Thz7, also quickly formed for LazAwt and comprised
over 40% of total substrate after a 5-min incubation with the full enzyme set (Fig. 2c–e). The
biosynthetic order leading up to this peptide was identical to LazAaux, including the bifurcation
event. Modification of residues 10–13 proceeded once the Ser4-Thz5-Dha6-Thz7 motif was
installed. With the exception of Ser12 dehydration, which took place prior to the
heterocyclization of either residue around it (Fig. S38), modification of residues 10–13
generally followed the sequence established for LazAmin, including the Dha-dependent
Oxn11 dehydrogenation.
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Altogether, these experiments unravel lactazole A biosynthesis one PTM at a time,
revealing a carefully orchestrated sequence of events, in which the actions of different
enzymes are intertwined to ensure the integrity of biosynthesis.
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Figure 4. Proposed lactazole A biosynthesis pathways. a) The order of PTM installation for
LazAmin. b) Modification of residues 4–7 as studied in LazAaux. c) Modular assembly of
lactazole A from LazAwt. See also S.I. 3.3 – 3.5.
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Primary macrocycle assembly for thiopeptides studied to date separates azole and Dha
formation into two stages, where Dha installation follows azole formation.22,23,38 Such a
separation can be due to the specificity of a Dha-installing enzyme toward the native azole
pattern,22 or due to an additional enzyme acting as a gatekeeper and preventing premature
Dha synthesis.23,38 Intertwined enzymatic action observed in the experiments above is
distinct from these models, indicating that lactazole biosynthesis integrity is maintained
differently. The time course study revealed some of these mechanisms (for instance, Dha-
dependent dehydrogenation at Oxn11 serving to prevent formation of underdehydrated
thiopeptides), but some issues, especially the nature of enzyme competition over Ser
residues, remained elusive. To gain deeper insight into the nature of cooperation and
competition during lactazole biosynthesis, we performed analysis of individual enzymes and
their innate substrate preferences. To this end, we sought to dissect and narrow down the
substrate recognition requirements for each enzyme.
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LazDE (azoline formation). Because the action of LazDE is mostly independent of other
enzymes, characterization of its substrate scope is relatively straightforward. First, we
investigated whether any specific sequence elements in LazA CP are critical for LazDE
activity, and prepared 4 LazA variants bearing randomized CPs with an Ala-Cys-Ala
tripeptide grafted in the middle (Fig. 5a, LazACP1-4). The peptides, produced with the FIT
system, were incubated with LazDEF for 2 h, and reactions were analyzed by LC-MS.
Efficient modification of 3 out of 4 tested substrates (Fig. 5a, LazACP1-3) indicated that LazDE
is a promiscuous enzyme able to act in “unfamiliar” sequence environments. Next, we
studied relative heterocyclization rates for Cys vs. Ser and Thr. Consistent with a number of
previously characterized YcaO enzymes,42–44 modification efficiency at Cys exceeded that
of Ser/Thr (Fig. 5a, LazACP1, LazACP1 C7T, LazACP1 C7S). Heterocyclization of Ser/Thr in
sequences bearing a Thz in position +2 proceeded similarly (Fig. S42a).
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The structure of lactazole A does not contain adjacent azoles, and even a prolonged
incubation of LazAwt with LazDEF does not result in consecutive heterocyclizations. To test
whether LazDE is unable to form consecutive azoles, we prepared two substrates containing
2 or 3 Cys in a row (Fig. 5a; LazACP1 2C and LazACP1 3C). Treating these peptides with
LazDEF led to one or two cyclodehydrations respectively, supporting our hypothesis. In
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contrast, substrates containing up to 4 Cys residues all separated by an Ala (Fig. S42b)
resulted in formation of Thz at every Cys residue.
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To ascertain whether the distance from the LP to the cyclizable amino acid affects
modification rate, we prepared 4 LazA variants containing a single Cys in position 2, 4, 6 or
8 (Fig. S42e). LazALP ruler Cys2 was unmodified after a 30-min treatment with LazDEF,
suggesting that similar to PatD, a YcaO enzyme from a cyanobactin BGC,43,45 LazDE
requires a spacer sequence between LP and residues undergoing modification, which
explains how Ser1 escapes cyclodehydration. LazALP ruler Cys4 and LazALP ruler Cys6 gave over
80% Thz product compared to the 28% heterocyclization yield for LazALP ruler Cys8, indicating
that reaction rate decreases with distance from the LP, which rationalizes fast modification
of Cys5 and Cys7 relative to Ser11 and Cys13 in LazAwt.
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Finally, we studied the effect of amino acids adjacent to the cyclodehydration site using 12
single-point mutants of LazACP1 (Fig. 5a). In position +1 (Cys-Xaa), LazDE preferred
small/hydrophobic residues such as Ala, Ser, Gly and Val, while modification next to charged
Asp and Arg as well as bulky Trp was impaired. In position –1, Ser or Ala were strongly
preferred, while other mutants suffered from inefficient processing. In addition, we also
grafted “native” tripeptides from LazAwt into LazACP1, and compared their relative
modification rates after a 30-min treatment with LazDEF (Fig. S42d). These data uncovered
a preference for Ser over Ala in position –1. The selectivity for Ser in position –1 was
especially apparent for Ser-Ser motifs. Whereas the Ala-Ser-Ala substrate remained
essentially unmodified after a 2 h treatment with LazDEF, Ser-Ser-Ala and Ser-Ser-Ser
peptides were quantitatively cyclodehydrated under the same conditions (Fig. S42c).
Altogether, this study narrows down the primary recognition sequence of LazDE to the
tripeptide Xaa1-(Cys/Thr/Ser)-Xaa2, where small Ser, Ala and Gly residues are strongly
preferred in Xaa1 and Xaa2 positions, and Cys is modified faster than Thr and Ser (Fig. 6a).
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Figure 5. Substrate scope of LazDE (panel a), LazF dehydrogenase (panels a and c), and
LazBF (panel b). a, b) In vitro translated LazA analogs with randomized CP sequences were
incubated with either LazDEF (panel a) or LazBF/GluRS/tRNAGlu (panel b) for 2 h. Reaction
outcomes were analyzed by LC-MS and quantified as described in S.I. 2.5. The data reveal
substrate preferences for individual Laz enzymes. The data for additionally tested substrates
are summarized in Fig. S42. c) Comparison of the reduction potentials between LazF and
several Oxz/Thz-containing tripeptides. The values for tripeptides were calculated as
described in S.I. 2.6; for LazF — experimentally determined as described in S.I. 2.7 and Fig.
S45. The oxidizing ability of LazF-bound FMN is sufficient to dehydrogenate all studied
substrates, but the reaction potential increases when Dha flanks the substrate Oxn in
position –1, i.e. Dha in position –1 promotes Oxn dehydrogenation.
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LazBF (Dha formation) has a two-fold activity profile. During lactazole biosynthesis, it
converts Ser1 to Dha1 in an azoline/azole-independent fashion, while formation of remaining
3 Dha is azoline/azole dependent. We first focused on a more tractable azoline/azole-
independent activity. As before, we began by preparing 3 LazA analogs with randomized
CPs and grafted a Ser1-Trp2-Gly3-Ala4 tetrapeptide adjacent to the LP for each sequence
(Fig. 5b; LazACP5–7); one more substrate had the CP truncated after Ala4 (Fig. 5b; LazACP8).
The peptides were produced with the FIT system and treated with LazBF/GluRS/tRNAGlu for
2 h. Efficient dehydration of all substrates, including the truncated peptide, indicated that
LazBF is also a promiscuous enzyme that acts “locally”, i.e. it recognizes at most a few
amino acids around the modification site. To further narrow down the recognition
requirement, we prepared a series of single point mutants of LazACP6 in positions 2 and 3
(Fig. 5b). In position +1, an aromatic residue was essential for good modification efficiency,
although residual dehydration occurred in all cases. Position +2 tolerated more variation,
and only a negatively charged Asp compromised processing. Additionally, we found that
LazA mutants bearing a randomized CP with Ala-Ser-Trp grafted in the middle also
underwent LazBF-catalyzed dehydration (Fig. 5b; LazACP9–11), indicating that proximity to
the LP is not a critical recognition element. Finally, we checked whether LazBF can accept
Thr as a substrate to generate dehydrobutyrine (Dhb) residues. After a 2 h treatment, two
Thr-containing substrates (Fig. 5b; LazACP6 S1T and LazACP7 S1T) were 7% and 3%
modified, suggesting that LazBF strongly prefers Ser as a substrate, although synthesis of
Dhb is possible. Combined, these results narrow down the primary recognition sequence of
the azoline/azole-independent LazBF activity to the Ser-Trp dipeptide (Fig. 6a).
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The substrate requirements for the azoline/azole-dependent Dha formation proved more
difficult to generalize. First, we studied modification of single point Ala mutants of LazAmin
and LazAaux by LazBDEF/GluRS/tRNAGlu using LazBF/GluRS/tRNAGlu and LazDEF
treatments as controls to gauge LazDE-dependent Dha formation (Fig. S43a, b). We found
that Thz-Ser, Oxn-Ser-Thn/Thz, and Ser-Oxn-Dha-Thz motifs were generally dehydrated,
but Ser-Thz, Oxn-Ser, Ser-Oxn, or Ser-Thz-Dha-Thz motifs were poor substrates (Fig. 6a).
Processing of randomized CPs containing similar local environments recapitulated these
findings (Fig. S43c, S44). However, based on these results, we were unable to generalize
the rules governing Ser dehydration around azole/azolines: it is an intricately controlled
activity, which would require a dedicated study. Nevertheless, these results help in
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rationalizing slow dehydration of Ser4 in Ser4-Thz5-Dha6-Thz7 motif during lactazole
biosynthesis.
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Notably, throughout the study, the product of LazB action, Ser(OGlu), was not detected so
long as LazF was present in the enzyme mixture. This observation suggests that the burden
of substrate discrimination lies primarily on LazB, because LazF appears to accept any
Ser(OGlu)-containing peptide as a substrate.
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LazF dehydrogenase (azole formation). The aforementioned LazDE study provided a
number of clues to the substrate scope of LazF dehydrogenase. First, like other Laz
enzymes, LazF acts in unfamiliar sequence environments. The enzyme prefers small side
chains such as Ala, Ser and Gly, on either side of the substrate azoline (Fig. 5a; Fig. 6a),
and dehydrogenation of Thn happens much faster than Oxn or 5-MeOxn. Only one Oxn
substrate not flanked by a Dha (Fig. S42c, LazACP1 2S) underwent up to 20%
dehydrogenation after 2 h.
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LazF-mediated dehydrogenation of Oxn11 emerged as the key step of lactazole
biosynthesis, and thus, we sought to explore it in greater detail. How does Dha facilitate Oxn
dehydrogenation given a relaxed specificity profile of LazF and its strong preference for Thn?
We hypothesized that π-conjugation between the double bond of Dha in position –1 and the
π-system of substrate Oxn may tune its reduction potential and facilitate dehydrogenation.
To see whether this hypothesis is plausible, we calculated reduction potentials for several
Oxz and Thz-containing tripeptides flanked by Dha or Ala on either side of the heterocycle
(Fig 5c). We utilized density functional theory at the B3LYP level of theory and used the 6-
311+G(d) basis set following previously established methods (S.I. 2.6).46–48
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According to our calculations, Ala-Oxz-Ala had E0’ = –273 mV (pH 8), some 19 mV above
a Thz analog, consistent with the notion that Thn undergoes dehydrogenation easier than
Oxn.49 We also found that an Ala to Dha substitution in position –1 for an Oxz-containing
peptide lowers its reduction potential by 34 mV, suggesting that the Dha-Oxn motif
undergoes dehydrogenation easier than Ala-Thn. Dha in position +1 had a minor effect, and
a tripeptide Dha-Oxz-Dha had E0’ = –281 mV (pH 8), halfway between Ala-Thz-Ala and Ala-
Oxz-Ala. These data support our original hypothesis. To see whether these calculations are
in line with the dehydrogenation ability of LazF, we experimentally determined reduction
potential for LazF-bound FMN following a modified method of Massey,50,51 and established
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E0’ = –244±1 mV (pH 8; Fig. S45, S.I. 2.7). This value indicates that LazF-bound FMN
provides sufficient oxidizing power to dehydrogenate all studied substrates, but the reaction
potential increases from Ala-Oxn-Ala to Dha-Oxn-Dha to Ala-Thn-Ala, which helps in
explaining Dha-dependent acceleration of Oxn oxidation. A similar mechanism might be at
play during oxidation of Thn7. A Thn inside the Ala-Thn-Gln motif (Fig. S43a) was not
dehydrogenated on a 30-min time scale, but during maturation of LazAaux and LazAwt, an
intermediate Thz5-Dha6-Thn7-Gln8 was too fast-lived to be captured.
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The reduction potential determined here matches well with FMN-dependent azoline
dehydrogenases from other RiPP classes,52 indicating that electrochemically LazF is not
unique. Although Oxz-containing thiopeptides are not common, a number of such structures
have been characterized.27,53,54 Most Oxz in thiopeptides, especially those in berninamycin-
like structures,55 are flanked by a Dha residue in position –1 (Fig. S46), suggesting that Dha-
assisted Oxn dehydrogenation may be a general phenomenon in biosynthesis of Dha/Oxz-
containing natural products.
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LazC (macrocyclization). Even though we did not study LazC in isolation, a number of
results clarify its function during lactazole assembly. First, LazC-catalyzed macrocyclization
is fast compared to other Laz enzymes: during the time course studies, the macrocyclization
substrate, LazA Dha1/Dha10-Oxz11-Dha12-Thz13 never accumulated to over 5% of total
(Table S1, S3). Conversely, maturation of a LazAmin variant containing 5 mutations in the CP
(Fig. S22c) stalled due to inefficient LazC action, with the macrocyclization precursor peptide
comprising over 70% of total after a 3 h incubation. These data suggest that LazC might be
more sensitive to the overall substrate structure than other Laz enzymes.
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More importantly, LazC exerts kinetic control over lactazole assembly. The minimal
recognition requirement around the 4π component of LazC (Oxz11-Dha12) ensures that as
soon as Dha10-Oxz11-Dha12-Thz13 modifications are installed, macrocyclization will
terminate the biosynthesis. This checkpoint controls the fate of Ser4, which in the absence
of LazC is slowly dehydrated by LazBF. When LazC is present in the enzyme mixture, it
macrocyclizes the substrate before this dehydration happens.
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Figure 6. Dissection of lactazole biosynthesis. a) Summary of innate substrate preferences
of LazDE, LazF dehydrogenase, and both LazBF Trp-dependent and azoline/azole-
dependent modes of actions. Laz biosynthetic enzymes are in general characterized by
“local” action, and require short 1–3 amino acid-long motifs for activity. b) Observed
interactions between enzymatic activities in lactazole A biosynthesis. Pointed-end arrows
indicate activation/promotion of the recipient activity at specified residues of LazAwt CP (for
instance, cyclodehydration at residue 5 enables dehydrogenation at residue 5, or
dehydration at Ser12 promotes dehydration at Ser10). Blunt-end arrows indicate inhibition
(in the broad meaning of the word) of the recipient activity at specified positions (for example,
†
macrocyclization utilizes residue 1 and 12 and prevents dehydration of Ser4). Although
Ser4 is not dehydrated in wild type lactazole A, formation of Dha4-lactazole is possible as
discussed elsewhere in the text. This analysis visualizes the central role of LazBF during
lactazole biosynthesis.
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Our results map a multienzyme biosynthesis process of a RiPP at a single PTM resolution,
and thus, provide important clues on how Laz enzymes maintain integrity of lactazole
assembly. LazBF dehydrates 4 out of 6 Ser in the CP of LazAwt, and, as we previously
found,37 depending on the order of enzyme addition, over- and underdehydrated
thiopeptides bearing 5 or 3 Dha respectively can also be produced, hinting at a deep-seated
cooperation between Laz enzymes. The results of this work reveal the extent of this
cooperation. Coordination of the dehydratase activity emerges as the central theme of
lactazole biosynthesis, and every enzyme is involved in the regulation of LazBF-mediated
Dha synthesis (Fig. 6b). Innate substrate preferences of LazDE — specifically, its inability
to cyclize amino acids adjacent to azoline/azoles, selectivity for Ser in position –1, and
preference of Cys over Ser as substrate — discriminate a single Ser residue (Ser11) for
cyclodehydration, leaving the remaining five Ser as potential LazBF substrates.
Concomitantly, cyclodehydration of Cys5, 7 and 13 prepares Ser4, 6, 10 and 12 for
azoline/azole-dependent dehydration. LazF dehydrogenase exerts a finer level control,
promoting dehydration at Ser10 and impeding facile Dha synthesis at Ser4 through
dehydrogenation of Thn13 and Thn5, respectively. Coupling of dehydrogenation to Dha
formation, observed during the oxidation of Dha6-Thn7 and Dha10-Oxn11-Dha12-Thz13
motifs, serves as another important control mechanism, as it accelerates the biosynthesis
and ensures that Dha10 is installed prior to LazC-catalyzed macrocyclization, preventing
formation of the underdehydrated thiopeptide. Finally, through macrocyclization and LP
cleavage, LazC emerges as a kinetic regulator of biosynthesis, restricting the action of
LazBF at Ser4, which would otherwise be slowly dehydrated to Dha4. These control
mechanisms are tightly coupled to the substrate preferences of LazBF, which — despite its
apparent promiscuity — evolved to accurately sense PTMs introduced by other enzymes.
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“Local” action also characterizes other Laz enzymes (Fig. 6a). Their primary substrate
requirements may be narrowed down to 1–3 amino acids around the modification site, which
allow them to act multiple times during lactazole biosynthesis and explain previously
observed successful modification of substrates with extensively mutated CPs. The local
action of the enzymes also explains the unusual punctuation of LazDE-mediated cyclizations
by LazBF-catalyzed dehydrations and azoline dehydrogenation throughout the process:
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every enzyme acts as soon as it finds a substrate in a suitable local environment, regardless
of the overall modification pattern. Nevertheless, enzymatic activities are tuned to the
substrate, and with the exception of Ser1 dehydration, in vitro lactazole biosynthesis
proceeds through a unified pathway, allowing a brief bifurcation during modification of Ser6
and Cys7.
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Our results can explain lactazole biosynthesis without invoking a supramolecular enzyme
complex, sometime hypothesized for thiopeptide assembly56 and often observed during
biosynthesis of other RiPPs.20,56,57 Instead, it appears that the enzymes communicate with
each other via the substrate, and interactions between installed PTMs influence the
assembly process in a major way. Such substrate-assisted assembly is emerging as a
general phenomenon in RiPPs biosynthesis.7,58 At the same time, a possibility for a
supramolecular assembly is not ruled out, as intricate protein-protein interactions occurring
in vivo might not be perfectly recapitulated in our in vitro study. Additionally, competitive
binding to substrate LP likely adds an extra level of complexity to how enzymes
communicate during lactazole biosynthesis, but further studies are required to substantiate
this discussion.
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Despite apparent structural similarities between thiopeptides, their biosynthetic logic
appears to be fairly divergent. For example, during micrococcin maturation, Thz installation
is separated from Ser dehydration by an obligatory C-terminal decarboxylation step,23 and
in thiomuracin biosynthesis, glutamyl transferase TbtB is highly selective for the overall azole
pattern.22,59 The laz BGC is also unique in its minimalistic size, unusual gene architecture
(for instance, the fusion between glutamate elimination and dehydrogenase domains) and
low sequence similarity to orthologous enzymes from other thiopeptide families.41 Perhaps,
lactazole-like thiopeptides evolved from a goadsporin-like linear azole/Dha-containing
peptide60,61 independently of other thiopeptide BGCs.
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In summary, by identifying several control mechanisms responsible for integrity of lactazole
assembly, this study begins to address how multiple promiscuous RiPP enzymes capable
of competing over their substrate cooperatively modify it to converge on a single natural
product. Our results rationalize the ability of laz BGC to synthesize diverse thiopeptides and
inform on future bioengineering applications of Laz enzymes, including functional
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reprogramming of lactazole achieved by screening of lactazole-inspired combinatorial
libraries.
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492 Acknowledgements
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We thank Dr. Kazuya Teramoto and Dr. Takayuki Kuge for their help with protein
expression. This work was supported by CREST for Molecular Technologies, JST to H.S.;
KAKENHI (JP16H06444 to H.S. and Y.G. and H.O.; JP17H04762, JP18H04382,
JP19K22243, and JP20H02866 to Y.G.) from the Japan Society for the Promotion of
Science (JSPS); a grant-in-aid from the Institute for Fermentation, Osaka (IFO) to H.O.;
Amano Enzyme, Inc. to H.O.; and the A3 Foresight Program, JSPS to H.O. DFT
computations were performed using Research Center for Computational Science, Okazaki,
Japan.
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501 Supporting Information. Experimental procedures, additional experimental data,
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nucleotide sequences and DNA template assembly schemes. This material is available free
of charge via the Internet at http://pubs.acs.org.
504 References
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