Biocatalysts from cyanobacterial hapalindole pathway afford antivirulent isonitriles against MRSA

Article abstract of DOI:10.1007/s12038-021-00156-4

Abstract: The emergence of resistance to frontline antibiotics has called for novel strategies to combat serious pathogenic infections. Methicillin-resistant Staphylococcus aureus [MRSA] is one such pathogen. As opposed to traditional antibiotics, bacteriostatic anti-virulent agents disarm MRSA, without exerting pressure, that cause resistance. Herein, we employed a thermophilic Thermotoga maritima tryptophan synthase (TmTrpB1) enzyme followed by an isonitrile synthase and Fe(II)-α-ketoglutarate-dependent oxygenase, in sequence as biocatalysts to produce antivirulent indole vinyl isonitriles. We report on conversion of simple derivatives of indoles to their C3-vinyl isonitriles, as the enzymes employed here demonstrated broader substrate tolerance. In toto, eight distinct L-Tryptophan derived α-amino acids (7) were converted to their bioactive vinyl isonitriles (3) by action of an isonitrile synthase (WelI1) and an Fe(II)-α-ketoglutarate-dependent oxygenase (WelI3) yielding structural variants possessing antivirulence against MRSA. These indole vinyl isonitriles at 10 μg/mL are effective as antivirulent compounds against MRSA, as evidenced through analysis of rabbit blood hemolysis assay. Based on a homology modelling exercise, of enzyme-substrate complexes, we deduced potential three dimensional alignments of active sites and glean mechanistic insights into the substrate tolerance of the Fe(II)-α-ketoglutarate-dependent oxygenase. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s12038-021-00156-4

J Biosci (2021)46:37
DOI: 10.1007/s12038-021-00156-4
Ó Indian Academy of Sciences
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Biocatalysts from cyanobacterial hapalindole pathway afford
antivirulent isonitriles against MRSA
1
2
3
BRITTNEY M BUNN , MIZHI XU , CHASE M WEBB and
4
RAJESH VISWANATHAN *
1Thermo Fisher Scientific, Cleveland, OH, USA
2Department of Chemistry, Georgia Institute of Technology, Atlanta, GA, USA
3Department of Pharmaceutical Chemistry, School of Pharmacy, University of California,
San Francisco, CA, USA
4Department of Chemistry and Biology, Indian Institute of Science Education and Research Tirupati,
Tirupati, India
*Corresponding author (Email, rajesh@iisertirupati.ac.in)
MS received 29 June 2020; accepted 22 February 2021
The emergence of resistance to frontline antibiotics has called for novel strategies to combat serious pathogenic
infections. Methicillin-resistant Staphylococcus aureus [MRSA] is one such pathogen. As opposed to tradi-
tional antibiotics, bacteriostatic anti-virulent agents disarm MRSA, without exerting pressure, that cause
resistance. Herein, we employed a thermophilic Thermotoga maritima tryptophan synthase (TmTrpB1) enzyme
followed by an isonitrile synthase and Fe(II)-a-ketoglutarate-dependent oxygenase, in sequence as biocatalysts
to produce antivirulent indole vinyl isonitriles. We report on conversion of simple derivatives of indoles to their
C3-vinyl isonitriles, as the enzymes employed here demonstrated broader substrate tolerance. In toto, eight
distinct L-Tryptophan derived a-amino acids (7) were converted to their bioactive vinyl isonitriles (3) by action
of an isonitrile synthase (WelI1) and an Fe(II)-a-ketoglutarate-dependent oxygenase (WelI3) yielding structural
variants possessing antivirulence against MRSA. These indole vinyl isonitriles at 10 lg/mL are effective as
antivirulent compounds against MRSA, as evidenced through analysis of rabbit blood hemolysis assay. Based
on a homology modelling exercise, of enzyme-substrate complexes, we deduced potential three dimensional
alignments of active sites and glean mechanistic insights into the substrate tolerance of the Fe(II)-a-ketoglu-
tarate-dependent oxygenase.
Keywords. Antivirulence; Enzymology; Isonitriles; MRSA; Natural products; Tryptophan-derived
bioactives
1. Introduction
entire world (Otto 2010). Antivirulence agents present
alternatives to conventional antibiotics (Yu et al. 2014).
Emergence of highly resistant strains to frontline In contrast to conventional antibiotics, antivirulence
antibiotics, coupled with the decline in the discovery of agents are typically not bactericidal. Antivirulence
new antibiotics has called in an urgent search for novel agents, by virtue of not threatening the survival of
agents to combat serious bacterial infections (Gordon bacteria, will elicit less evolutionary pressure against
and Lowy 2008). Methicillin-Resistant Staphylococcus the pathogen to develop resistance (Kuo et al. 2015).
Aureus (MRSA), is one among the most widespread The mechanism of action is based upon curtailing the
bacterial pathogens in the United States (Tommasi pathogen’s ability to elicit toxins against the host’s
et al. 2015) and this trend is slowly spreading to the immune system. An unimpaired immune system may
Electronic Supplementary Information: The online version contains supplementary material available at https://doi.org/10.
1007/s12038-021-00156-4.
http://www.ias.ac.in/jbiosci

37 Page 2 of 13
Brittney M Bunn et al.
then be able to fight off the infection on its own. benchmark for the development of selective methods to
Alternatively, a boost in the form of a low-dose con- orchestrate concise bond-forming chemistries (Bhat
ventional antibiotic in combination with an antiviru- et al. 2014; Lu et al. 2014; Maimone et al. 2015).
lence agent may become a successful strategy against Though several groups advanced their biosynthetic
more invasive infections. Antivirulence therapy offers hypothesis underlying nature’s strategy for hapalindole
the attractive prospect of bringing back conventional construction based on elegant chemistries (Park et al.
and affordable antibiotics into the clinic. We have 1992; Richter et al. 2008), it wasn’t until recently that
recently reported the design and evaluation of novel the genetic and biochemical basis of their biosynthesis
antivirulent agents and showed that the dual measure- have begun to emerge (Micallef et al. 2014; Li et al.
ment of pathogen’s growth progress in tandem with the 2015; Liu et al. 2016; Hillwig et al. 2016). Strain-
agent’s ability to disrupt hemolysis (causing a reduced specific gene clusters were identified and biochemically
amount of hemoglobin to be ruptured) can serve as a characterized via heterologous expression of select
reporter assay to evaluate a novel agent’s ability to enzymes, that include a highly conserved set of genes
reduce the pathogen’s virulent release of a-toxins (Yu encoding for isonitrile biosynthesis (Scheme 1B).
et al. 2014). In this regard, due to previously known These studies collectively have thrown light at the
antibiotic properties, we hypothesized that the novel biocatalytic roles of isonitrile synthases (Ittiamornkul
indole vinyl isonitriles may possess activity worthy of et al. 2015), indole N-methyl transferases, free-stand-
evaluating for potential antivirulence against MRSA.
ing halogenases (Hillwig and Liu 2014; Hillwig et al.
Synthetic biology continues to enhance the structural 2016) and prenyltransferases with their associated
and functional novelty of natural products. By cyclase enzymes (Li et al. 2015) from the biosynthetic
recruiting a diverse set of enzymes from secondary pathway of the hapalindoles (Scheme 1C).
metabolic pathways, the repertoire of this field is
We aimed at the production of novel indole vinyl
rapidly expanding (Keasling 2010; Keasling et al. isonitrile analogs using genes from hapalindole path-
2012). While total chemical synthesis continues to be a way with potential routes to their novel unnatural
tour-de-force for access to scalable quantities of unique derivatives. Herein, we report a biocatalytic approach
chemicals (especially pharmaceuticals), recent advan- by sequential expression of genes from T. maritima and
ces offers complementary synthetic biological tools to Westiella intricata to construct enzymes that produce
execute chemoenzymatic production of complex novel indole vinyl isonitriles (3) and their unnatural
molecules (Savile et al. 2010; Kwon et al. 2012; derivatives (Bunn 2016). Figure 1 illustrates multiple
Ongley et al. 2013). Cyanide or isonitrile-containing beneficial features of biocatalytic conversions for
natural products and their intriguing biosynthetic constructing bioactive molecules, in particular the vinyl
pathways continue to generate synthetic interest indole isonitriles such as 3 (Hoppe and Scho¨llkopf
because of their chemical complexity and versatility 1984; Spallarossa et al. 2016).
(Scheuer 1992; Simpson and Garson 2004; Wilson
The isonitrile synthase and Fe(II)-a-ketoglutarate-
et al. 2012). Furthermore, isonitriles have shown pro- dependent oxygenase genes (welI1 and welI3) are
mise as imaging probes and have distinct use in encoding for indole vinyl isonitrile biosynthesis and are
chemical biology approaches (Wainman et al. 2013).
conserved in every hapalindole-producing cyanobac-
Since their original discovery by Moore in the terial genome sequenced thus far (hpi, fam or amb gene
1980’s, (Moore et al. 1984, 1987) the structurally clusters). Previously, we and others have shown that
complex hapalindole isonitriles have expanded to a the indole-isonitrile (3) (derived from L-tryptophan) is
super family of more than 80 highly bioactive natural the product of the sequential biocatalysis transforma-
products identified to date (Scheme 1A). Based on tions carried out by isonitrile synthase and Fe(II)-a-
established molecular and biochemical targets, these ketoglutarate-dependent oxygenase enzymes. (Micallef
alkaloids display an array of bioactivities ranging from et al. 2014; Hillwig et al. 2014) Specifically, in these
antibacterial, antimycotic and multiple drug resistant two steps, the first enzyme is an isonitrile synthase that
(MDR) reversal, microtubulin inhibition, vasopressin converts an amino group into its isocyano group and
receptor blockage, RNA polymerase inhibition, NFjb WelI3 is an Fe(II)-a-ketoglutarate-dependent oxyge-
inhibition and as sodium channel modulators (Walton nase that causes decarboxylative desaturation of the
and Berry 2016). This relatively large family of iso- Trp backbone. Homologs of these two enzymes (also
prenoid-indole alkaloids, with exquisite structural found from eDNA) are highly conserved in all
diversity, has inspired numerous total synthesis pursuits hapalindole gene clusters identified so far (Brady and
([ 50 independent routes) and have served as a Clardy 2005; Brady et al. 2007). These enzymes work

Biocatalysts afford antivirulent isonitriles against MRSA
Page 3 of 13 37
A
B
C
Scheme 1. (A) Representative hapalindole alkaloid family members. Conventional ring numbering is provided for core
structure only. (B) Conserved portion of the gene clusters characterized from four distinct cyanobacterial strains. The
isonitrile biosynthetic genes are colored in red; prenyltransferase is colored blue and a proposed cyclase associated with ring
closure is colored purple. (C) Currently accepted operating mechanism for hapalindole core biosynthesis involving early
stage isonitrile generation followed by prenyltransfer and a Cope rearrangement of the terpene skeleton ending with an Aza-
Prins cyclization to result in the hapalindole core. Cis-3 is a vital intermediate and is also a known antibiotic.
in tandem to synthesize indole-isonitriles (3) and indole-isonitriles are bona fide pathway intermediates
thereby afford a biocatalytic strategy for their synthesis. in cyanobacteria (Micallef et al. 2014; Bunn 2016).
By isolating metabolites directly from hapalindole- Considering their chemical and biological versatility of
producing Fischerella strains and unambiguously enzymes producing vinyl isonitriles, we probed the
characterizing their structural identity, we established innate substrate promiscuity of indole vinyl isonitrile

37 Page 4 of 13
Brittney M Bunn et al.
A
B
Figure 1. Premise of this work. (A) Descriptions and details of chemical synthesis leading to vinyl isonitriles. (B) Green and
sustainable biocatalysis that can provide access to bioactive isonitriles using enzymes.
synthase and Fe(II)-a-ketoglutarate-dependent dioxy- vinyl isonitrile analogs are evaluated revealing an
genase involved in hapalindole biosynthesis as a impressive structural motif capable of curtailing
promising pathway for producing isonitrile natural Methicillin Resistant S. aureus (MRSA) virulence.
products.
Together, these results offer promising avenues to
To establish feasibility toward a streamlined assem- develop new antibiotic strategies for the future.
bly of these alkaloids, we developed a two-enzyme-
single-assay system with the isonitrile synthase and
2. Results and discussion
Fe(II)-a-ketoglutarate-dependent dioxygenase. By
sequencing the pathway with a third hyperthermophilic
Thermotoga maritima tryptophan synthase (TmTrpB1)
enzyme (Hettwer and Sterner 2002) we report on
conversion of indoles to yield 3. We assembled C2-
methyl L-tryptophan analog (as an intermediate) from
C2-methyl indole and L-Serine, en route to novel
indole isonitriles. Results from exploring the substrate
tolerance of isonitrile synthase (WelI1) and Fe(II)-a-
ketoglutarate-dependent dioxygenase (WelI3) through
a direct tandem enzymatic assay is reported. Interest-
ingly, antivirulence potential of these novel indole
Indole vinyl isonitrile synthase and Fe(II)-a-ketoglu-
tarate-dependent dioxygenase were expressed in E. coli
and were purified to homogeneity (figure 2). We
streamlined biosynthesizing analogues such as
2-methyl-L-tryptophan (7b) using an additional in vitro
enzymatic reaction, by TrpB1 (recombinant tryptophan
synthase) from the hyperthermophilic bacterium Ther-
motoga maritima. (Hettwer and Sterner 2002) In the
presence of L-serine, an indole derivative, and PLP,
TmTrpB1 catalyzes the formation of a tryptophan

Biocatalysts afford antivirulent isonitriles against MRSA
Page 5 of 13 37
Figure 2. (A) SDS-PAGE analysis of the Ni-NTA purification of WelI1. (B) SDS-PAGE analysis of the Ni-NTA purification
of WelI3. (C) SDS-PAGE analysis of the Ni-NTA purificed of WelI1 and WelI3. WelI1 is 37205.6 Da and 31239.3 Da
derivative and accepts a variety of indole precursors as employed in this investigation, including the use of
substrates. Therefore, E. coli BL21-(DE3)-RIPL cells TmTrpB1 for generation of 7b.
transformed with a plasmid encoding TmTrpB1
For each assay, HPLC analysis of the organic
(pET28a-tmtrpB1) were cultured and induced to over- extracts from the isonitrile synthase and Fe(II)-a-ke-
express TmTrpB1, which was subsequently purified by toglutarate-dependent dioxygenase catalyzed reaction
Ni-NTA chromatography (figure S1). An assay was set mixtures showed the formation of corresponding
up containing 2-methyl indole and allowed to incubate products (figure 3B). The extracts were simultaneously
at 80°C for approximately 48 hours. The reaction was analyzed by high resolution mass spectrometry
monitored by thin layer chromatography and the pro- (figure 3B, entries 1–8). Following chromatographic
duct was purified by silica gel flash chromatography.
separation, we obtained high % yields in the range of
A single-pot enzymatic assay was set up as outlined 60-90% depending on each substitution on the indole
in figure 3A. This tandem assay was based on previous ring. We were able to scale up the synthesis of 3a, 3c,
experiments with L-Tryptophan as the natural substrate 3e and 3g to 850-920 lmol levels without losing
(Micallef et al. 2014). In this study, we probed seven biocatalytic efficiency (table S1). By increasing L-Trp
distinct L-tryptophan derivatives as possible analogs of (or its analog 7c, 7e or 7g) quantities and correspond-
the natural substrate. Assay components included the ingly increasing the amount of ribose-5-phosphate and
enzymes (21–28 lM isonitrile synthase and 11-19 lM that of a-KG, the scaled-up synthetic assays occurred
Fe(II)-a-ketoglutarate-dependent dioxygenase) in with similar efficiencies. Product structures of 3a, 3c,
addition to 2 mg of L-tryptophan or its respective 3e and 3g as the major product in each case as were
analog. The assay buffer consisted of 2.5 mM ribulose- verified through NMR data analyses presented in the
5-phosphate (exchangeable with its keto-enol tautomer supporting information.
ribose-5-phosphate), equimolar amount of a-ketoglu-
Overall, as revealed in figure 3B, each of the seven
taric acid disodium salt dehydrate, 100 lM (NH4)2- distinct substrate analogs was recruited successfully
Fe(SO4)2), and 5% (v/v) glycerol. After incubation at resulting in their corresponding indole-isonitrile ana-
37°C for 18 h , the assays were extracted with ethyl logs efficiently. HPLC analysis indicated the presence
acetate (v:v: 1:1) and analog production was evaluated of both cis and trans isomers of the natural product
by LC-HRMS, MS-MS fingerprinting and NMR anal- analogs, and their ratios were quantified through HPLC
yses. Figure 3A displays the biocatalytic sequence analyses. A C2-methyl substituent (7b) upon turnovers

37 Page 6 of 13
Brittney M Bunn et al.
Figure 3. (A) Sequential biocatalysis involving three enzymatic steps. (B) List of substrate analogs incorporated into the
WelI1 and WelI3 catalysis, resulting in isonitriles 3 a-h. Ratios of cis:trans isomers measured from HPLC.

Biocatalysts afford antivirulent isonitriles against MRSA
Page 7 of 13 37
by isonitrile synthase and Fe(II)-a-ketoglutarate-de- for its intensity, referring to highly antivirulent com-
pendent dioxygenase caused smooth production of pound activity.
indole vinyl isonitrile with a methyl substituent, as
DMSO served as a vehicle with no detectable growth
shown in 3b. With the exception of 5-methoxy-sub- inhibition, and no inhibition of hemolysis. Diflunisal, a
stituted 7g, all other L-Trp derivatives (7c, d, e, f and well-established control served as a standard. None of
h) produced product isonitrile analogs that favored the the indole vinyl isonitriles tested at concentrations up
cis isomer marginally (\ 10:1) (figure 2B). Interest- to 50 lg/mL had any detectable bactericidal effect.
ingly, substitutions at C5 position with a methoxy They allowed MRSA to grow sustainably, thereby
group, or at C2 position with a methyl group or having posing less of a chance for the pathogen to develop
no substitution at all, produces a [10:1 ratio favoring resistance. Interestingly, C2-methyl substituted isoni-
the cis isonitrile. All other analogs produced corre- trile (3b) possessed a detectable inhibition of a-toxin
sponding products in about a *5:1 ratio, or even an production while displaying favorable combination of
equimolar mixture as in 3h.
growth and hemolysis inhibitory effects. C5- methyl
Elemental composition of major products and MS2 substituted isonitrile (3e) had a similar effect. The
fragmentation patterns (table S2) further supported the parent isonitrile 3a was slightly less effective than 3b
production of these seven analogs of indole-isonitriles and 3e suggesting that the methyl substitution favor-
(7b-h). Unambiguous characterization of major prod- ably increases the antivirulence property of these
ucts from each of the scaled-up reactions are carried out compounds. We compared these positive results to a
1
with NMR of the respective H and 13C nuclei as series of rigorous controls. Analogs where all structural
shown in the supporting information. Furthermore, the motifs were identical except for the isonitrile func-
LC retention times, followed by MS information fin- tionality were tested. A nitrile in place of the isonitrile,
gerprinting their fragmentation patterns are also cor- an ester in place of the isonitrile and its halogenated
roborating and are provided in the supporting analogs, and aldehyde functionality in place of the
information (figures MS1-MS16, SI). Based on these isonitrile were tested as controls (figure 4, pairs of bars
results, it is clear that isonitrile synthase and Fe(II)-a- 6–9 from left). Significantly these functional groups
ketoglutarate-dependent dioxygenase tolerate a broad offered no antivirulent property evident by the lack of
range of substitutions on the indole ring portion of hemolysis inhibitory effect. The C3-aldehyde contain-
L-Trp. As the efficiency of these transfomations was ing indole also did not possess any antivirulence to the
good enough for access to diverse bioactive molecules, degree as the isonitrile functional group indicating
we setup our next phase of study to probe their clearly that the isonitrile functionality renders bioac-
antivirulence activity.
tivity. The N1-methyl substituted analogs (3c) gave
impressive hemolysis inhibitory activity and in this
case alone, we were able to independently measure this
activity for each geometrical isomer. Upon evaluating
these individually, we found the trans isomer was
slightly more efficacious than its cis isomer. Overall,
while indole isonitriles were previously investigated for
their ability to serve as antibiotics, their antivirulent
property were not investigated before. This study
illustrates that they possess the potential and therefore
future medicinal chemistry is worthy to delineate their
mode of efficacy.
3. Novel indole vinyl isonitriles show antivirulence
property against MRSA
We next addressed the possibility of these novel
isonitrile-containing analogues possessing valuable
pharmacological property. As shown in figure 4, we
measured the MRSA growth and hemolysis inhibitory
effects. We followed methods that were reported earlier
(Yu et al. 2014) and also tested a series of new indole
vinyl isonitriles produced through our earlier investi-
gations. In figure 4, the green bar refers to MRSA
growth, and for antivirulent property measurement, one 4. Mechanistic details of indole vinyl isonitrile
would ideally like to see this bar at near 100% growth, biosynthesis
to ensure that it is not pressurizing resistance devel-
opment. The red bar indicates extent to which hemol- In order to rationalize the broad substrate tolerance of
ysis, caused by the virulence of the pathogen, is still isonitrile synthase and Fe(II)-a-ketoglutarate-depen-
observed in the presence of the antivirulent drug like dent dioxygenase, we turned to homology-based
compound. Therefore, we like to ideally see a low % modelling and in silico docking studies, given that

37 Page 8 of 13
Brittney M Bunn et al.
Figure 4. Evaluation of antivirulence potential for indole-isonitriles produced in this study. Green bars in plot above refer to
% MRSA bacterial growth and red bars indicate % of hemolysis inhibited in the presence of the indole vinyl isonitrile or a
control agent.
neither WelI1 nor WelI3 have been crystallized yet. The and it unambiguously revealed that the 1C source for
pathway to indole isonitrile 3 was characterized from isonitrile incorporation was a C5-sugar-phosphate:
eDNA-derived metagenomes by Brady and Clardy Ribulose-5-P. At the time, these were intriguing results
(2005). Expression of the isocyanide (isn) biosynthetic wherein a C5-sugar-phosphate provided the source for
cluster from eDNA led to identification of the roles of isonitrile carbon, as opposed to glycine as originally
IsnA and IsnB (homologs of isonitrile synthase and interpreted (from isotope feeding experiments) by
Fe(II)-a-ketoglutarate-dependent
dioxygenase Moore et al. (Bornemann et al. 1988). Subsequent
respectively).
studies have revealed a generally accepted mechanism
The study by Brady et al. about the eDNA-derived for the the formation of Trp-isonitrile from L-Trp
isonitrile synthase (IsnA) utilized a C13-labeled system through the formation of a Schiff’s base with Ribulose-

Biocatalysts afford antivirulent isonitriles against MRSA
Page 9 of 13 37
Scheme 2. Currently accepted mechanism of isonitrile biosynthesis from a-amino acid and a 1C donor (Ribulose-5-
phosphate). Ref: EC: 4-1-99-25 (https://www.qmul.ac.uk/sbcs/iubmb/enzyme/EC4/1/99/25.html).
5-P followed by degradative elimination of Pi, H2O, a- and PaPvcB have structural similarity, because if so,
hydroxyacetone and formaldehyde as shown in we could use structural similarities and differences to
Scheme 2 (Chang et al. 2017). The structure of paer- explain the relaxed substrate specificities observed in
cumarin, an isn-derived metabolite was identified in this study.
Pseudomonas aureginosa and the mechanistically
Therefore, we first built a homology model of Fe(II)-
related IsnA-B homologs PaPvcA and PaPvcB were a-ketoglutarate-dependent dioxygenase (based on
then biochemically characterized. (Clarke-Pearson and PaPvcB) and docked its substrate to create a three
Brady 2008) Three dimensional structures of PaPvcA dimensional model to explain the mode of substrate
and PaPvcB were reported later by Drake and Gulick binding to the active site (figure 5). Similar to Tipton
(Drake and Gulick 2008). Recent mechanistic investi- and Gulick’s study, we used clavaminate synthase
gations and an improved crystallographic structure of (Zhang et al. 2000) as a model for extracting coordi-
PaPvcB discovered additional homologs XnPvcB and nates of Fe(II) and we reconstructed a homology model
EaPvcB with divergent biochemical pathways to dis- of Fe(II)-a-ketoglutarate-dependent dioxygenase,
˚
tinct products. (Zhu et al. 2015)
based on PaPvcB’s 2.1 A structure. The model built for
Isonitrile synthase and Fe(II)-a-ketoglutarate-depen- Fe(II)-a-ketoglutarate-dependent dioxygenase signifi-
dent dioxygenase display a high degree of sequence cantly superimposed with the structure of PaPvcB with
conservation with its respective homologs ([ 90%, a Phyre TM-score of 0.9 (figure S2, SI). This combined
BLASTP), among Group V strains, indicating their with the pairwise multiple sequence alignment offered
shared biogenesis to the hapalindole core assembly. a high degree of confidence in predicting molecular
Furthermore, isonitrile synthase and Fe(II)-a-ketoglu- level interactions. Figure 5 shows the docked poses
tarate-dependent dioxygenase share significant active generated through the Schrodinger suite, after the
site similarity to PaPvcA and PaPvcB respectively model generated for Fe(II)-a-ketoglutarate-dependent
(table S3, SI). This observation allowed us to question dioxygenase was reconstructed with active site Fe(II)
whether Fe(II)-a-ketoglutarate-dependent dioxygenase and a-KG. Residues that help define substrate binding

37 Page 10 of 13
Brittney M Bunn et al.
Figure 5. Side view of WelI3 bound to L-tryptophan isonitrile (9) with benzylic methane proximal to Fe(II), shown in
brown; a-KG, His110; His259 and Asp112 shown in magenta; Substrate isonitrile 2 is shown in orange. Non-binding
residues that impact the size of the active site pocket are labelled.
and channeling, as defined for PaPvcB were highly proposed pathway to formation of cis and trans iso-
conserved. These included: Trp83, Arg274, Leu89, mers of indole isonitrile 3, to be caused by constraints
Leu91, Arg270, His110, His259 and Asp112 bind to of stereospecifity imposed by Fe(II)-a-ketoglutarate-
Fe(II) and are conserved in WelI3. The distinct and dependent dioxygenase during the decarboxylative,
significant difference between PaPvcB and WelI3 desaturation step (indicated by elimination of the b-OH
based on Clustalx alignments is absence of the group) as represented in Scheme 3.
hydrophobic pocket comprising of Met114-Tyr115-
Overall, in the presence of ribose-5-phosphate (or its
Leu-116 (PaPvcB numbering) in WelI3. Instead, in tautomer, ribulose-5-phosphate), the isonitrile synthase
Fe(II)-a-ketoglutarate-dependent dioxygenase we find catalyzes the incorporation of the isonitrile carbon to
a larger pocket filled with relatively smaller residues the L-tryptophan N-terminus, resulting in a (relatively
such as Ala114-Phe115-Ala116 as a triad. We then unstable) tryptophan-isonitrile intermediate (2). This
docked L-tryptophan isonitrile (2) and identified key intermediate is then presumably hydroxylated in situ at
poses wherein the substrate 2 presents its pro-S-hy- Cb-position, by Fe(II)-a-ketoglutarate-dependent
˚
drogen on b-carbon within 3.0 A from Fe(II).
dioxygenase, a non-heme oxygenase, and subse-
This result derived from bioinformatic alignment and quently, decarboxylated and desaturated (concomi-
docking, allows us to reason that the expanded sub- tantly) resulting in indole-isonitrile products (3).
strate profile displayed by WelI3 could be seen as an
Indole vinyl isonitriles are antibacterial as were
effect of having three (114–116) relatively smaller established long before. Their antivirulence property is
residues that open up the site for indole-derived, ster- revealed here for the first time. Also, as a relevant
ically bulkier groups; and consequently, that the aspect, these isonitiriles are the first stable intermedi-
mechanism of Fe(II)-a-ketoglutarate-dependent dioxy- ates in the biosynthetic pathway leading to the
genase resembles those observed for PvcB homolog hapalindoles. Natural products biogenetically arising
XnPvcB. Based on this reasoning, we present our from L-Tryptophan derived metabolic pathways

Biocatalysts afford antivirulent isonitriles against MRSA
Page 11 of 13 37
Scheme 3. Putative mechanism of Fe(II)-a-ketoglutarate-dependent dioxygenase (WelI3) based on its analogy to PaPvcB.
continue to spur novel concepts related to enzymatic 5. Conclusions
mechanism. (Alkhalaf and Ryan 2015) In that regard,
the hapalindole pathway has offered a suite of mech- Biocatalysts in general are green and cost-effective
anistically novel enzymes. The fact that both isonitrile options to recruit in synthesis. Enzymes performing
synthase and Fe(II)-a-ketoglutarate-dependent dioxy- unique transformations could serve as alternatives and
genase from Hapalindole pathway, allow a broader could complement transition metal-based chemistries,
substrate diversity opens doors for mechanistic inves- if only the robustness of newer enzymes with unique
tigations in the future. Milligram quantities of com- bond-forming repertoires are firmly established. Plas-
mercially available tryptophan derivatives can be quite mid-induced production of enzymes and their catalysis
costly, in addition, the prospect of a green method employed strategically to solve synthetic problems
employing three biocatalysts to produce hapalindole- hold promise toward development of sustainable syn-
type analogs from indole derivatives (much less thesis of complex molecules. In this study, we obtained
expensive compared to tryptophan derivatives), indole isonitriles with distinct substitution patterns on
sparked our interest. This investigation communicates the heterocyclic ring in high efficiency by employing
one of the early reports toward biosynthetically pro- both isonitrile synthase and Fe(II)-a-ketoglutarate-de-
ducing hapalindole-type analogs in addition to pro- pendent dioxygenase as sequential biocatalysts. The
viding the first support towards utilizing a tri-catalytic convenience of scaled up reactions provide quantitative
biosynthetic methodology to produce such analogs in a access to indole isonitriles that are accessed otherwise
more environmentally friendly, cost-effective, time through multi-step operations. Through a sequential
efficient manner. Since the isonitrile synthase and three-step biocatalytic variant, catalytic action of a
Fe(II)-a-ketoglutarate-dependent dioxygenase system hyperthermophilic TmTrpB1 was employed for syn-
has already been shown to work under cell lysate thesis of C2-methyl indole-isonitrile in 80% yield.
conditions (Micallef et al. 2014), the TmTrpB1 cat- Multiple members of this product class offer a mech-
alyzed reaction could be attempted at the lysate stage as anistically unique probe to curtail antibiotic resistance,
well. Side-stepping the purification of all three proteins against MRSA. Using homology modeling and sub-
˚
would only cause this biosynthetic methodology to strate docking, based on a 2.1 A crystal structure of
become even more time and cost effective. Modifica- PaPvcB, the relaxed substrate specificity is rational-
tions made to this early biosynthetic intermediate could ized. Given that hapalindole-type of alkaloid natural
eventually serve as a template for novel hapalindole products are extremely diverse and pharmacologically
natural product analogs with promising therapeutic promising, studies on establishing substrate promiscu-
value.
ity for enzymes involved in hapalindole biosynthesis

37 Page 12 of 13
Brittney M Bunn et al.
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study will pave ways for combinatorial biosynthesis of
hapalindole alkaloids, a biosynthetically diverse col-
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Hettwer S and Sterner R 2002 A novel tryptophan synthase
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Hillwig ML and Liu X 2014 A new family of iron-
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Hillwig ML, Zhu Q, Ittiamornkul K and Liu X 2016
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The authors thank Prof. C Dale Poulter for generous
gift of TmTrpB1 plasmid. The authors thank the Indi-
ana University Mass Spectrometry facility for assis-
tance with LC-MS and MS-MS data acquisition. BMB
thanks the Department of Education (DOE) grant
support through a GAANN fellowship (2012–2013).
RV is grateful for funding from the Case Western
Reserve University’s Start-Up award and support from
the Dean’s Office for Inclusion Diversity Faculty
Excellence achievement award. RV acknowledges the
support from IISER Tirupati towards this publication.
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Corresponding editor: BJ RAO