Structure-guided product determination of the bacterial type ii diterpene synthase tpn2

ABSTRACT A grand challenge in terpene synthase (TS) enzymology is the ability to predict function from protein sequence. Given the limited number of characterized bacterial TSs and

significant sequence diversities between them and their eukaryotic counterparts, this is currently impossible. To contribute towards understanding the sequence-structure-function

relationships of type II bacterial TSs, we determined the structure of the terpentedienyl diphosphate synthase Tpn2 from _Kitasatospora_ sp. CB02891 by X-ray crystallography and made

structure-guided mutants to probe its mechanism. Substitution of a glycine into a basic residue changed the product preference from the clerodane skeleton to a _syn-_labdane skeleton,

resulting in the first _syn_-labdane identified from a bacterial TS. Understanding how a single residue can dictate the cyclization pattern in Tpn2, along with detailed bioinformatics

analysis of bacterial type II TSs, sets the stage for the investigation of the functional scope of bacterial type II TSs and the discovery of novel bacterial terpenoids. SIMILAR CONTENT

BEING VIEWED BY OTHERS MOLECULAR INSIGHTS INTO THE CATALYTIC PROMISCUITY OF A BACTERIAL DITERPENE SYNTHASE Article Open access 06 July 2023 FUNCTIONAL CHARACTERIZATION AND STRUCTURAL BASES

OF TWO CLASS I DITERPENE SYNTHASES IN PIMARANE-TYPE DITERPENE BIOSYNTHESIS Article Open access 30 September 2021 STRUCTURAL INSIGHT ON ASSEMBLY-LINE CATALYSIS IN TERPENE BIOSYNTHESIS Article

Open access 09 June 2021 INTRODUCTION Terpenoids are a large family of natural products biosynthesized from five-carbon building blocks. Acyclic precursors, such as the universal precursor

for the C20 diterpenes geranylgeranyl diphosphate (GGPP, 1), are often cyclized by terpene synthases (TSs) and the resulting mono- or polycyclic hydrocarbon skeletons are subsequently

modified by a variety of tailoring enzymes leading to the extraordinary diversity of structures and biological functions seen in this class of natural products1,2,3. Terpenoids are one of

the largest and most diverse families of natural products, with over 84,000 members currently characterized4. However, terpenoids of bacterial origin represent only a small fraction (<2%)

of this family of natural products1. For example, there are over 2300 clerodane natural products known in nature, but only four are from bacteria4,5,6,7. TSs employ carbocation chemistry to

convert acyclic prenyl diphosphates into diverse terpene skeletons. These reactions are quite complex, frequently involving changes in bonding, hybridization, or configuration for most the

carbons in the substrate through a variety of reactions including carbon-carbon bond formation, hydride and alkyl shifts, and eliminations8. The conformational flexibility of terpene

substrates paired with the inherent reactivity of each cationic intermediate allows TSs to provide a diverse array of molecular templates for folding the substrate into catalytically

relevant poses and stabilizing intermediates9,10,11. Canonical bacterial TSs are categorized into type I or type II depending on how they initiate catalysis. Type I TSs, which have conserved

metal ion-binding motifs DDxxD and NSE/DTE, abstract the diphosphate group from the substrate8,12,13,14. Type II TSs utilize a highly conserved DxDD motif, in which the central Asp residue

acts as a general acid, to protonate an alkene or epoxide8,15. Due to the limited number of known bacterial terpenoids, there is a lack of general sequence-structure-function knowledge

regarding the biosynthetic machinery for bacterial TSs. Only a handful of bacterial type II TSs have been functionally characterized and only three diterpene skeletons, labdane, clerodane,

and halimane, are currently known to be produced by these enzymes (Fig. 1a and Supplementary Fig. 1). Enzymes that form labdane skeletons include the _ent_-copalyl diphosphate (CPP) synthase

PtmT2 from platensimycin and platencin biosynthesis in _Streptomyces platensis_16,17, as well as other _ent_-CPP18,19,20 and _n_-CPP21,22 synthases. The clerodane synthase Cyc1, found in

the biosynthetic gene cluster (BGC) of the antitumor antibiotic terpentecin, is proposed to bicyclize GGPP into _syn_-CPP+ before a cationic cascade that ends with deprotonation at C3 to

form terpentedienyl diphosphate (TPP, 2), or _ent_-_neo_-_trans_-_trans_-clerodienyl diphosphate (CLPP, Supplementary Fig. 1)23. Haur_2145 forms (+)-kolavenyl diphosphate, the

_ent_-_neo_-_trans-cis_-CLPP diastereomer from the _n_-CPP+ intermediate24. Rv3377c, from _Mycobacterium tuberculosis_, also forms _n_-CPP+ but quenches the cationic cascade by deprotonation

at C6 on a halimane skeleton to yield tuberculosinyl diphosphate (TbPP)25. Only two of these enzymes, PtmT2 and Rv3377c, have been structurally characterized16,26. In this study, we aimed

to understand what controls labdane, halimane, or clerodane skeletal formation in bacterial TSs. To achieve this, we structurally and biochemically investigated Tpn2, a homolog of Cyc1. We

experimentally verified Tpn2 as a TPP synthase and determined its structure by X-ray crystallography at a resolution of 2.57 Å. This is only the third structurally determined bacterial type

II TS and the first from any organism that produces a clerodane skeleton. Using the structural data, we performed a series of mutations to probe its mechanism and identified a single-residue

switch, G485D, that controls the selectivity between clerodane and labdane formation. Finally, we used bioinformatics to categorize four major families of type II TSs in bacteria, predict

labdane- vs clerodane-forming TSs in actinobacteria, and support that uncharacterized BGCs harbor novel terpenoids. This study illustrates the need for additional characterization of

bacterial TSs to expand the current scope of terpene enzymology and improve functional prediction of TSs, ultimately providing opportunities for genome mining and discovery of otherwise

cryptic novel terpenoids. RESULTS AND DISCUSSION TPN2 IS A TERPENTEDIENYL DIPHOSPHATE SYNTHASE We identified a BGC (_tpn_) in _Kitasatospora_ sp. CB02891 identical in organization to the one

reported in _Kitasatospora griseola_ MF730-N6, the original terpentecin producer5,23,27. The _tpn_ BGC includes a GGPP synthase, two TSs, two cytochrome P450 enzymes, and a ferredoxin (Fig.

 1b). Both TSs, the type II TS Tpn2 and the type I TS Tpn3, are homologous to Cyc1 and Cyc2 from _K. griseola_23,28 with 96 and 97% sequence identities, respectively, and are proposed to

form terpentetriene (3) from GGPP via TPP (2) (Fig. 1c). To confirm that Tpn2 is a TPP (2) synthase, we cloned and expressed _tpn2_ in _E. coli_ for protein production and purification

(Supplementary Fig. 2). Purified Tpn2 was incubated with GGPP and HPLC and GC-MS analysis of the enzyme reaction revealed one major product with an MS fragmentation pattern supporting a

clerodane diterpene (Supplementary Fig. 3). To simplify detection, reaction products were dephosphorylated and detected as their alcohol derivatives 1′ (geranylgeraniol, GGOH) and 2′

(_syn_-kolavenol) (Fig. 1d). To unambiguously confirm the function of Tpn2, 2 was isolated from large scale in vitro reactions and its structure was confirmed by 1H and 31P NMR analysis

(Supplementary Figs. S4 and S5 and Supplementary Table 4). FIRST CRYSTAL STRUCTURE OF A CLERODIENYL DIPHOSPHATE SYNTHASE We determined the crystal structure of Tpn2 at a resolution of 2.57 Å

(Fig. 2 and Supplementary Table 6). As seen in PtmT2 and Rv3377c16,26, Tpn2 adopts the characteristic double α-barrel βγ fold with the active site residing in the interfacial cavity. As is

typical for TSs from both eukaryotes and prokaryotes8, the active site cavity of Tpn2 is lined with both aromatic (H180, F250, W254, Y329, W384, W483, and Y489) and aliphatic (V116, A117,

T177, T181, G244, G287, L291, and G485) residues to form a hydrophobic pocket suitable for binding the long nonpolar tail of the substrate and preventing water or other nucleophiles from

prematurely quenching the reaction (Fig. 2b). At the bottom of the active site pocket lies the catalytically essential Asp-rich motif D294xDD. Docking experiments support that the central

Asp, D296, is properly positioned (3.2 Å from C14) for both the protonation of GGPP (Fig. 2c) and the deprotonation of the _syn_-cleroda-13_E_-en-4-yl+ intermediate (TPP+, Supplementary Fig.

 1). D296 is also suitably positioned (2.8 Å) near H342 for activation. Tpn2 also includes other conserved residues found in type II TSs including K383, which was previously proposed to

interact with the negatively charged diphosphate group on the substrate in both plant and bacterial _ent_-CPPSs16,29. One structural feature of type II diterpene synthases that remains

ambiguous is the location of metal ions in type II TSs. Previous studies support that type II TSs are dependent on the presence of divalent ions with their absence either completely

inactivating or severely diminishing activity in both plant and bacterial TSs16,30,31. However, because divalent metal ions are not directly involved in catalysis, it is proposed that they

instead play an important role in binding the negatively charged diphosphate group of the substrate29. Tpn2, PtmT2, and Rv3377c each contain a D114xxxxE motif that resides near the opening

of the active site and is proposed to be responsible for metal-binding in bacterial type II TSs (Supplementary Fig. 6)16,26 This is supported by molecular modeling in both Tpn2 (Fig. 2c) and

Rv3377c26, as well as site-directed mutagenesis in PtmT216. As with PtmT2 and Rv3377c, no bound divalent metal ions can be inferred in the electron density of the crystal structure of Tpn2,

leaving the exact binding mechanism of divalent ions in bacterial type II TSs undetermined. Overall, Tpn2 is structurally homologous to both PtmT2 and Rv3377c with root-mean-square

deviations (rmsd) of 1.25 and 1.89 Å, respectively, for Cα atoms (Fig. 2a). Tpn2 shares 33% sequence identity and 45% similarity with PtmT2 and 26% identity and 38% similarity with Rv3377c

(Supplementary Fig. 6). A few key differences between these three enzymes give rise to changes in product selectivity. Analysis of the active site cavity of Tpn2 revealed that Tpn2 and PtmT2

possess cavities of similar size and shape; however, its pocket is much narrower than the one seen in Rv3377c (Fig. 2c–e). Most notably, W384 and L291 in Tpn2 (W380 and L290 in Rv3377c)

protrude into the domain interface resulting in a narrower cavity that likely restricts the conformation of GGPP. This establishes a template for _syn_-CPP+ formation, positioning residues

that may facilitate deprotonation near the proposed location of the cationic intermediates. Previous structural, docking, and mutation studies support that Y479 is responsible for

deprotonation of C6 of the halima-13-en-5-yl+ intermediate (TbPP+) to form TbPP (Supplementary Fig. 1)26,32. While this residue is strictly conserved across type II TSs in bacteria, its

function is not. Despite the presence of an analogous Tyr residue in Tpn2 (Y489), no detectable halimadienyl diphosphate is formed in the Tpn2 reaction. Structurally, this is supported by

the docking pose of GGPP and a distance of 6.2 Å between Y489 and C9 of GGPP (C6 of the halimane scaffold); this distance is only 2.7 Å in Rv3377c (Fig. 2c)26. With no other basic residues

appropriately positioned for deprotonation, Tpn2 forces _syn_-CPP+ through a series of methyl and hydride shifts to access the recently deprotonated D296 (Fig. 3). This mechanism, where the

central Asp acts as both initiating acid and quenching base, is consistent with labeling studies of type II mutant TSs that produce CLPPs33,34. Given the high conservation in active site

cavity shape and residues between Tpn2 and PtmT2, we reasoned that the residue at 485 controls product determination. In PtmT2 and _At_CPS, deprotonation of the CPP+ at C17 is proposed to

occur by a water molecule that is hydrogen bonded to D50216,29. Lacking an analogous Asp on the side opposing the DxDD motif, Tpn2 instead has a G485 situated 3.6 Å from C19 of the substrate

(Fig. 2c). Given the similarities in active site cavity and residues, and the position of G485 in relation to C7 of GGPP (C8 of the labdane scaffold), we hypothesized that a basic residue

at 485 would result in deprotonation of C19 and formation of CPP. STRUCTURE-GUIDED MUTAGENESIS CONVERTS TPN2 INTO A _SYN_-COPALYL DIPHOSPHATE SYNTHASE To assess whether we could stimulate

labdane production by Tpn2, we created a G485D mutant. Preliminary in vitro reactions supported that the G485D mutant produced at least two new products compared to native Tpn2

(Supplementary Fig. 7). Due to its low activity, we were unable to isolate enough product for structure determination. To overcome this challenge, we employed a terpenoid overproduction

system in _E. coli_. First, while it has been previously shown that endogenous _E. coli_ phosphatases can remove the diphosphate moiety25,35, we envisaged that using Tpn3 to eliminate the

diphosphate group via its native type I TS activity may provide a more efficient route for the production and isolation of diterpenes. In addition, it was reported that the Tpn3 homolog Cyc2

accepts both labdane and clerodane scaffolds36, demonstrating the potential substrate promiscuity needed for the investigation of Tpn2 mutants. Second, we utilized our recently reported

MKI4 system, a GGPP overproduction system that leverages two promiscuous kinases to phosphorylate exogenously added isoprenol37,38, to further enhance diterpene production in vivo

(Supplementary Fig. 8). We built a series of plasmids with _tpn2_ and _tpn3_, including a genetically fused _tpn2_-_tpn3_ construct39, to test for terpentetriene (3) production in _E. coli_

(Supplementary Table 3). Bacterial TSs are excellent candidates for building multidomain enzymes as fusing a type II and type I TS mimics the structure of naturally occurring bifunctional

TSs from plants and fungi8,40,41. Our initial screen found that the _tpn2-tpn3_ fusion led to the highest production of 3, likely due to the increased proximity of the TS active sites

(Supplementary Fig. 9). As expected, the major product was 3, isolated at a yield of 9.0 mg L−1 and structurally confirmed via 1H and 13C NMR (Supplementary Table 4, Supplementary Figs. 3,

10, and 11). This confirmed the function of Tpn3 as a terpentetriene synthase. Two additional products, 6 and 7, were also isolated with GC-MS and NMR confirming them as α- and β-springene,

respectively (Supplementary Figs. 12–14). Consistent with our results, it was previously shown that Cyc2 eliminates the diphosphate moiety from GGPP, resulting in the formation of various

acyclic diterpenes36. With an efficient diterpene production strategy in place, we cloned the Tpn2G485D-Tpn3 fusion construct and expressed it in the MKI4 system. GC-MS analysis of the crude

extract showed the production of three new products in addition to 3, 6, and 7 (Supplementary Fig. 12). All three new products showed fragmentation patterns representative of labdane

diterpenes (Supplementary Fig. 15). The major product, 5, exhibited an _m/z_ of 272.15; the two minor products exhibited _m/_z values of 290.39 and 272.28, respectively. NMR and optical

rotation analysis of 5, which was isolated at a yield of 8.0 mg L−1, revealed it to be _syn_-sclarene (Fig. 3, Supplementary Figs. 15–20, and Supplementary Table 5, also referred to as

griseolaene)36,42. The formation of _syn_-sclarene by Tpn2G485D-Tpn3 indicates that Tpn2G485D preferentially forms _syn_-CPP (4). Thus, G485D acts as a single-residue switch, exhibiting sole

control over the formation of the clerodane TPP or labdane _syn_-CPP (Fig. 3). We also probed if mutation to a Glu residue would affect the formation of 5. Tpn2G485E-Tpn3 also produced both

3 and 5, but at a ratio of 2.3:1 (Supplementary Fig. 21); Tpn2G485D-Tpn3 gave a ratio of 1.6:1. It is interesting that Tpn2G485D and Tpn2G485E are still able to produce 2, albeit not

surprising given that they retain both the active site contour and D296. Previous quantum chemical calculations also suggested that there is not a large energetic barrier between the initial

bicyclization of GGPP into _syn_-CPP+ and the final methyl shift to form the _syn_-cleroda-13_E_-en-4-yl+ final intermediate43. Conversely, although Tpn2 has all the machinery necessary to

produce the halimane diterpene TbPP, i.e., D296 for initiation, a non-basic residue at G485 (the analogous residue in Rv3377c is A475), and the proposed base Y489 (Y479 in Rv3377c), Tpn2

does not produce any halimane compounds. In addition, the Tpn2Y489A mutant is completely inactive (Supplementary Fig. 7). This supports that Y489 is not involved as a general base in the

Tpn2 reaction, but instead may play an important role in forming the active site contour. Therefore, it is evident that the structural differences between Tpn2 and Rv3377c lead to

significant differences in substrate binding and therefore control product specificity in these two TSs. SITE-DIRECTED MUTAGENESIS SUPPORTS THE MECHANISM OF CLERODANE BIOSYNTHESIS IN

BACTERIA While the residue analogous to H180 in Tpn2 is conserved among labdane synthases from plants, clerodane synthases in plants generally include a Phe or Tyr residue in this

position44,45. Exchanging this His in some plant TSs switched product selectivity from CPP to a CLPP or halimane diphosphate (HPP). For example, AtCPSH263Y switched activity from _ent_-CPP

to (–)-kolavenyl diphosphate (_neo_-_trans-cis_-CLPP)34 and OsCPS4H501F from _syn_-CPP to _syn_-HPP43. Correspondingly, SdCPS2F255H from _Salvia divinorum_ switched its activity from

neo-_trans_-_cis_-TPP to _ent_-CPP46. It is therefore intriguing that Tpn2 possesses a His residue at this position despite being a clerodane synthase. We created the Tpn2H180F and Tpn2H180V

mutants to examine if it would affect product formation; however, these mutants were inactive (Supplementary Fig. 7). These results imply that while this residue may give insight into

product selectivity in plant TSs, the same logic is not applicable to bacterial TSs. We made additional mutations in the Tpn2 active site to probe the importance of various residues for

catalysis (Supplementary Fig. 7). As expected, mutation of the central Asp, D296A, results in complete inactivation of the enzyme. It has been proposed that the central Asp is activated by

water in cyclases such as SHC or by an adjacent His residue in PtmT216,47. Accordingly, mutation of H342 to Ala or Phe abolished any product formation. These results, along with the

structural data, support that D296 is responsible for initiation via protonation and that H342 is required to activate D296 for catalysis. Finally, mutation of the conserved K383 also

resulted in inactivation of the enzyme (Supplementary Fig. 7), consistent with previous studies that suggested this residue is involved in diphosphate binding16. BIOINFORMATICS ANALYSIS OF

TYPE II TSS HIGHLIGHTS TERPENOID BIOSYNTHETIC POTENTIAL IN BACTERIA With a single residue mutation, Tpn2G485D became the first _syn_-CPP synthase from bacteria. Such a simple change supports

the notion that bacteria possess uncharacterized type II TSs that possess new functions. To assess this hypothesis, we performed a comprehensive bioinformatics analysis of all canonical

type II TSs in bacteria. Using the Enzyme Function Initiative - Enzyme Similarity Tool48, a total of 964 type II TSs were identified from a BLAST search of Tpn2 given a minimum e-value

threshold of 5; 876 of these were unique sequences. These TSs separated into four major subfamilies in a sequence similarity network (SSN) at an e-value of 10−60 (Fig. 4a). Tpn2 clustered

with actinobacterial CPP synthases (384 members), gibberellin-associated _ent_-CPP synthases clustered with Haur_2145 (336 members), Rv3377c was found in its own subfamily consisting of

terrabacterial TSs (126 members), and one family, of mainly actinobacterial TSs, has no characterized enzymes (108 members) but is highly similar to squalene-hopene cyclases (SHCs) from

other bacteria49. At an e-value of 10−88, Tpn2, the _n-_CPP synthases, and Haur_2145 were each found within distinct clusters (Fig. 4b). Sequence analysis of these four major subfamilies

revealed both conservation and variation of key motifs and residues (Fig. 4). As expected for type II TSs, the central Asp is almost 100% conserved in all families. In the Tpn2/CPP synthase

family, H180 (95.6%) and Y489 (96.9%) are all highly conserved; this is consistent with mutations at these positions rendering Tpn2 inactive. The residue at position 485 is either Asp

(84.1%) or Gly (13.3%) implicating that most of these TSs form labdanes. In the Rv3377c family, the basic Y479 (92.1%) is conserved; A475, the structurally analogous position to G485 in

Tpn2, shows more variation with Gly (58.7%), Ala (25.4%), Asp (6.3%), and Ser (3.2%). Overall, this is consistent with most members of this family forming a halimane skeleton. Contradictory

to the actinobacterial CPP synthases, the cluster containing the gibberellin-associated _ent_-CPP synthases and Haur_2145 mostly possess Gly (91.1%) at the position analogous to G485. While

this makes sense for the clerodane-forming Haur_2145, this suggests that the active site pocket of the gibberellin-associated TSs may be altered from the actinobacterial orthologues.

Finally, the putative SHCs have significantly different sequences (≅30% identity with ≅30% coverage) than the characterized TSs although they have strictly conserved (>99%) Glu and Tyr in

a similar C-terminal motif. To assess the diversity of BGCs containing the 964 type II TSs, we constructed a genome neighborhood network (GNN) from the SSN at an e-value of 10−88

(Supplementary Fig. 22)48. The subfamily with the most genetic diversity is the actinobacterial _ent_-CPPS cluster. Of the 342 BGCs, 67% encode a type I TS and 14% encode a likely UbiA

cyclase; this implies that the remaining 67 BGCs do not use a second cyclase, unless a noncanonical TS is utilized14. Many (44%) of the BGCs that contain homologs of Rv3377c have equivalents

of Rv3378c, the adenosine prenylation enzyme50, although 33% of these contain type I TSs. The family containing Haur_2145 is similar with 21% containing Rv3378c homologs and 36% containing

type I TSs. The BGCs containing Tpn2/Cyc1, _n_-CPP synthases, gibberellin-associated _ent_-CPP synthases, and SHCs are all extremely conserved in gene presence and organization supporting

production of tepentecin, cyslabdan/labdanmycins, gibberellins and hopanoids, respectively23,51,52,53,54. These findings suggest that there is more biosynthetic diversity than what is

currently known for bacterial diterpenoids, e.g., _ent_-CPP may not always be transformed into the polycyclic kauranes, atisanes, and pimaranes of currently known bacterial diterpenoids. Add

this to the recent discoveries of a monodomain type II TS from cyanobacteria55 and a bifunctional sesqui-TS that belongs to the haloacid dehalogenase-like hydrolase superfamily56 and the

terpenoid biosynthetic potential of bacteria is likely severely underestimated. In summary, we determined the structure of the TPP synthase Tpn2, a clerodane-forming TS, and mapped out

residues that are essential for catalysis and those that control the selectivity of product formation. We identified a single-residue switch, G485D, that converted a clerodane-specific TS

into a labdane-selective TS. This residue is different from those previously seen in plant TSs that controlled clerodane vs labdane formation, highlighting the sequence-function differences

between eukaryotic and prokaryotic TSs and illustrating the need to continue characterizing known and new bacterial TSs. Notably, TpnG485D is the first _syn-_CPP synthase from bacteria. This

strongly implies that the functional scope of bacterial TSs is much broader than currently known and supports continued discovery of novel TSs and terpenoids in bacteria. METHODS GENERAL

EXPERIMENTAL PROCEDURES 1H and 13C NMR experiments were performed in D2O or CDCl3 at 600 MHz for 1H and 151 MHz for 13C nuclei on a Bruker Avance III Ultrashield 600. 2D NOE experiments were

performed on a Bruker Avance-III-800 console equipped with a Bruker 18.8 T/54 mm Ascend Magnet at 25 °C. Preparative HPLC was conducted using an Agilent 1260 Infinity II LC equipped with an

Agilent Eclipse XDB-C18 column (250 mm × 21.2 mm, 7 μm). Analytical HPLC was performed on an Agilent 1260 Infinity II system equipped with an Agilent 5 HC-C18(2) column (150 × 4.6 mm, 5 

μm). GC-MS experiments were run on a ThermoScientific Trace GC Ultra spectrometer equipped with an Rxi-5MS column (Restek Corp., 30 m × 0.25 mm i.d., 0.25 μm df). The injection temperature

was 250 °C, electron ionization was performed with 70 eV, ion source temperature was 250 °C, transfer line temperature was 280 °C, and mass scan range was from m/z 30–500 at 1500 μ s−1. The

program held at 50 °C for 3 min, increased the temperature at a rate of 20 °C min−1 up to 300 °C, and then was maintained at 300 °C for 3 min. Optical rotations were measured on a JASCO

P-1010 polarimeter. GENE CLONING Strains and plasmids used in this study are listed in Supplementary Tables 1 and 2, respectively. The _tpn2_ gene from _Kitasatospora_ sp. CB02891 (UniProt

accession A0A2M9LDX2, cloned starting at Met9) was amplified by PCR from genomic DNA using Q5 DNA polymerase (NEB) with primers pRSFtpn2_F and pRSFtpn2_R (Supplementary Table 3) following

the manufacturer’s protocols. The PCR product was purified and treated with T4 polymerase for cloning into pBS3080 (ref. 57) following ligation independent procedures58 to create pJR2001.

For site-directed mutagenesis, the _tpn2_ gene was amplified in two steps by primer extension59 using primers pRSFtpn2_F and pRSFtpn2_R, with internal primers containing the desired

mutation(s) as listed in Supplementary Table 3. The mutant genes were then cloned into pBS3080 as described previously to yield pJR2002–pJR2012. The _tpn2_ G485E mutant was amplified by

primer extension using primers Tpn2-3_F and Tpn2_HindIII_R and internal primers containing the mutation. The gene was then cloned into pCDF-Duet at the _Asc_I and _Hind_III sites using T4

DNA Ligase to afford pJR2019. For in vivo production, _tpn2_ (Tpn2 = A0A2M9LDX2), _tpn3_ (Tpn3 = A0A2M9LE16), and _tpn6_ (Tpn6 = A0A2M9LDW8) were amplified from _Kitasatospora_ sp. CB02891

genomic DNA using Q5 DNA polymerase and primers Tpn2-3_F and Tpn1-3_R for _tpn2_ and _tpn3_ and primers Tpn6_F and Tpn6_R for _tpn6_. _tpn2/tpn3_ and _tpn6_ were inserted into separate

multiple cloning sites in pET-Duet via restriction enzyme digestion using _Asc_I and _Hind_III for the former and _Nde_I and _Pac_I for the latter, followed by ligation via T4 DNA Ligase.

For the addition of a second ribosome-binding site and fusion protein construction, _tpn2_ was amplified from pJR2001 using Q5 DNA polymerase and primers NcoI_ala_Tpn2_F and either

Tpn2_RBSOP_R or Tpn2_GSlink_R, respectively. The full length _tpn3_ gene was similarly amplified using either primer RBSOP_Tpn3_F or GSlink_Tpn3_F and Tpn3_Hind_R for addition of a second

ribosome binding site or flexible linker upstream of Tpn3. Each construct was amplified by primer extension with primers NcoI_ala_Tpn2_F and Tpn3_Hind_R and inserted into pCDF-Duet

linearized with _Nco_I using T5 exonuclease-dependent assembly (TEDA)60, affording pJR2015. For antibiotic selection compatibility, wild-type (WT) _tpn2_ and the G485D mutant were subcloned

by digestion of pJR2001 and pJR2011 with _Bam_HI and _Pac_I and ligated into pCDF-Duet to form pJR2014 and pJR2015, respectively. For construction of the _tpn2_ G485D and G485E mutant fusion

proteins, mutant _tpn2_ was amplified from pJR2011 and pJR2019, respectively, using primers Tpn2-3_F and Tpn2_GSlink_R. The full length _tpn3_ gene was amplified from pJR2015 using primers

GSlink_Tpn3_F and Tpn1-3_R. The _tpn2_ mutants and _tpn3_ were amplified together by primer extension using primers Tpn2-3_F and Tpn1-3_R and similarly cloned into the _Asc_I and _Hind_III

sites affording pJR2018 and pJR2020. GENE EXPRESSION AND PROTEIN PRODUCTION For in vitro activity assays, pJR2001 was transformed into _E. coli_ NiCo cells (NEB) and grown in 500 mL of

lysogeny broth (LB) at 37 °C with shaking at 200 rpm until an OD600 of 0.6 was reached. The culture was cooled on ice before gene expression was induced by addition of 0.5 mM isopropyl

β-d-1-thiogalactopyranoside (IPTG). Flasks were grown overnight at 16 °C before the cells were harvested by centrifugation at 5000 × _g_ for 20 min at 4 °C. The cell pellet was prepared for

purification by resuspending in lysis buffer (50 mM Tris, 300 mM NaCl, and 10 mM imidazole, pH 8.0) and 1 mg mL−1 lysozyme before incubating on ice for 30 min. Cells were then sonicated and

centrifuged at 10,000 × _g_ for 30 min to separate soluble proteins before filtering with a 0.8 µM filter. The lysate was purified by nickel affinity chromatography and size exclusion

chromatography using an AKTA FPLC with a 5 mL HisTrap column and Superdex HiScale 16/40 80 mL column (GE Healthcare), respectively. Purified Tpn2 was concentrated using an Amicon Ultra-15

concentrator (Millipore) in SEC buffer (50 mM Tris, pH 8.0) before storing aliquots at −80 °C. Protein concentration was calculated at 280 nm using a molar absorptivity constant of 98,320 

M−1 cm−1. Each of the Tpn2 site-directed mutants was produced and purified as described above using nickel affinity chromatography. CRYSTALLIZATION, DATA COLLECTION, AND STRUCTURE

DETERMINATION OF TPN2 Purified Tpn2 was concentrated to 30.0 mg mL−1 in 20 mM Tris buffer (pH 8.0) with 100 mM NaCl and crystallized using the hanging drop vapor-diffusion method in a screen

condition: 10 mM zinc sulfate heptahydrate, 25% v/v polyethylene glycol monomethyl ether 550, and 100 mM MES monohydrate, pH 6.5. The Tpn2 crystals were observed after two weeks. The

crystals were transferred to cryoprotectant solution containing 20% glycerol prior to the X-ray data collection. The diffraction data of Tpn2 were collected at National Synchrotron Radiation

Research Center (Taiwan) on the 15A1 beamline using a wavelength of 0.9732 Å with the ADSC QUANTUM 315r CCD detector. Diffraction data were indexed and scaled with HKL200061. The structure

of Tpn2 was solved by the molecular replacement using the structure of PtmT2 (PDB entry 5BP8) as a search model16. The model was refined with REFMAC62. The atomic coordinates and structure

factors of Tpn2 were deposited in the Protein Data Bank (PDB) with the accession code 7XKX (Supplementary Data 1). Data processing and refinement statistics are summarized in Supplementary

Table 6. ENZYMATIC ACTIVITY OF TPN2 GGPP was synthesized as previously described in the literature63,64. In vitro reactions were performed in 50 mM Tris pH 6.8, containing 1 mM MgCl2, 5 mM

β-mercaptoethanol (BME), 10% glycerol, 4 mM GGPP, and 20 µM Tpn2 in a total volume of 50 µL. Reactions were incubated for 16 h at 30 °C before undergoing dephosphorylation of reaction

products via addition of Quick-CIP (NEB) and incubation following the manufacturer’s protocols. The reaction mixture was then gently extracted with 100 µL acetonitrile and 50 µL saturated

NaCl before centrifugation for 1 min. The organic layer was then removed and injected on an HPLC system using an isocratic method of 95% acetonitrile in H2O. The substrate and product were

detected at 210 nm as the dephosphorylated analogs 1′ and 2′ and eluted at 5.4 min and 6.0 min, respectively. TPP (2) was isolated by scaling up in vitro reactions to yield approximately 0.5

 mg of the product. Reactions were incubated for 16 h at 30 °C before extraction with equal volume of acetonitrile and placing on ice for 1 h. TPP (2) was purified via preparative HPLC using

a solvent gradient of 5–50% acetonitrile in NH4HCO3. Fractions containing 2 were combined and dried down via rotary evaporator (Supplementary Figs. 3, 4). IN VIVO PRODUCT ISOLATION AND

DETERMINATION For in vivo product isolation, pJR2014–pJR2018 were individually co-transformed along with pJR1064 into _E. coli_ BL21 Star (DE3) (Invitrogen) and grown in 9–12 L terrific

broth (TB) at 37 °C with shaking at 200 rpm until an OD600 of 1.0 was reached. The cultures were cooled on ice before gene expression was induced by the addition of 0.5 mM IPTG and 5 mM

isoprenol was supplemented in the culture. Flasks were shaken for 72 h at 28 °C before harvesting the cells by centrifugation. For the isolation 2′, 3, 5, 6, and 7, the cell pellets were

extracted with 1:1 methanol: acetone and the organic layer dried under air at room temperature. The extract was then redissolved in ethyl acetate and purified via silica chromatography with

a hexanes mobile phase before additional purification via preparative HPLC as previously described. Fractions containing the desired products were combined and dried down on a rotary

evaporator at 70 mbar at 20 °C. Compounds 3, 5, 6, and 7 were confirmed via NMR. The 1H and 13C spectra of 3 matched literature values (Supplementary Figs. 11, 12)28. The structure of

_syn_-sclarene (5) was confirmed via additional NMR experiments, including 1H and 13C, which matched literature values36, 1H-1H COSY, HSQC, and HMBC (Supplementary Figs. 13–16, Supplementary

Table 4). The absolute stereochemistry of 5 was confirmed by 2D NOESY and optical rotation experiments (Fig. 3 and Supplementary Fig. 17), the latter of which gave an \({[\alpha

]}_{{{{{{\rm{D}}}}}}}^{21}\)  = +2.269 (c 0.005, CHCl3). Compound 2′ was confirmed via purification and comparison to in vitro reaction data via analysis by HPLC. Yields were calculated by

making a calibration curve using purified 3 and 5 and injecting on HPLC. Area under the curve and injection concentration was used to plot a standard curve. Tpn2-Tpn3 and Tpn2G485D-Tpn3

fusion proteins were fermented and extracted as previously described. Crude extracts were injected on HPLC and the area under the curve at λ = 210 nm was used to calculate yield.

COMPUTATIONAL MODELING The structure of the ligand, GGPP, was optimized for docking using the default parameters of MM2 energy minimization in ChemDraw 3D. This was saved in SDF format, then

converted to pdbqt in OpenBabel65. The receptor was prepared for docking in AutoDockTools 1.5.6 (ref. 66); hydrogen atoms were added and merged and Gasteiger charges calculated before

generating the final pdbqt file. The ligand and receptor were docked using AutoDock Vina67. Docking results were generated for a grid box of 30 × 25 × 20, default grid spacing of 0.375 Å,

and exhaustiveness = 20, using the coordinates of the central Asp side chain oxygen atom as the grid box center to ensure the entire active site was encompassed within the grid box.

BIOINFORMATICS The sequence alignment of plant and bacterial type II TSs obtained by aligning sequences from UniProt using ClustalW68 and visualized in ESPript 3.0 (ref. 69). Consensus

sequence logos of motifs were depicted with partial aligned sequences using WebLogo 3 (ref. 70). The collection of bacterial type II TSs for sequence similar network (SSN) analysis was

achieved using the Enzyme Function Initiative (EFI) Enzyme Similarity Tool (EST)48 with Tpn2 as the query sequence, taxonomy filter of “bacteria”, and an e-value of 5. The SSNs and genome

neighborhood networks (GNNs), created using the SSN Cluster Hub-Nodes function, were generated using EFI-EST and visualized in Cytoscape 3.9.1 (ref. 71). Taxonomic distribution of each

protein family was determined using the taxonomy function on EFI-EST. REPORTING SUMMARY Further information on research design is available in the Nature Research Reporting Summary linked to

this article. DATA AVAILABILITY All data generated during this study are available either in the main text, supplementary materials, or are deposited in online repositories. The atomic

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Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This work funded in part by NIH Grants R00 GM124461 and R35 GM142574 (JDR) and MOST Grant

110-2113-M-A49-026-MY3 (CYC). We wish to thank James Rocca and Ion Ghiviriga for excellent NMR support. We acknowledge the University of Florida’s Center for Nuclear Magnetic Resonance

Spectroscopy and the McKnight Brain Institute at the National High Magnetic Field Laboratory’s Advanced Magnetic Resonance Imaging and Spectroscopy (AMRIS) Facility, which is supported by

the US NSF Cooperative Agreement No. DMR-1644779 and the State of Florida. Some NMR spectra were acquired using a unique 1.5 mm High Temperature Superconducting Cryogenic Probe developed

with support from the NIH (R01 EB009772). We also thank Jodie Johnson and the University of Florida’s Mass Spectrometry Research and Education Center (MSREC), which is supported by the NIH

S10 OD021758-01A1, for GC-MS support. We thank the experimental facility and technical services provided by the National Synchrotron Radiation Research Center (NSRRC) in Taiwan. We thank Ben

Shen for bacterial strains. Finally, we acknowledge Alisha Das, Adriana LaVopa, and Santiago Velez for their assistance in cloning TpnG485E as part of the University of Florida’s

Course-Based Undergraduate Research Experience (CURE). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Chemistry, University of Florida, Gainesville, FL, 32611, USA Emma A.

Stowell, Michelle A. Ehrenberger & Jeffrey D. Rudolf * Department of Biological Science and Technology, National Yang Ming Chiao Tung University, Hsinchu, 30010, Taiwan, ROC Ya-Lin Lin 

& Chin-Yuan Chang * Center for Intelligent Drug Systems and Smart Bio-devices, National Yang Ming Chiao Tung University, Hsinchu, 30010, Taiwan, ROC Chin-Yuan Chang Authors * Emma A.

Stowell View author publications You can also search for this author inPubMed Google Scholar * Michelle A. Ehrenberger View author publications You can also search for this author inPubMed 

Google Scholar * Ya-Lin Lin View author publications You can also search for this author inPubMed Google Scholar * Chin-Yuan Chang View author publications You can also search for this

author inPubMed Google Scholar * Jeffrey D. Rudolf View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS J.D.R. conceived the project; E.A.S.,

M.A.E., C.-Y.C., and J.D.R. designed the experiments; E.A.S., M.A.E., Y.-L.L, C.-Y.C., and J.D.R. performed the experiments; E.A.S., M.A.E., Y.-L.L, C.-Y.C., and J.D.R. analyzed the results;

and E.A.S., C.-Y.C., and J.D.R. wrote the manuscript with inputs from all co-authors. CORRESPONDING AUTHORS Correspondence to Chin-Yuan Chang or Jeffrey D. Rudolf. ETHICS DECLARATIONS

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CITE THIS ARTICLE Stowell, E.A., Ehrenberger, M.A., Lin, YL. _et al._ Structure-guided product determination of the bacterial type II diterpene synthase Tpn2. _Commun Chem_ 5, 146 (2022).

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