Structural and functional insight into an unexpectedly selective N-methyltransferase involved in plantazolicin biosynthesis

Enzymatic Conversion of DesmethylPZN to PZN.

To assess enzymatic activity, we cloned, expressed, and purified BamL as a fusion with maltose-binding protein (MBP). MBP-BamL was then added to reactions containing desmethylPZN, SAM, and a nucleosidase that converts the SAH by-product to adenine and S-ribosyl-l-homocysteine, minimizing potential product inhibition (Pfs) (15). Reactions were initiated by the addition of SAM, quenched at desired time points, and analyzed by MALDI-MS (Fig. 2). In the presence of desmethylPZN and SAM, MBP-BamL catalyzed the formation of PZN (m/z 1336). Omitting either SAM or BamL resulted in no product formation. Identical results were achieved with the BpumL methyltransferase (from B. pumilus), another bona fide producer of PZN (3) (Fig. 2). Removal of the MBP tag from BamL led to a lower level of PZN formation, suggesting that MBP does not interfere with methyltransferase activity (SI Appendix, Table S1). The higher level of PZN formation with MBP was likely due to increased stability conferred by the tag (16). Using the MALDI-MS end point assay, we further evaluated the activity of MBP-BamL by testing the effect of pH, buffer, and a variety of other additives (SI Appendix, Table S1). Omission of Pfs from the reaction led to only a modest decrease (∼25%) in product formation. Using isothermal titration calorimetry (ITC), we obtained a dissociation constant between SAM and BamL of 7.5 ± 0.8 µM (SI Appendix, Fig. S1). In accord with lack of SAH by-product inhibition, identical ITC runs with SAH generated unusual titration curves that could not be mathematically fit to any reasonable binding model. To confirm the site of BamL dimethylation, collision-induced dissociation (CID) spectra were obtained using Fourier transform MS. A previously characterized diagnostic fragment ion, m/z 1277.4291 Da, was observed, indicating that the site of methylation was on the expected N-terminal α amine (SI Appendix, Fig. S2) (3).

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Fig. 2.

Activity of purified methyltransferases. MALDI-MS was used to monitor the conversion of desmethylPZN (m/z 1308) to PZN (m/z 1336). The m/z 1326 and 1354 species represent hydrolyzed desmethylPZN and PZN, respectively. Samples containing all reaction components used either the methyltransferase from BamL or BpumL. The SAM cosubstrate and BamL enzyme were omitted from the samples labeled −SAM and −BamL, respectively. PznL, PZN methyltransferase (either BamL or BpumL); rxn, reaction.

Despite the variety of in vitro reconstitution reactions screened (SI Appendix, Table S1), monomethylPZN was never detected (m/z 1322). Even at shorter time points, when consumption of desmethylPZN was incomplete, monomethylPZN was not observed. In an additional effort to detect this species, we performed BamL reactions under pseudosingle turnover conditions using variable concentrations of BamL, desmethylPZN, and SAM (SI Appendix, Fig. S3). Under no condition was monomethylPZN (m/z 1322) detected. However, when all reaction components were supplied at 100 µM, we observed a minor peak consistent with hydrolyzed (+18 Da) monomethylPZN (m/z 1340). Unfortunately, the low signal-to-noise ratio did not allow for structural confirmation.

DesmethylPZN Substrate Analogs.

Because of the hydrolytic instability of PZN, we observed by MALDI-MS hydrolyzed desmethylPZN (m/z 1326) and hydrolyzed PZN (m/z 1354) in all in vitro reactions (Fig. 2 and SI Appendix, Fig. S3). These species arise from the hydrolysis of the sole methyloxazoline ring. We have previously reported on the lability of this heterocycle, which reinstates the most C-terminal Thr (Fig. 1) (3). To establish whether BamL could directly accept hydrolyzed desmethylPZN (m/z 1326) as a substrate to produce hydrolyzed PZN (m/z 1354), we subjected desmethylPZN to conditions that yielded quantitative conversion to hydrolyzed desmethylPZN for BamL substrate testing (SI Appendix, Materials and Methods). Analysis of reaction products run under identical conditions showed that BamL processed hydrolyzed desmethylPZN with efficiency approximately equal to that of desmethylPZN (Figs. 2 and 3 A and B). From earlier work, we noted that another desmethylPZN variant harboring two methyloxazolines (dihydrodesmethylPZN, m/z 1310) was readily accessible by using oxygen-saturated cultivation (3). Because of separation difficulties, we tested a mixture of m/z 1308, 1310, 1326, and 1328 (desmethylPZN analog mixture) for processing by BamL. Analysis of reaction products by MALDI-MS showed the presence of PZN (m/z 1336), dihydroPZN (m/z 1338, note isotopic ratio), hydrolyzed PZN (m/z 1354), and hydrolyzed dihydroPZN (m/z 1356, Fig. 3C). These data indicate BamL successfully processed dihydrodesmethylPZN.

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Fig. 3.

Substrate tolerance of BamL. MALDI-MS was used to assess the ability of BamL to process substrates other than desmethylPZN. (A) Subset of peptides tested as substrates with the N-terminal Arg shown in bold. Heterocyclized residues of desmethylPZN are underlined. The most C-terminal heterocycle is reverted to Thr in hydrolyzed desmethylPZN. (B–F) Bottom spectra, unreacted peptides; top spectra, after reaction with BamL. (B) Hydrolyzed desmethylPZN (m/z 1326). (C) DesmethylPZN analog mixture that contained desmethylPZN (m/z 1308), dihydrodesmethylPZN (m/z 1310), and the hydrolyzed forms (m/z 1326 and 1328, respectively). (D) Bradykinin (m/z 1061). (E) “RPG” peptide (m/z 1358). (F) “RAT” peptide (m/z 733).

Unusual Substrate Selectivity of BamL.

To further explore substrate permissiveness, we tested bradykinin and a synthetic peptide termed “RPG” (sequence given in Fig. 3) as BamL substrates. To a first approximation, bradykinin and the RPG peptide mimic desmethylPZN. Both harbor an N-terminal Arg, are similar in size to desmethylPZN, and contain Pro where several azole heterocycles are found in desmethylPZN (Fig. 3A). As with Pro, azoles are five-membered, nitrogen-containing heterocycles that serve as peptide conformational restraints. Although not a perfect match for an azole, the ability of Pro to structurally and functionally substitute in TOMM natural products has been documented (17). Upon treatment of bradykinin and the RPG peptide with BamL and SAM, no reaction products were detected, even after increasing the enzyme concentration and allowing for extended reaction times (Fig. 3 D and E). Similarly, adrenocorticotropic hormone peptide 18–39, with an N-terminal amino acid sequence of RPV, was not processed by BamL. Because Pro is an imperfect match for an azole, we also tested whether BamL would process an additional synthetic peptide “RAT” (sequence given in Fig. 3), which contained the left segment of the native PZN precursor sequence except that the Cys residues were replaced with Ala. Again, no reaction product was observed, even after increasing the enzyme concentration to near single turnover levels (Fig. 3F). Two tetrapeptides, RAAA and RGGG, were also evaluated as BamL substrates, but no reaction products were detected. As a final test, BamL accepted Arg amide (carboxylate replaced with a neutral amide) as a substrate, albeit a very inefficient one. After increased reaction time with elevated substrate concentration, high-resolution MS identified a mass consistent with dimethylArg amide (experimental 202.1677 Da, theoretical 202.1688 Da, error 4.5 ppm). The very low intensity of this peak suggested that <5% of the starting material was converted. These results suggested that BamL does not simply recognize peptides with an N-terminal Arg and furthermore is intolerant to substrates not containing desmethylPZN-like (i.e., polyheterocyclic) functionality. In contrast to the specificity exhibited by BamL, other characterized SAM-dependent methyltransferases acting on small molecules and peptides have much broader substrate scopes. For example, the methyltransferases involved in rebeccamycin (18, 19), dehydrophos (20), aminocoumarin (21), and CypM (14) biosynthesis methylate a wide range of substrates.

Structure Determination of Two PZN Methyltransferases.

To provide insight into the unexpected lack of activity toward nonheterocyclized peptide substrates, we determined the crystal structures of BamL (1.75 Å resolution) and BpumL (2.0 Å resolution), each in complex with SAH. Initial crystallographic phases were determined at 3.2 Å resolution using SeMet-labeled crystals of BpumL, followed by model building and refinement against higher resolution data until convergence. Phases for BamL were determined by molecular replacement using a partial model of BpumL. Relevant data collection and refinement statistics are given in SI Appendix, Table S2. The BamL and BpumL structures consist of a core Rossman-fold domain composed of seven β-strands surrounded by six α-helices, with an overall architecture similar to other methyltransferases (Fig. 4 A and B). As expected from the 48% sequence identity between BamL and BpumL, the structures are superimposable with an rmsd of 1.1 Å over 256 aligned α carbons. A DALI search against the Protein Data Bank (PDB) identifies the closest structural homologs as the bacterial Hen1 methyltransferase (PDB ID code, 3JWG; Z-score, 16.8; rmsd, 2.5 Å over 174 aligned α carbons) and the hypothetical bacterial YecO protein (PDB ID code, 1IM8; Z-score, 16.7; rmsd, 2.9 Å over 194 aligned α carbons). The conservation in structure is restricted to the core Rossman fold domain and persists despite minimal similarities (roughly 10–13% identity) across the primary structure.

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Fig. 4.

Ribbon diagrams derived from the X-ray crystal structures of (A) BamL and (B) BpumL, with the bound SAH ligand shown in yellow ball-and-stick representation. (C) Electron density maps calculated using Fourier coefficients (F_obs – F_calc) with phases derived from the refined 1.75 Å resolution structure of BamL calculated with the coordinates for SAH omitted before one round of crystallographic refinement. The map is contoured at 2.6σ (blue mesh) and 8σ (red mesh) and the final refined coordinates are superimposed. The coordinates for SAH and polar active site residues that are important in catalysis are shown as yellow (ball-and-stick).

Unlike the structures of typical small molecule methyltransferases, the architectures of BamL and BpumL are minimalistic and lack additional secondary structure decorations. One molecule of SAH is bound across the interior of BamL with the adenine ring enclosed in a cavity defined along the sides by S92, F137, and a loop encompassing residues S112–A114 across the top. The side chain of D91 is within interaction distance with the 2′ hydroxyl of the ribose (Fig. 4C). BamL residues R42 and H131 provide additional contacts with SAH via the homocysteine carboxylate. Notably, the trajectory of the homocysteine moiety of SAH is not collinear with the adenine but is deflected toward the center of the polypeptide by F21 and a loop encompassing G68–Q71. This would position the electrophilic methyl group of SAM at the base of a long tunnel running to the surface of the protein. The walls of this tunnel are defined by a number of hydrophobic residues including F21, Y33, T38, L132, L162, Y182, and L183. These residues are strictly conserved between BamL and BpumL, with the exception of L183, which is I181 in BpumL (SI Appendix, Fig. S4). Because the depth of this tunnel is of sufficient length to accommodate the first four to five residues of desmethylPZN, we presume that it defines the substrate-binding pocket (SI Appendix, Fig. S5). At the base of the tunnel, a shorter, perpendicular cavity is found. This shorter cavity is immediately adjacent to the presumptive position of the electrophilic methyl group of SAM and is of suitable size to accommodate the N-terminal Arg side chain of (desmethyl)PZN. Residues D34, Y182, and Q186 of BamL presumably stabilize the N-terminal amine of the substrate (Fig. 4C). Evidence for the role of these residues in substrate engagement is borne out by the mutational analyses described in the following section.

Selection and Enzymatic Activity of BamL Mutants.

To better understand catalysis, we aligned the amino acid sequence of BamL with a series of well-characterized methyltransferases. With the exception of identifying residues that comprise the SAM-binding pocket, this exercise shed little light on what residues may be important for BamL catalysis because of the poor conservation across the methyltransferase superfamily (22). Fortunately, sequence alignment of the five known PZN methyltransferases (including BpumL) identified in our earlier work (3) revealed a manageable number of conserved residues with which to begin the enzymological dissection of BamL (SI Appendix, Fig. S4). We thus targeted eight residues of BamL to be replaced with Ala (SI Appendix, Table S3). We then analyzed the structures of BamL and BpumL cocrystalized with SAH and mutated seven additional positions hypothesized to be critical either for substrate binding (desmethylPZN and SAM) or catalysis. At four of these locations, the wild-type (WT) residue was replaced with Ala and an additional alternative residue, ultimately yielding a database of 19 mutant proteins (Table 1).

To establish if any point mutant reduced enzymatic activity or structural instability, we first expressed and purified all 19 mutant BamL proteins from Escherichia coli under identical conditions (SI Appendix, Materials and Methods). BamL W20A, F21A, D91A, and D126A underwent extensive proteolytic degradation and were low yielding, indicating that these positions play an important role in structural stability (SI Appendix, Fig. S6). In an effort to obtain values for the kinetic constants k_cat and K_m for the mutant panel, we attempted quantitative liquid chromatography MS, coupled-enzymatic assays, and tritiated SAM assays. Despite many attempts, a number of problems arose that limited our ability to determine the desired kinetic parameters. As an alternative, we used internally calibrated MALDI-MS to compare the ion intensities of the desmethylPZN starting material with the PZN product under three reaction conditions (Table 1). In what is referred to as reaction condition A [10 µM MBP-BamL, 10 µM Pfs (SAH nucleosidase), 50 µM desmethylPZN, and 3 mM SAM for 16 h at 37 °C], all tested mutants except for BamL T38A, T38F, L132A, L132F, Y182F, and S190A showed impaired activity. In a more stringent test of activity, the concentrations of MBP-BamL, Pfs, and desmethylPZN were all lowered 10-fold, and the reaction time was limited to 1 h at 22 °C (referred to as reaction condition B). Under condition B, we only observed product formation from BamL T38A and S190A. Importantly, the SAM concentration was intentionally kept high (3 mM) in reaction condition B. To evaluate if a BamL point mutant affected the ability to properly handle SAM, we examined a subset of the mutants in reaction condition C. Here, the desmethylPZN concentration was returned to 50 µM and the SAM concentration was reduced to 500 µM (other variables were the same as in condition B). Upon comparison with condition B, detectable product formation was resurrected from reactions with BamL T38F, L132A, L162A, and Y182F (Table 1). This tripartite assay allowed for the generalized assessment of which residues were required for proper handling of the desmethylPZN and SAM cosubstrates.

Detection of Monoalkylated PZN Analogs.

Although our earlier attempts to identify monomethylPZN as a BamL reaction intermediate were unsuccessful, we observed a strong indication that this species was formed upon analysis of the BamL Y182F reaction using condition A (SI Appendix, Fig. S7). After supplying WT BamL to the Y182F reaction mixture containing m/z 1308, 1322, and 1336, all species converged to m/z 1336 (PZN). Convergence provided definitive evidence that m/z 1322 was indeed monomethylPZN, an on-pathway reaction intermediate as expected.

A close inspection of the SAM-binding pocket within BamL/BpumL suggested that slightly larger alkyl substituents might be tolerated (Fig. 4 and SI Appendix, Fig. S5). To evaluate if an ethyl group could be transferred from a SAM analog bearing an electrophilic ethyl group, rather than the naturally occurring methyl substituent, we synthesized “ethyl SAM” using an established procedure (23). Enzymatic reactions were carried out as performed previously. Analysis of the reaction product by MALDI-MS revealed the transfer of a single ethyl group to desmethylPZN, yielding monoethylPZN (SI Appendix, Fig. S7). This variant is isobaric with native PZN (m/z 1336), which bears two methyl groups. Subjecting this species to CID gave a diagnostic fragment ion of m/z 1277, which localized the ethyl modification to the N terminus. Intriguingly, despite increased reaction times, addition of fresh enzyme, and the use of either ethyl SAM or natural SAM, the corresponding dialkylated PZN derivative was not observed. This suggests that there are insurmountable steric barriers to the formation of monomethyl-monoethylPZN (m/z 1350) and diethylPZN (m/z 1364).