Cody
Lemke
,
Owen
Whitham
and
Reuben J.
Peters
*
Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, IA 50011, USA. E-mail: rjpeters@iastate.edu
First published on 10th July 2020
The class II diterpene cyclase (DTC) from pleuromutilin biosynthesis uniquely mediates ‘A’ ring contraction of the initially formed decalin bicycle, yielding mutildienyl diphosphate (MPP). Catalysis requires a divalent metal cation co-factor. Intriguingly, selectively with magnesium, this DTC catalyzes ring expansion/contraction between MPP and halimadienyl diphosphate, providing some catalytic insight.
Intriguingly, while after induction these recombinant cultures initially only produce 2 (observed as mutildien-15-ol, 2′, derived from dephosphorylation of 2 catalysed by endogenous phosphatases),4 with sufficient time (>1 day) another DTC product was observed in slowly increasing amounts (Fig. 1 and S1†). Comparison to authentic standards readily identified this as syn-halima-5,13E-dienyl diphosphate (HPP, 3, also observed as the dephosphorylated derivative – i.e., syn-halimadien-15-ol, 3′). Notably, formation of 3 represents deprotonation of the penultimate intermediate, syn-halima-13E-15-PP-5-yl+ (3+), just before the unique ring contraction step, in the reaction leading to 2. However, the delayed appearance of 3 suggests that it might be produced from 2, which would represent ‘A’ ring expansion, essentially reversing the final ring contraction step in the reaction catalysed from 1.
This surprising production of 3 by CpPS:D649L was first investigated by co-expression experiments using the D311A mutant of CpPS that blocks DTC activity.4 Notably, whereas co-expression of CpPS:D311A with CpPS:D649L in E. coli also engineered to produce 1 led to production of premutilin (4) with no detection of 3, analogous co-expression of a DTC that produces 3 (OsCPS4:H501D)6 with CpPS:D311A did not. Instead, as identified by comparison to an authentic standard,7 isotuberculosinol/nosyberkol (5) was observed (Fig. S2†). This is derived from addition of water to the tertiary carbocation formed by lysis of the allylic diphosphate ester bond in 3, consistent with the known class I activity of CpPS (i.e., the hydroxyl group found in premutilin). Thus, 3 is not a precursor to 4 and, accordingly, CpPS:D649L does not seem to produce 3 directly from 1 (i.e., as 3 is not detected upon co-expression with CpPS:D311A). This suggests that the CpPS DTC active site seems be able to produce 3 from its usual product 2, albeit this ring expansion reaction is clearly much less efficient than that catalysed by its class I active site (i.e., to produce 4).
To further investigate the hypothesis that the CpPS DTC active site is capable of catalysing this intriguing partial reverse (ring expansion) reaction in vitro assays were carried out. Consistent with the production of 3 from 2, accumulation of 3 was delayed relative to 2 (Fig. S3†). Notably, in the course of optimizing conditions for the in vitro assays it was found that, while altering pH and buffer or salt did not significantly alter production of 3 (Fig. S4†) increasing the concentration of the magnesium (Mg2+) co-factor led to higher rates of production (Fig. S5†). By contrast, assays in the presence of a variety of alternative divalent metal ions (0.1 mM Ca2+, Co2+ or Ni2+) all produced only 2, with no 3 observed (Fig. S6†). Moreover, further transformation of 2 into 3 required active CpPS as well as Mg2+ (Fig. S7†). These results indicate that CpPS specifically can use Mg2+ to catalyse ring expansion of 2 to produce 3, albeit at a much slower rate than the production of 2 from 1. Nevertheless, this finding enabled separate examination of the production of 2versus both this and 3.
The production of 3 from 2 requires an additional protonation step beyond that necessitated by the formation of 2 from 1 (see Scheme 2). This mechanistic implication was investigated by deuterium labelling studies. Specifically, assays carried out in 2H2O (D2O) versus H2O. As expected, when CpPS was limited to the production of just 2 (by use of Ni2+), in D2O the resulting 2 contained a single deuterium (Fig. 2A). Intriguingly, in the presence of Mg2+ the resulting 2 and 3 were both multiply labelled as evidenced by increases in the m/z of the observed fragments (Fig. 2B and C). Accordingly, not only does this provide strong support for the production of 3 from 2 rather than directly from 1, it further indicates that, selectively in the presence of Mg2+, the CpPS DTC active site can catalyse interconversion of 2 and 3. Note that the observed essentially equivalent labelling is consistent with the previously demonstrated (methyl) specific deprotonation that yields 2,8,9 as well as expected stereospecific protonation/deprotonation at C6 of the endo olefin in 3 (Scheme S1†).
Scheme 2 CpPS DTC catalysed reactions from 1 to 2, and (inefficient) ring expansion/contraction interconverting 2 and 3. |
Footnote |
† Electronic supplementary information (ESI) available: Experimental methods and supplemental figures. See DOI: 10.1039/d0ob01422b |
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