Structure of Isoprene Synthase Illuminates the Chemical Mechanism of Teragram Atmospheric Carbon Emission

Abstract

The X-ray crystal structure of recombinant isoprene synthase from grey poplar leaves (Populus x canescens) (PcISPS) has been determined at 2.7 Å resolution, and the structure of its complex with three Mg 2+ ions and the unreactive substrate analogue dimethylallyl-S-thiolodiphosphate has been determined at 2.8 Å resolution. Analysis of these structures suggests that the generation of isoprene from substrate dimethylallyl diphosphate occurs via a syn-periplanar elimination mechanism in which the diphosphate leaving group serves as a general base. This chemical mechanism is responsible for the annual atmospheric emission of 100 billion kilograms of isoprene by terrestrial plant life. Importantly, the PcISPS structure promises to guide future protein engineering studies potentially leading to hydrocarbon fuels and products that do not rely on traditional petrochemical sources.

Bisphosphonate Inhibitors Reveal a Large Elasticity of Plastidic Isoprenoid Synthesis Pathway in Isoprene-Emitting Hybrid Aspen

“…Existence of such a direct inhibition could be expected given that, in the case of inhibition of GDP, FDP, and GGDP synthases, alendronate and zoledronate bind to the three-Mg 2+ cluster in the allylbinding (DMADP-binding) pocket of the enzyme active site (Jahnke et al, 2010;No et al, 2012;Lindert et al, 2013;Liu et al, 2014). The active site of isoprene synthase also coordinates the diphosphate tail of DMADP by three Mg 2+ atoms (Köksal et al, 2010), and is thus analogous to the allyl-binding active site in prenyltransferases. However, the active site for isoprene synthase is shallower and more rigid than the active site in prenyltransferases as indicated by lack of enzyme conformational changes upon DMADP binding by isoprene synthase (Köksal et al, 2010), which is different from prenyltransferases where DMADP and bisphosphonate binding results in enzyme conformational changes Figure 10.…”

“…The active site of isoprene synthase also coordinates the diphosphate tail of DMADP by three Mg 2+ atoms (Köksal et al, 2010), and is thus analogous to the allyl-binding active site in prenyltransferases. However, the active site for isoprene synthase is shallower and more rigid than the active site in prenyltransferases as indicated by lack of enzyme conformational changes upon DMADP binding by isoprene synthase (Köksal et al, 2010), which is different from prenyltransferases where DMADP and bisphosphonate binding results in enzyme conformational changes Figure 10. Illustration of dark-release kinetics of C6 lipoxygenase (LOX) pathway volatiles in control and alendronate-inhibited leaves measured with proton transfer reaction-mass spectrometry (PTR-MS; A), and integrated pools of DMADP converted to isoprene and LOX volatiles during a dark period of 15 min in control and zoledronateinhibited leaves measured by gas chromatography-mass spectrometry (GC-MS; B) in leaves of hybrid aspen.…”

“…A slow rate of inhibition of isoprene synthase does not rule out a strong binding once the inhibitor reaches the active site. Given further that K m for DMADP of isoprene synthase is very high (Köksal et al, 2010;Rasulov et al, 2014), a low value of inhibitory constant would imply that bisphosphonate inhibition could be considered essentially irreversible over a physiological range of DMADP pool sizes, explaining the apparent reduction in isoprene synthase V max values.…”

Identification, Functional Characterization, and Evolution of Terpene Synthases from a Basal Dicot

“…For LnTPS1, 60 models (LnTPS2 96, LnTPS3 50) were created based on different alignments using more than 10 x-ray templates in each case. For LnTPS1, an isoprene synthase (Protein Data Bank code: 3N0F; Köksal et al, 2010) appeared as best suited templates from which the final model was built. For LnTPS2, the x-ray structure of a 5-epi-aristolochene synthase (Starks et al, 1997) was preferentially used, but finally a hybrid model was formed by replacing in this model some small better folded loop regions from models created from 3M02 (Noel et al, 2010), 4GAX, and 4FJQ (Li et al, 2013).…”

A Glimpse into the Biosynthesis of Terpenoids

“…The structure of ISPS reveals a shallower active site cavity compared to other class I terpene synthases, even the monoterpene synthases. This corresponds with its specificity for the smaller substrate DMAPP [28,35].…”