Backbone 1H, 13C, 15N NMR assignments of the unliganded and substrate ternary complex forms of mevalonate diphosphate decarboxylase from Streptococcus pneumoniae

Abstract

Mevalonate diphosphate decarboxylase (MDD) catalyzes the ATP-dependent decarboxylation of diphosphomevalonate (DPM) to produce isopentenyl diphosphate (IPP), the molecular "building block" for more than 25,000 distinct isoprenoids, including cholesterol, steroid hormones and terpenoids. Here, we present the first backbone assignment of Streptococcus pneumoniae MDD in the unliganded state and in a ternary complex with DPM and AMPPCP--a nucleotide analogue unable to transfer the γ-phosphoryl group. The secondary chemical shifts for the unliganded form are in good agreement with the crystal structure of Streptococcus pyogenes (~70% sequence identity). The addition of substrate and nucleotide to the enzyme results in chemical shift changes of cross peaks that correspond to residues in the binding pocket.

Fig. 1

Assigned 2D ¹H,¹⁵N TROSY spectra of [²H,¹³C,¹⁵N] MDD. a,c Unliganded MDD. b,d MDD in complex with DPM and AMPPCP. Sample conditions: 90% H₂O, 10% D₂O, Hepes (25 mM, pH 7.5), 298 K

Fig. 2

MDD Secondary Structure. a Secondary structure predicted from a model of the pneumoniae enzyme derived, by homology modeling, from the crystal structure of the S. pyogenes MDD (Kelley and Sternberg 2009), which is 70% identical to the pneumoniae MDD. α-helices are represented by barrels; β-sheets by arrows. b Difference of chemical-shift deviation from random coil ¹³Cα and ¹³Cβ. Values are averaged over three consecutive residues. Unassigned residues were given a value of zero. c The consensus result from CSI (Wishart and Sykes 1994)

Fig. 3

Average chemical shift change ${\delta }_{av}=\sqrt{(\Delta H^2+1∕5\Delta N^2)∕2}$ of amide cross peaks between unliganded and ternary-complex forms of of MDD (pH 7.5 and 298 K). Zero values correspond to unassigned residues