Abstract
Isoprenyl chains are found in many important metabolites. These are derived from precursors of the appropriate length produced by isoprenyl diphosphate synthases (IDSs). The human pathogen Mycobacterium tuberculosis makes various isoprenoids/terpenoids, with important roles in their biosynthesis played by two closely related IDSs, encoded by grcC1 (Rv0562) and grcC2 (Rv0989c), with Rv0989c generating the 10-carbon precursor (E)-geranyl diphosphate (GPP), and Rv0562 the 20-carbon precursor (E,E,E)-geranylgeranyl diphosphate (GGPP). Intriguingly, while Rv0562 contains the prototypical trans-IDS first and second aspartate-rich (DDxxD) motifs (FARM and SARM, respectively), Rv0989c uniquely contains arginine in place of the second Asp in the FARM and first Asp in the SARM. Here site-directed mutagenesis of the corresponding residues in both Rv0562 and Rv0989c reveals that these play a role in determination of product chain length. Specifically, substitution of Asp for the Arg in the FARM and SARM of Rv0989c leads to increased production of the longer 15-carbon farnesyl diphosphate (FPP), while substitution of Arg for the corresponding Asp in Rv0562 leads to increased release of shorter products, both FPP and GPP. Accordingly, while the primary role of the FARM and SARM is known to be chelation of the divalent magnesium ion co-factors that assist substrate binding and catalysis, the Arg substitutions found in Rv0989c seem to provide a novel means by which product chain length is moderated, at least in these M. tuberculosis IDSs.
Highlights
Isoprenoids play essential roles in many metabolic pathways
A common determinant of product chain length in trans-isoprenyl diphosphate synthases (IDSs) is the identity of the amino acid residue in the fifth position before the FARM [22]
With smaller residues typically found in geranylgeranyl diphosphate (GGPP) synthases and larger residues in those trans-IDS with shorter products
Summary
Isoprenoids play essential roles in many metabolic pathways. In large part, due to the hydrophobic nature of polyprenyl chains, these are used to anchor proteins to the membrane or, after cyclization, form the sterols or hopanoids that are integral membrane molecules. In production of the polyprenyl phosphate serving as a glyco-carrier required for cell wall biosynthesis [6] This 10 isoprenyl-unit carrier is produced via an unusual (Z,E)-farnesyl diphosphate (FPP) intermediate, which serves as the initial allylic substrate for the long-chain cis-IDS Rv2361c [12]. The 15-carbon (Z,E)-FPP is produced by the short-chain cis-IDS Rv1086, which uses (E)-geranyl diphosphate (GPP) as its allylic substrate [13]. This unusual bacterial production of the 10-carbon GPP is mediated by the trans-IDS Rv0989c (grcC2) [14]. This is presumably due to the presence of an additional trans-IDS that produces the 20-carbon GGPP in M. tuberculosis, Rv0562 (grcC1) [21]
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