## 2 Results  
As indicated by their shared genetic nomenclature (i.e., \(grc\)C1 and \(grc\)C2), Rv0562 and Rv0989c are closely related (e.g., particularly among the _M. tuberculosis_ IDSs; Figure S1), sharing >56% amino acid sequence identity, despite their divergent product lengths. A common determinant of product chain length in _trans_-IDSs is the identity of the amino acid residue in the fifth position before the FARM [22]. In particular, with smaller residues typically found in GGPP synthases and larger (aromatic) residues in those _trans_-IDS with shorter products. However, Rv0562 and Rv0989 both have small residues at this position (i.e., alanine and glycine, respectively; Figure 2). Thus, there must be an alternative determinant for the difference in product chain length between these two _trans_-IDSs. Notably, while Rv0562 contains the canonical DDxxD sequence in both its FARM and SARM, Rv0989c instead contains arginine in place of the second Asp in its FARM and first Asp in its SARM (Figure 2). Although Rv0989c also contains an Ala in place of the last Asp of its SARM, this Asp has been indicated to be less important for _trans_-IDS activity [23]. Indeed, the _M. tuberculosis trans_-IDS that produces (\(E\),\(E\))-FPP, Rv3398c (idsA1) [24], has a Gly at this position. By contrast, all the other Asp in these motifs have been shown to be important for catalytic activity [4]. Perhaps not surprisingly then, Arg has not been reported in the FARM or SARM of other _trans_-IDS. Accordingly, it was hypothesized that these unique substitutions in Rv0989c might influence product chain length. This was investigated by iteratively swapping the corresponding active site residues via site-directed mutagenesis between Rv0989c and the closely related Rv0562. The resulting mutants were purified and assayed in vitro with DMAPP and IPP. Products were observed during 30-min assays, indicating that these mutants retain reasonable amounts of catalytic activity. However, to ensure thorough representation of all synthesized products, the product ratios reported here are from overnight assays (Table 1). With such extended incubation, wild-type (WT) Rv0989c produces some (\(E\),\(E\))-FPP unless IPP concentrations are reduced (Table S1). Thus, the product  
Figure 2: Partial protein sequence alignment of Rv0989c and Rv0562. Rv0562 contains the canonical _trans_-IDS first and second aspartate-rich DDxxD motifs (FARM and SARM; highlighted in green), but these are both disrupted by Arg substitutions in Rv0989c. The fifth residue upstream of the FARM, which is typically responsible for product length determination in _trans_-IDS, is also highlighted here (orange). ratio quantification assays were run with 100 \(\upmu\)M DMAPP and 10 \(\upmu\)M IPP overnight (Table 1). Under these conditions, WT Rv0989c produces only GPP, and WT Rv0562 still produces only GGPP. By contrast, all of the mutants yielded a variety of products. In general, substitution of Asp for the Arg in Rv0989c led to the production of longer chain product, specifically (_E,E_)-FPP (but not GGPP), in addition to GPP. Conversely, substitution of Arg for the corresponding Asp in Rv0562 led to the appearance of the shorter chain products, both (_E,E_)-FPP and GPP, with only small amounts of GGPP produced. With both of these _M. tuberculosis trans_-IDSs, while the single mutants certainly affect product outcome, the double-mutant led to the greatest change in product outcome i.e., these exhibit additive effects. These results indicate that the unique Arg observed in the FARM and SARM of Rv0989c play a role in the unusual production of the 10-carbon GPP by this _M. tuberculosis trans_-IDS. This hypothesis is supported not only by the ability of substituting Asp for these Arg to increase Rv0989c product chain length, but also the inverse decrease in product chain length observed upon substituting Arg for the corresponding Asp in the closely related Rv0562 (Table 1).