We report the X-ray structures of two bacterial terpene synthases involved in virulence factor or biofilm formation: tuberculosinol/(13R,S)-iso-tuberculosinol synthase (Rv3378c) from Mycobacterium tuberculosis, involved in virulence factor formation, and YisP from Bacillus subtilis, involved in biofilm formation. Both enzymes contain the DDXXD domains found in most enzymes involved in terpene biosynthesis and act on terpene diphosphate substrates.  Rv3378c acts as a phosphatase and its structure is unique for a terpene synthase, closely resembling that of the cis-isoprenoid diphosphate synthases involved in bacterial cell wall biosynthesis. We solved structures with bound substrates and an inhibitor and these results combined with site-directed mutagenesis lead to a mechanism of action in which two Tyr residues activate water molecules for nucleophilic attack on the tuberculosinol diphosphate substrate. The BsYisP structure closely resembles that of dehydrosqualene synthase (CrtM, used in formation of the S. aureus virulence factor staphyloxanthin), but it also acts as a phosphatase and produces farnesol. We show that both tuberculosinol and farnesol (as well as staphyloxanthin) affect membrane structure in a cholesterol-like manner that may be associated with their effects on virulence or biofilm formation. The results are of broad general interest in the context of mechanistic enzymology as well as in drug discovery in which membrane structure modulators are targeted.

Structures of bacterial diterpene and isoprenoid synthases: Targeting virulence and biofilm formation

246th National Meeting (2013)

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