The presence of tryptophan biosynthesis in pathogenic microorganisms, such as bacteria, enables them to evade the host immune response and promotes bacterial survival. Anthranilate phosphoribosyltransferase, also known as AnPRT or TrpD, belongs to the phosphoribosyltransferase superfamily and serves as a catalyst for the second step in tryptophan biosynthesis. Notably, this enzyme has emerged as a potential drug target, supported by prior research. For instance, the removal of the trpD gene in the Mycobacterium tuberculosis knockout strain (Mtb ΔtrpD) has been shown to be avirulent even in immunodeficient mice, or the trpD encoding gene has been identified as virulence factor for Pseudomonas aeruginosa.
TrpD is a homodimeric enzyme dependent on magnesium cation and is responsible for catalyzing a nucleophilic substitution reaction, replacing the inorganic pyrophosphate from anomeric carbon of 5-phospho-D-ribose 1-diphosphate (PRPP) with anthranilate, resulting in the formation of N-(5-phosphoribosyl)-anthranilate (PRA). It is important to highlight that PRA, is relatively unstable, which introduces certain limitations when conducting existing biochemical assays. In our approach, we expressed and purified the wild-type AnPRT enzyme (TrpD) from both Serratia Marcesans (Sm-AnRPT) and Mycobacterium tuberculosis (Mtb-AnRPT) species. We utilized salicylic acid (λ_max = 298 nm) as an alternative substrate for these enzymes, leading to production of N-(5-phosphoribosyl)- salicylate (PRS) when reacted with PRPP. PRS is a stable product (λ_max = 276 nm), which we characterized using MS-TOF and NMR spectroscopy. The stability of PRS enabled us to develop a real-time absorbance-based assay to determine the kinetic parameters of Sm-AnPRT and Mtb-AnPRT. Also, we evaluated the inhibitory effects of previously reported AnPRT inhibitors, one with an anthranilate-like moiety (ACS172) and another with a non-anthranilate-like moiety (TAMU-A7) to determine the IC₅₀ and K_i values for Sm-AnRPT and Mtb-AnRPT in the presence of PRPP and salicylic acid.

Scheme 1: Nucleophilic substitution reaction catalyzed by AnPRT

Steven Van Lanen

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