Alternative substrates reveal catalytic cycle and key binding events in the reaction catalysed by anthranilate phosphoribosyltransferase from Mycobacterium tuberculosis

Abstract

AnPRT (anthranilate phosphoribosyltransferase), required for the biosynthesis of tryptophan, is essential for the virulence of Mycobacterium tuberculosis (Mtb). AnPRT catalyses the Mg2+-dependent transfer of a phosphoribosyl group from PRPP (5'-phosphoribosyl-1'-pyrophosphate) to anthranilate to form PRA (5'-phosphoribosyl anthranilate). Mtb-AnPRT was shown to catalyse a sequential reaction and significant substrate inhibition by anthranilate was observed. Antimycobacterial fluoroanthranilates and methyl-substituted analogues were shown to act as alternative substrates for Mtb-AnPRT, producing the corresponding substituted PRA products. Structures of the enzyme complexed with anthranilate analogues reveal two distinct binding sites for anthranilate. One site is located over 8 Å (1 Å=0.1 nm) from PRPP at the entrance to a tunnel leading to the active site, whereas in the second, inner, site anthranilate is adjacent to PRPP, in a catalytically relevant position. Soaking the analogues for variable periods of time provides evidence for anthranilate located at transient positions during transfer from the outer site to the inner catalytic site. PRPP and Mg2+ binding have been shown to be associated with the rearrangement of two flexible loops, which is required to complete the inner anthranilate-binding site. It is proposed that anthranilate first binds to the outer site, providing an unusual mechanism for substrate capture and efficient transfer to the catalytic site following the binding of PRPP.

Anthranilate phosphoribosyltransferase from the hyperthermophilic archaeon Thermococcus kodakarensis shows maximum activity with zinc and forms a unique dimeric structure

“…Similarly, the second substrate, anthranilate, was kept constant at 4 lM when the kinetic parameters toward PRPP were measured. Anthranilate concentrations above 4 lM resulted in reduced enzymatic activity, suggesting substrate inhibition by anthranilate as observed also in M. tuberculosis TrpD [21]. Apparent K m values for anthranilate and PRPP were 2.2 lM and 250 lM, respectively ( Fig.…”

A Three-Ring Circus: Metabolism of the Three Proteogenic Aromatic Amino Acids and Their Role in the Health of Plants and Animals

“…AnPRT is a member of the phosphoribosyl transferase (PRT) involved in nucleotide biosynthesis and salvage apart from AAA biosynthesis (Sinha and Smith, 2001 ). A number of microbial AnPRT have been studied in detail, with the M. tuberculosis enzyme showing some interesting structural features (Castell et al, 2013 ; Cookson et al, 2014 , 2015 ). In this case, an unusual channel, which could deliver anthranilate to the active site, has been observed.…”

“…Anthranilates fluorinated at the 5- or 6- position of the benzene ring were demonstrated to be antibacterial, with modest in vivo activity in mouse models (Zhang et al, 2013 ). Since the fluoro-anthranilates were not shown to be TrpD inhibitors for the E. coli enzyme, but alternate substrates leading to formation of fluorinated IGP in vitro (Cookson et al, 2014 ), the anti-tubercular activity of fluoro-anthranilates might stem from two possibilities. Either, the biosynthesis is inhibited downstream of TrpD, or the fluorinated tryptophan formed at the end of this pathway is toxic to the bacterium.…”

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

“…Three sites for anthranilate are responsible for tunnel movement of anthranilate, with a concomitant 180°flipping of the compound toward MgPRPP, which at this point is presumably already present. However, anthranilate may bind to the outer end of the tunnel before MgPRPP is bound, and binding of the latter compound may participate in tunnel formation (290). Interestingly, in S. solfataricus anthranilate phosphoribosyltransferase Gly79 also forms a hydrogen bond to the anthranilate nitrogen.…”

“…Since the organism's host, humans, are unable to synthesize tryptophan, a search for inhibitors of tryptophan biosynthetic enzymes, including for anthranilate phosphoribosyltransferase, has been conducted in hopes of identifying an antimicrobial agent (287,290,292).…”