Benzimidazole analogs of l‐tryptophan are substrates and inhibitors of tryptophan indole lyase from Escherichia coli

Abstract

Tryptophan indole lyase (TIL), an enzyme found in Escherichia coli and related enterobacteria, produces indole from L-tryptophan (L-Trp). Indole is a signaling molecule in bacteria, affecting biofilm formation, pathogenicity and antibiotic resistance. b-(Benzimidazol-1-yl)-L-alanine (BZI-Ala), 2-amino-4-(benzimidazol-1-yl)butyric acid (homo-BZI-Ala) and 2-amino-5-(benzimidazol-1-yl)pentanoic acid (bishomo-BZI-Ala) were synthesized and tested as substrates and inhibitors of TIL. BZI-Ala is a good substrate of TIL, with K m = 300 lM, k cat = 5.6 s À1 and k cat /K m = 1.9 9 10 4 , similar to L-Trp. BZI-Ala is also a good substrate for H463F mutant TIL, which has very low activity with L-Trp. In contrast, homo-BZI-Ala was found to be a potent competitive inhibitor of TIL, with a K i of 13.4 lM. However, the higher homolog, bishomo-BZI-Ala, was inactive as an inhibitor of TIL at a concentration of 600 lM, and is thus a much weaker inhibitor. The reaction of TIL with BZI-Ala was too fast to be observed in the stopped-flow spectrophotometer, and shows an aldimine intermediate in the steady state. However, H463F TIL shows equilibrating mixtures of aldimine and quinonoid complexes in the steady state. The spectra of the reaction of TIL with homo-BZI-Ala show a rapidly formed intermediate absorbing at 340 nm, probably a gem-diamine, that decays slowly to form a quinonoid complex absorbing at 494 nm. The potent binding of homo-BZI-Ala may be due to it being a 'bi-product' analog of the indole-a-aminoacrylate complex. These results demonstrate that an amino acid substrate may be converted to a potent inhibitor of TIL simply by homologation, which may be useful in the design of other potent TIL inhibitors.

Gut microbiome-derived phenyl sulfate contributes to albuminuria in diabetic kidney disease

“…It is conceivable that TPL inhibitors also partially inhibit TIL, resulting in a simultaneous reduction in PS and IS concentrations. To clarify this, we examined the effect of TPL inhibitors on TIL activity 33 . The specific TIL inhibitor 2-amino-4-(benzimidazol-1-yl)butyric acid (homo-BZI-Ala) at 100 μM, significantly inhibited TIL (apotryptophanase) activity as reported 33 (Fig.…”

“…PS and 13 C 6 labeled PS were synthesized by Sundia MediTech (Shanghai, China) and ISOTEC (Sigma-Aldrich), respectively. 2-Aza-tyrosione 27 and 2-amino-4-(benzimidazol-1-yl)butyric acid (Homo-BZI-Ala) 33 were chemically synthesized. l -meta-tyrosine was purchased from Astatech (Bristol, PA).…”

Fermentative Production of Halogenated Tryptophan Derivatives with Corynebacterium glutamicum Overexpressing Tryptophanase or Decarboxylase Genes

“…Another promising E. coli TnaA mutant was generated by exchanging histidine 463 by phenylalanine, which is located in the substrate‐binding portion of the active site [56] . As consequence, the high activity and substrate specificity towards tryptophan were lost, whereas the substrate spectrum was extended to the tryptophan benzimidazole analog β‐(benzimidazol‐1‐yl)‐ l ‐alanine [57] . Shifting substrate specificity from the natural substrate tryptophan towards tryptophan analogues may also be beneficial for conversion of halogenated tryptophans.…”

“…[56] As consequence, the high activity and substrate specificity towards tryptophan were lost, whereas the substrate spectrum was extended to the tryptophan benzimidazole analog β-(benzimidazol-1-yl)-l-alanine. [57] Shifting substrate specificity from the natural substrate tryptophan towards tryptophan analogues may also be beneficial for conversion of halogenated tryptophans.…”

The Catalytic Mechanisms of the Reactions between Tryptophan Indole-Lyase and Nonstandard Substrates: The Role of the Ionic State of the Catalytic Group Accepting the Cα Proton of the Substrate

“…The other substrates of TIL are tryptophan analogs substituted at various positions of the indole ring [2, 3], benzimidazole analogs of tryptophan [4], as well as amino acids containing suitable leaving groups at the β-carbon atom, including S-(o-nitrophenyl)-L-cysteine (SOPC) [5], S-alkyl-L-cysteine analogs [6], β-chloro-Lalanine [5], and L-serine [6] and O-acyl-L-serines [7].…”