Conformational Modulation of a Mobile Loop Controls Catalysis in the (βα)8-Barrel Enzyme of Histidine Biosynthesis HisF
Hupfeld, Enrico ; Schlee, Sandra ; Wurm, Jan Philip ; Rajendran, Chitra ; Yehorova, Dariia ; Vos, Eva ; Ravindra Raju, Dinesh ; Kamerlin, Shina Caroline Lynn LU
; Sprangers, Remco
and Sterner, Reinhard
(2024)
In JACS Au
4(8).
p.3258-3276
- Abstract
The overall significance of loop motions for enzymatic activity is generally accepted. However, it has largely remained unclear whether and how such motions can control different steps of catalysis. We have studied this problem on the example of the mobile active site β1α1-loop (loop1) of the (βα)8-barrel enzyme HisF, which is the cyclase subunit of imidazole glycerol phosphate synthase. Loop1 variants containing single mutations of conserved amino acids showed drastically reduced rates for the turnover of the substrates N'-[(5'-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PrFAR) and ammonia to the products imidazole glycerol phosphate (ImGP) and 5-aminoimidazole-4-carboxamide-ribotide (AICAR). A... (More)
The overall significance of loop motions for enzymatic activity is generally accepted. However, it has largely remained unclear whether and how such motions can control different steps of catalysis. We have studied this problem on the example of the mobile active site β1α1-loop (loop1) of the (βα)8-barrel enzyme HisF, which is the cyclase subunit of imidazole glycerol phosphate synthase. Loop1 variants containing single mutations of conserved amino acids showed drastically reduced rates for the turnover of the substrates N'-[(5'-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PrFAR) and ammonia to the products imidazole glycerol phosphate (ImGP) and 5-aminoimidazole-4-carboxamide-ribotide (AICAR). A comprehensive mechanistic analysis including stopped-flow kinetics, X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations detected three conformations of loop1 (open, detached, closed) whose populations differed between wild-type HisF and functionally affected loop1 variants. Transient stopped-flow kinetic experiments demonstrated that wt-HisF binds PrFAR by an induced-fit mechanism whereas catalytically impaired loop1 variants bind PrFAR by a simple two-state mechanism. Our findings suggest that PrFAR-induced formation of the closed conformation of loop1 brings active site residues in a productive orientation for chemical turnover, which we show to be the rate-limiting step of HisF catalysis. After the cyclase reaction, the closed loop conformation is destabilized, which favors the formation of detached and open conformations and hence facilitates the release of the products ImGP and AICAR. Our data demonstrate how different conformations of active site loops contribute to different catalytic steps, a finding that is presumably of broad relevance for the reaction mechanisms of (βα)8-barrel enzymes and beyond.
(Less)
- author
- Hupfeld, Enrico
; Schlee, Sandra
; Wurm, Jan Philip
; Rajendran, Chitra
; Yehorova, Dariia
; Vos, Eva
; Ravindra Raju, Dinesh
; Kamerlin, Shina Caroline Lynn
LU
; Sprangers, Remco
and Sterner, Reinhard
- publishing date
- 2024-08-26
- type
- Contribution to journal
- publication status
- published
- in
- JACS Au
- volume
- 4
- issue
- 8
- pages
- 19 pages
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- scopus:85201421413
- pmid:39211614
- ISSN
- 2691-3704
- DOI
- 10.1021/jacsau.4c00558
- language
- English
- LU publication?
- no
- additional info
- © 2024 The Authors. Published by American Chemical Society.
- id
- 760d9b1a-8246-4eaf-a1d7-d78aebfd8d45
- date added to LUP
- 2025-01-11 18:12:24
- date last changed
- 2025-07-27 19:30:45
@article{760d9b1a-8246-4eaf-a1d7-d78aebfd8d45,
abstract = {{<p>The overall significance of loop motions for enzymatic activity is generally accepted. However, it has largely remained unclear whether and how such motions can control different steps of catalysis. We have studied this problem on the example of the mobile active site β1α1-loop (loop1) of the (βα)8-barrel enzyme HisF, which is the cyclase subunit of imidazole glycerol phosphate synthase. Loop1 variants containing single mutations of conserved amino acids showed drastically reduced rates for the turnover of the substrates N'-[(5'-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PrFAR) and ammonia to the products imidazole glycerol phosphate (ImGP) and 5-aminoimidazole-4-carboxamide-ribotide (AICAR). A comprehensive mechanistic analysis including stopped-flow kinetics, X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations detected three conformations of loop1 (open, detached, closed) whose populations differed between wild-type HisF and functionally affected loop1 variants. Transient stopped-flow kinetic experiments demonstrated that wt-HisF binds PrFAR by an induced-fit mechanism whereas catalytically impaired loop1 variants bind PrFAR by a simple two-state mechanism. Our findings suggest that PrFAR-induced formation of the closed conformation of loop1 brings active site residues in a productive orientation for chemical turnover, which we show to be the rate-limiting step of HisF catalysis. After the cyclase reaction, the closed loop conformation is destabilized, which favors the formation of detached and open conformations and hence facilitates the release of the products ImGP and AICAR. Our data demonstrate how different conformations of active site loops contribute to different catalytic steps, a finding that is presumably of broad relevance for the reaction mechanisms of (βα)8-barrel enzymes and beyond.</p>}},
author = {{Hupfeld, Enrico and Schlee, Sandra and Wurm, Jan Philip and Rajendran, Chitra and Yehorova, Dariia and Vos, Eva and Ravindra Raju, Dinesh and Kamerlin, Shina Caroline Lynn and Sprangers, Remco and Sterner, Reinhard}},
issn = {{2691-3704}},
language = {{eng}},
month = {{08}},
number = {{8}},
pages = {{3258--3276}},
publisher = {{The American Chemical Society (ACS)}},
series = {{JACS Au}},
title = {{Conformational Modulation of a Mobile Loop Controls Catalysis in the (βα)<sub>8</sub>-Barrel Enzyme of Histidine Biosynthesis HisF}},
url = {{http://dx.doi.org/10.1021/jacsau.4c00558}},
doi = {{10.1021/jacsau.4c00558}},
volume = {{4}},
year = {{2024}},
}