8s8r
An induced-fit motion of a mobile loopAn induced-fit motion of a mobile loop
Structural highlights
FunctionHIS6_THEMA IGPS catalyzes the conversion of PRFAR and glutamine to IGP, AICAR and glutamate. The HisF subunit catalyzes the cyclization activity that produces IGP and AICAR from PRFAR using the ammonia provided by the HisH subunit.[HAMAP-Rule:MF_01013] Publication Abstract from PubMedThe 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 beta(1)alpha(1)-loop (loop1) of the (betaalpha)(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 (betaalpha)(8)-barrel enzymes and beyond. Conformational Modulation of a Mobile Loop Controls Catalysis in the (betaalpha)(8)-Barrel Enzyme of Histidine Biosynthesis HisF.,Hupfeld E, Schlee S, Wurm JP, Rajendran C, Yehorova D, Vos E, Ravindra Raju D, Kamerlin SCL, Sprangers R, Sterner R JACS Au. 2024 Aug 15;4(8):3258-3276. doi: 10.1021/jacsau.4c00558. eCollection , 2024 Aug 26. PMID:39211614[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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