2012
DOI: 10.1093/protein/gzs015
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Directed evolution of (  )8-barrel enzymes: establishing phosphoribosylanthranilate isomerisation activity on the scaffold of the tryptophan synthase  -subunit

Abstract: Phosphoribosylanthranilate (PRA) isomerase (TrpF) and tryptophan synthase α-subunit (TrpA) are (βα)(8)-barrel enzymes that are involved in the biosynthesis of tryptophan. They contain a conserved phosphate binding site, which indicates a common evolutionary origin. In order to experimentally back this hypothesis, we have established TrpF activity on the scaffold of TrpA from Salmonella typhimurium using protein engineering. Based on the superposition of crystal structures with bound ligands, two residues in th… Show more

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Cited by 15 publications
(15 citation statements)
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“…Of these, about 1% (15/1457) could confer TrpF function. This suggests that there are many different pathways toward obtaining a new activity within HisA, illustrating the versatility of the (βα) 8 -barrel fold to acquire novel activities ( Jespersen et al, 1993 ; Fong et al, 2000 ; Wierenga, 2001 ; Henn-Sax et al, 2002 ; Schmidt et al, 2003 ; Vega et al, 2003 ; Sterner and Höcker, 2005 ; Li et al, 2008 ; Evran et al, 2012 ).…”
Section: Resultsmentioning
confidence: 99%
“…Of these, about 1% (15/1457) could confer TrpF function. This suggests that there are many different pathways toward obtaining a new activity within HisA, illustrating the versatility of the (βα) 8 -barrel fold to acquire novel activities ( Jespersen et al, 1993 ; Fong et al, 2000 ; Wierenga, 2001 ; Henn-Sax et al, 2002 ; Schmidt et al, 2003 ; Vega et al, 2003 ; Sterner and Höcker, 2005 ; Li et al, 2008 ; Evran et al, 2012 ).…”
Section: Resultsmentioning
confidence: 99%
“…The N-terminal domain consists of three anti-parallel β-strands, followed by four α-helices that are connected by the flexible loop L1 (residues 38–45). The C-terminal domain contains the widely occurring 8-stranded β/α barrel architecture [ 44 , 45 ]. The barrel, however, does not contain the typical (βα) 8 fold as found in triosephosphate isomerase (TIM)-barrel enzymes [ 46 ], but rather comprises a β 2 α 2 (βα) 6 fold that is also seen in enolases from yeast or other bacterial origins [ 15 , 47 , 48 ].…”
Section: Resultsmentioning
confidence: 99%
“…Nevertheless, HisA can gain specificity for the TrpF substrate, PRA, after random gain-of-function mutations selected for in a Δ trpF background [1,3]. Moreover, PRA specificity has been found to be highly evolvable in other mechanistically unrelated protein folds [4,5]. This poses PRA isomerase activity as a suitable enzymatic function to investigate functional shifts involving co-occurring analogous enzymes.…”
Section: Introductionmentioning
confidence: 99%