6am8

Engineered tryptophan synthase b-subunit from Pyrococcus furiosus, PfTrpB2B9 with Trp bound as E(Aex2)

spinqualitylabelsall models 6am8, resolution 1.83Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 6am8 is a 4 chain structure with sequence from Pyrococcus furiosus DSM 3638. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.83Å Ligands: LLP, NA, PLT, TRP Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function TRPB1_PYRFU The beta subunit is responsible for the synthesis of L-tryptophan from indole and L-serine (By similarity). Publication Abstract from PubMed Allosteric enzymes contain a wealth of catalytic diversity that remains distinctly underutilized for biocatalysis. Tryptophan synthase is a model allosteric system and a valuable enzyme for the synthesis of non-canonical amino acids (ncAA). Previ-ously, we evolved the beta-subunit from Pyrococcus furiosus, PfTrpB, for ncAA synthase activity in the absence of its native partner protein PfTrpA. However, the precise mechanism by which mutation activated TrpB to afford a stand-alone cata-lyst remained enigmatic. Here, we show that directed evolution caused a gradual change in the rate-limiting step of the cata-lytic cycle. Concomitantly, the steady-state distribution of intermediates shifts to favor covalently bound Trp adducts, which is associated with increased thermodynamic stability of these species. The biochemical properties of these evolved, stand-alone TrpBs converge on those induced in the native system by allosteric activation. High resolution crystal structures of the wild-type enzyme, an intermediate in the lineage, and the final variant, encompassing five distinct chemical states, show that activating mutations have only minor structural effects on their immediate environment. Instead, mutation stabi-lizes the large-scale motion of a sub-domain to favor an otherwise transiently populated closed conformational state. This increase in stability enabled the first structural description of Trp covalently bound in a catalytically active TrpB, confirming key features of catalysis. These data combine to show that sophisticated models of allostery are not a prerequisite to reca-pitulating its complex effects via directed evolution, opening the way to engineering stand-alone versions of diverse allosteric enzymes. Directed evolution mimics allosteric activation by stepwise tuning of the conformational ensemble.,Buller AR, van Roye P, Cahn JKB, Scheele RA, Herger M, Arnold FH J Am Chem Soc. 2018 Apr 30. doi: 10.1021/jacs.8b03490. PMID:29712420 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Tryptophan synthase 3D structures

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