PDBsum entry 6amc

Biosynthetic protein

PDB id: 6amc

Contents

Protein chains

387 a.a.

Metals

_NA ×4

Waters ×263

PDB id:

6amc

Name:

Biosynthetic protein

Title:

Engineered tryptophan synthase b-subunit from pyrococcus furiosus, pftrpb4d11

Structure:

Tryptophan synthase beta chain 1. Chain: a, b, c, d. Engineered: yes. Mutation: yes

Source:

Pyrococcus furiosus (strain atcc 43587 / dsm 3638 / jcm 8422 / vc1). Organism_taxid: 186497. Strain: atcc 43587 / dsm 3638 / jcm 8422 / vc1. Gene: trpb1, pf1706. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008

Resolution:

1.93Å R-factor: 0.218 R-free: 0.255

Authors:

A.R.Buller,M.Herger

Key ref:

A.R.Buller et al. (2018). Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble. J Am Chem Soc, 140, 7256-7266. PubMed id: 29712420

Date:

09-Aug-17 Release date: 16-May-18

Protein chains

Q8U093  (TRPB1_PYRFU) -  Tryptophan synthase beta chain 1 from Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)

Seq: 388 a.a.

Struc: 387 a.a.*

* PDB and UniProt seqs differ at 6 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.4.2.1.20 - tryptophan synthase.
• Pathway: Tryptophan Biosynthesis
• Reaction: (1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate + L-serine = D-glyceraldehyde 3-phosphate + L-tryptophan + H2O
• Cofactor: Pyridoxal 5'-phosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

J Am Chem Soc 140:7256-7266 (2018)

PubMed id: 29712420

Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble.

A.R.Buller, P.van Roye, J.K.B.Cahn, R.A.Scheele, M.Herger, F.H.Arnold.

ABSTRACT

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 noncanonical amino acids (ncAA). Previously, we evolved the β-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 catalyst remained enigmatic. Here, we show that directed evolution caused a gradual change in the rate-limiting step of the catalytic cycle. Concomitantly, the steady-state distribution of the intermediates shifts to favor covalently bound Trp adducts, which have increased thermodynamic stability. 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 stabilizes the large-scale motion of a subdomain 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 recapitulating its complex effects via directed evolution, opening the way to engineering stand-alone versions of diverse allosteric enzymes.