Structural studies on folding intermediates of serine hydroxymethyltransferase using fluorescence resonance energy transfer.


Abstract

Previous studies have demonstrated that the in vitro folding pathway of Escherichia coli serine hydroxymethyltransferase has both monomer and dimer intermediates that are stable for periods of minutes to hours at 4 degrees C (Cai K., Schirch, D., and Schirch, V. (1995) J. Biol. Chem. 270, 19294-19299). Single Trp mutant enzymes were constructed and used in combination with other methods to show that on the folding pathway of this enzyme two domains rapidly fold to form a monomer in which the amino-terminal 55 amino acid residues and a segment around the active site region of Lys229 remain in a largely disordered form. This partially folded enzyme can form dimers and slowly undergoes a rate-determining conformational change in which the unstructured segments assume their native state (Cai, K. , and Schirch, V. (1996) J. Biol. Chem. 271, 2987-2994). To further assess the kinetics and structural details of the intermediates during folding, fluorescence energy transfer and fluorescence anisotropy measurements were made of the three Trp residues and pyridoxal 5'-phosphate, attached covalently to the active site by reduction to a secondary amine by sodium cyanoborohydride. These studies confirmed that the basic kinetic folding pathway remained the same in the reduced enzyme as compared to the earlier studies with the apoenzyme. Both equilibrium and kinetic intermediates were identified and their structural characteristics determined. The results show that the active site Lys229-bound pyridoxyl 5'-phosphate remains more than 50 angstroms from any Trp residues until the final rate-determining conformational change when it approaches each Trp residue at the same rate. The environment of each Trp residue and the pyridoxyl phosphate in both an equilibrium folding intermediate and a kinetic folding intermediate are described. Study holds ProTherm entries: 5260, 5261, 5262, 5263, 5264, 5265 Extra Details: additive : EDTA(1 mM),transition 1 hydroxymethyltransferase; Trp mutant; folding pathway;,conformational change; structural characteristics

Submission Details

ID: 7pFqeK744

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:29 p.m.

Version: 1

Publication Details
Cai K;Schirch V,J. Biol. Chem. (1996) Structural studies on folding intermediates of serine hydroxymethyltransferase using fluorescence resonance energy transfer. PMID:8910307
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3GBX 2009-03-10 1.8 Serine hydroxymethyltransferase from Salmonella typhimurium
1DFO 1999-12-10 2.4 CRYSTAL STRUCTURE AT 2.4 ANGSTROM RESOLUTION OF E. COLI SERINE HYDROXYMETHYLTRANSFERASE IN COMPLEX WITH GLYCINE AND 5-FORMYL TETRAHYDROFOLATE
1EQB 2000-04-19 2.7 X-RAY CRYSTAL STRUCTURE AT 2.7 ANGSTROMS RESOLUTION OF TERNARY COMPLEX BETWEEN THE Y65F MUTANT OF E-COLI SERINE HYDROXYMETHYLTRANSFERASE, GLYCINE AND 5-FORMYL TETRAHYDROFOLATE
3G8M 2009-11-10 3.3 Serine Hydroxymethyltransferase Y55F Mutant

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.2 Serine hydroxymethyltransferase A7FFW1 GLYA_YERP3
90.4 Serine hydroxymethyltransferase A1JKP3 GLYA_YERE8
91.6 Serine hydroxymethyltransferase A8GHZ4 GLYA_SERP5
93.3 Serine hydroxymethyltransferase C0PYJ5 GLYA_SALPC
93.0 Serine hydroxymethyltransferase A9MHI3 GLYA_SALAR
93.3 Serine hydroxymethyltransferase P0A2E1 GLYA_SALTY
93.3 Serine hydroxymethyltransferase B5BAV4 GLYA_SALPK
93.3 Serine hydroxymethyltransferase A9N1W0 GLYA_SALPB
93.3 Serine hydroxymethyltransferase Q5PII3 GLYA_SALPA
93.3 Serine hydroxymethyltransferase B4T1D1 GLYA_SALNS
93.3 Serine hydroxymethyltransferase B4TDC8 GLYA_SALHS
93.3 Serine hydroxymethyltransferase Q57LF7 GLYA_SALCH
93.3 Serine hydroxymethyltransferase B5F1D2 GLYA_SALA4
93.8 Serine hydroxymethyltransferase A8AD38 GLYA_CITK8
93.3 Serine hydroxymethyltransferase P0A2E2 GLYA1_SALTI
93.5 Serine hydroxymethyltransferase B4TRY8 GLYA_SALSV
93.5 Serine hydroxymethyltransferase A4WDC0 GLYA_ENT38
93.3 Serine hydroxymethyltransferase A6TCG5 GLYA_KLEP7
93.5 Serine hydroxymethyltransferase B5XNI6 GLYA_KLEP3
94.0 Serine hydroxymethyltransferase A7MGY5 GLYA_CROS8
99.8 Serine hydroxymethyltransferase Q3YZ04 GLYA_SHISS
99.8 Serine hydroxymethyltransferase Q32D21 GLYA_SHIDS
99.8 Serine hydroxymethyltransferase B7N6D8 GLYA_ECOLU
99.8 Serine hydroxymethyltransferase B5Z123 GLYA_ECO5E
99.8 Serine hydroxymethyltransferase Q8XA55 GLYA_ECO57
100.0 Serine hydroxymethyltransferase P0A827 GLYA_SHIFL
100.0 Serine hydroxymethyltransferase Q0T1W9 GLYA_SHIF8
100.0 Serine hydroxymethyltransferase Q31XT6 GLYA_SHIBS
100.0 Serine hydroxymethyltransferase B2TXW4 GLYA_SHIB3
100.0 Serine hydroxymethyltransferase Q1R8I4 GLYA_ECOUT
100.0 Serine hydroxymethyltransferase B1LNK7 GLYA_ECOSM
100.0 Serine hydroxymethyltransferase B6I5C4 GLYA_ECOSE
100.0 Serine hydroxymethyltransferase P0A825 GLYA_ECOLI
100.0 Serine hydroxymethyltransferase B1IVS6 GLYA_ECOLC
100.0 Serine hydroxymethyltransferase P0A826 GLYA_ECOL6
100.0 Serine hydroxymethyltransferase Q0TET8 GLYA_ECOL5
100.0 Serine hydroxymethyltransferase A1AE82 GLYA_ECOK1
100.0 Serine hydroxymethyltransferase A8A359 GLYA_ECOHS
100.0 Serine hydroxymethyltransferase B1XB26 GLYA_ECODH
100.0 Serine hydroxymethyltransferase C4ZXC6 GLYA_ECOBW
100.0 Serine hydroxymethyltransferase B7M8A7 GLYA_ECO8A
100.0 Serine hydroxymethyltransferase B7MYI0 GLYA_ECO81
100.0 Serine hydroxymethyltransferase B7NRK2 GLYA_ECO7I
100.0 Serine hydroxymethyltransferase B7LDE3 GLYA_ECO55
100.0 Serine hydroxymethyltransferase B7MIN5 GLYA_ECO45
100.0 Serine hydroxymethyltransferase B7UGZ1 GLYA_ECO27
100.0 Serine hydroxymethyltransferase A7ZPZ4 GLYA_ECO24