Site-directed mutagenesis of the alpha subunit of tryptophan synthase from Salmonella typhimurium.


Abstract

Site-specific mutagenesis has been used to prepare two mutant forms of the alpha subunit of tryptophan synthase from Salmonella typhimurium in which either cysteine-81 or cysteine-118 is replaced by a serine residue. These mutant proteins are potentially useful for x-ray crystallographic studies since a heavy metal binding site is specifically eliminated in each mutant. The purified mutant proteins are fully active in four reactions catalyzed by the wild type alpha 2 beta 2 complex of tryptophan synthase. However, the mutant alpha 2 beta 2 complexes dissociate more readily and are less heat-stable than the wild type alpha 2 beta 2 complex. Thus, cysteine-81 and cysteine-118 of the alpha subunit serve structural but not functional roles. Study holds ProTherm entries: 102, 103, 104 Extra Details: 2-mercaptoethanol (10 mM) and EDTA(1 mM) are added as additives. alpha subunit of tryptophan synthase; site-specific mutagenesis;,structural role; heavy metal binding site

Submission Details

ID: fVMDGtY23

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:14 p.m.

Version: 1

Publication Details
Ahmed SA;Kawasaki H;Bauerle R;Morita H;Miles EW,Biochem. Biophys. Res. Commun. (1988) Site-directed mutagenesis of the alpha subunit of tryptophan synthase from Salmonella typhimurium. PMID:3126743
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
5CGQ 2016-07-13 1.18 Crystal structure of Tryptophan Synthase from Salmonella typhimurium in complex with F9 ligand in the alpha-site and the product L-Tryptophan in the beta-site.
4HT3 2014-01-01 1.3 The crystal structure of Salmonella typhimurium Tryptophan Synthase at 1.30A complexed with N-(4'-TRIFLUOROMETHOXYBENZENESULFONYL)-2-AMINO-1-ETHYLPHOSPHATE (F9) inhibitor in the alpha site, internal aldimine
1QOP 2000-11-10 1.4 CRYSTAL STRUCTURE OF WILD-TYPE TRYPTOPHAN SYNTHASE COMPLEXED WITH INDOLE PROPANOL PHOSPHATE
6DZ4 2019-07-10 1.45 Crystal structure of Salmonella typhimurium Tryptophan Synthase with sodium ion at the metal coordination site and [3-hydroxy-2-methyl-5-phosphonooxymethyl-pyridin-4-ylmethyl]-serine (PLS) at the beta-site.
4HPJ 2013-12-25 1.45 Crystal structure of Tryptophan Synthase at 1.45 A resolution in complex with 2-aminophenol quinonoid in the beta site and the F9 inhibitor in the alpha site
2CLE 2007-06-12 1.5 Tryptophan Synthase in complex with N-(4'-trifluoromethoxybenzoyl)-2- amino-1-ethylphosphate (F6) - lowF6 complex
1TJP 2005-12-27 1.5 Crystal Structure Of Wild-Type Tryptophan Synthase Complexed With 1-[(2-hydroxylphenyl)amino]3-glycerolphosphate
1WBJ 2006-05-24 1.5 wildtype tryptophan synthase complexed with glycerol phosphate
2CLK 2007-06-12 1.5 Tryptophan Synthase in complex with D-glyceraldehyde 3-phosphate (G3P)
1KFC 2003-01-07 1.5 CRYSTAL STRUCTURE OF ALPHAT183V MUTANT OF TRYPTOPHAN SYNTHASE FROM SALMONELLA TYPHIMURIUM With Indole Propanol Phosphate
1K8Y 2002-06-19 1.5 CRYSTAL STRUCTURE OF THE TRYPTOPHAN SYNTHASE BETA-SER178PRO MUTANT COMPLEXED WITH D,L-ALPHA-GLYCEROL-3-PHOSPHATE
2CLO 2007-06-12 1.5 Tryptophan Synthase (external aldimine state) in complex with (naphthalene-2'-sulfonyl)-2-amino-1-ethylphosphate (F19)
2CLM 2007-06-12 1.51 Tryptophan Synthase (external aldimine state) in complex with N-(4'- trifluoromethoxybenzoyl)-2-amino-1-ethylphosphate (F6F)
4ZQC 2016-02-10 1.54 Tryptophan Synthase from Salmonella typhimurium in complex with two molecules of N-(4'-trifluoromethoxybenzoyl)-2-amino-1-ethylphosphate (F6F) inhibitor in the alpha-site and a single F6F molecule in the beta-site at 1.54 Angstrom resolution.
2CLL 2007-06-12 1.6 Tryptophan Synthase (external aldimine state) in complex with N-(4'- trifluoromethoxybenzenesulfonyl)-2-amino-1-ethylphosphate (F9)
6D0V 2019-04-17 1.64 Tryptophan synthase Q114A mutant in complex with inhibitor N-(4'-trifluoromethoxybenzenesulfonyl)-2-amino-1-ethylphosphate (F9F) at the alpha-site, aminoacrylate (P1T) at the beta site, and cesium ion at the metal coordination site
6DZO 2019-07-10 1.64 Crystal structure of Salmonella typhimurium Tryptophan Synthase mutant beta-Q114A with 2-({[4-(trifluoromethoxy)phenyl]sulfonyl}amino)ethyl dihydrogen phosphate (F9F) at the alpha-site, Cesium ion at the metal coordination site, and [3-hydroxy-2-methyl-5-phosphonooxymethyl-pyridin-4-ylmethyl]-serine (PLS) at the beta-site.
4HN4 2013-12-25 1.64 Tryptophan synthase in complex with alpha aminoacrylate E(A-A) form and the F9 inhibitor in the alpha site
6C73 2019-01-23 1.65 Tryptophan synthase Q114A mutant (internal aldimine state) in complex with N-(4'-trifluoromethoxybenzenesulfonyl)-2-amino-1-ethylphosphate (F9F) with cesium ion bound in the metal coordination site
4HPX 2013-12-18 1.65 Crystal structure of Tryptophan Synthase at 1.65 A resolution in complex with alpha aminoacrylate E(A-A) and benzimidazole in the beta site and the F9 inhibitor in the alpha site
4XUG 2016-01-27 1.65 Crystal structure of Tryptophan Synthase from Salmonella typhimurium in complex with 2-({[4-(Trifluoromethoxy)Phenyl]Sulfonyl}Amino)Ethyl Dihydrogen Phosphate (F9F) inhibitor in the alpha site and ammonium ion in the metal coordination site.
4Y6G 2016-02-10 1.65 Crystal structure of Tryptophan Synthase from Salmonella typhimurium in complex with N-(4'-trifluoromethoxybenzoyl)-2-amino-1-ethylphosphate (F6F) inhibitor in the alpha-site and beta-site.
1K8Z 2002-06-19 1.7 CRYSTAL STRUCTURE OF THE TRYPTOPHAN SYNTHASE BETA-SER178PRO MUTANT COMPLEXED WITH N-[1H-INDOL-3-YL-ACETYL]GLYCINE ACID
2CLI 2007-06-12 1.7 Tryptophan Synthase in complex with N-(4'- trifluoromethoxybenzenesulfonyl)-2-amino-1-ethylphosphate (F9)
1K3U 2002-07-03 1.7 CRYSTAL STRUCTURE OF WILD-TYPE TRYPTOPHAN SYNTHASE COMPLEXED WITH N-[1H-INDOL-3-YL-ACETYL]ASPARTIC ACID
1K7X 2002-06-19 1.7 CRYSTAL STRUCTURE OF THE BETA-SER178PRO MUTANT OF TRYPTOPHAN SYNTHASE
2CLF 2007-06-26 1.7 Tryptophan Synthase in complex with N-(4'-trifluoromethoxybenzoyl)-2- amino-1-ethylphosphate (F6) - highF6 complex
2RH9 2007-11-06 1.7 Tryptophan synthase complexed with IGP, internal aldimine, pH 9.0
2CLH 2007-06-12 1.7 Tryptophan Synthase in complex with (naphthalene-2'-sulfonyl)-2-amino- 1-ethylphosphate (F19)
4WX2 2015-11-18 1.75 Crystal structure of Tryptophan Synthase from Salmonella typhimurium in complex with two F6F molecules in the alpha-site and one F6F molecule in the beta-site
1KFE 2003-01-07 1.75 CRYSTAL STRUCTURE OF ALPHAT183V MUTANT OF TRYPTOPHAN SYNTHASE FROM SALMONELLA TYPHIMURIUM WITH L-Ser Bound To The Beta Site
4KKX 2014-01-01 1.77 Crystal structure of Tryptophan Synthase from Salmonella typhimurium with 2-aminophenol quinonoid in the beta site and the F6 inhibitor in the alpha site
1QOQ 2000-11-10 1.8 CRYSTAL STRUCTURE OF WILD-TYPE TRYPTOPHAN SYNTHASE COMPLEXED WITH INDOLE GLYCEROL PHOSPHATE
2J9Y 2007-12-04 1.8 Tryptophan Synthase Q114N mutant in complex with Compound II
2J9Z 2007-12-04 1.8 Tryptophan Synthase T110 mutant complex
1KFJ 2003-10-21 1.8 CRYSTAL STRUCTURE OF WILD-TYPE TRYPTOPHAN SYNTHASE COMPLEXED WITH L-SERINE
5BW6 2016-02-10 1.82 Tryptophan Synthase from Salmonella typhimurium in complex with a single molecule of 2-({[4-(trifluoromethoxy)phenyl]carbonyl}amino)ethyl dihydrogen phosphate (F6) in the alpha-site.
3PR2 2011-02-09 1.85 Tryptophan synthase indoline quinonoid structure with F9 inhibitor in alpha site
2J9X 2007-06-26 1.9 Tryptophan Synthase in complex with GP, alpha-D,L-glycerol-phosphate, Cs, pH6.5 - alpha aminoacrylate form - (GP)E(A-A)
1UBS 1996-03-08 1.9 TRYPTOPHAN SYNTHASE (E.C.4.2.1.20) WITH A MUTATION OF LYS 87->THR IN THE B SUBUNIT AND IN THE PRESENCE OF LIGAND L-SERINE
1K7F 2002-07-10 1.9 CRYSTAL STRUCTURE OF WILD-TYPE TRYPTOPHAN SYNTHASE COMPLEXED WITH N-[1H-INDOL-3-YL-ACETYL]VALINE ACID
1KFB 2003-01-07 1.9 CRYSTAL STRUCTURE OF ALPHAT183V MUTANT OF TRYPTOPHAN SYNTHASE FROM SALMONELLA TYPHIMURIUM WITH Indole Glycerol Phosphate
2TYS 1997-04-01 1.9 CRYSTAL STRUCTURES OF MUTANT (BETAK87T) TRYPTOPHAN SYNTHASE ALPHA2 BETA2 COMPLEX WITH LIGANDS BOUND TO THE ACTIVE SITES OF THE ALPHA AND BETA SUBUNITS REVEAL LIGAND-INDUCED CONFORMATIONAL CHANGES
1K8X 2002-12-18 1.9 Crystal Structure Of AlphaT183V Mutant Of Tryptophan Synthase From Salmonella Typhimurium
1BEU 1998-08-12 1.9 TRP SYNTHASE (D60N-IPP-SER) WITH K+
1A5A 1998-05-27 1.9 CRYO-CRYSTALLOGRAPHY OF A TRUE SUBSTRATE, INDOLE-3-GLYCEROL PHOSPHATE, BOUND TO A MUTANT (ALPHAD60N) TRYPTOPHAN SYNTHASE ALPHA2BETA2 COMPLEX REVEALS THE CORRECT ORIENTATION OF ACTIVE SITE ALPHA GLU 49
1CW2 1999-12-21 2.0 CRYSTAL STRUCTURE OF THE COMPLEX OF BACTERIAL TRYPTOPHAN SYNTHASE WITH THE TRANSITION STATE ANALOGUE INHIBITOR 4-(2-HYDROXYPHENYLSULFINYL)-BUTYLPHOSPHONIC ACID
2RHG 2007-11-20 2.0 Tryptophan synthase complexed with IGP, pH 7.0, internal aldimine
1A5B 1998-05-27 2.0 CRYO-CRYSTALLOGRAPHY OF A TRUE SUBSTRATE, INDOLE-3-GLYCEROL PHOSPHATE, BOUND TO A MUTANT (ALPHA D60N) TRYPTOPHAN SYNTHASE ALPHA2BETA2 COMPLEX REVEALS THE CORRECT ORIENTATION OF ACTIVE SITE ALPHA GLU 49
1TTQ 1996-03-08 2.0 TRYPTOPHAN SYNTHASE (E.C.4.2.1.20) IN THE PRESENCE OF POTASSIUM AT ROOM TEMPERATURE
2TRS 1997-04-01 2.04 CRYSTAL STRUCTURES OF MUTANT (BETAK87T) TRYPTOPHAN SYNTHASE ALPHA2 BETA2 COMPLEX WITH LIGANDS BOUND TO THE ACTIVE SITES OF THE ALPHA AND BETA SUBUNITS REVEAL LIGAND-INDUCED CONFORMATIONAL CHANGES
3CEP 2008-04-01 2.1 Structure of a tryptophan synthase quinonoid intermediate
1BKS 1999-03-23 2.2 TRYPTOPHAN SYNTHASE (E.C.4.2.1.20) FROM SALMONELLA TYPHIMURIUM
1C8V 2000-01-13 2.2 CRYSTAL STRUCTURE OF THE COMPLEX OF BACTERIAL TRYPTOPHAN SYNTHASE WITH THE TRANSITION STATE ANALOGUE INHIBITOR 4-(2-HYDROXYPHENYLTHIO)-BUTYLPHOSPHONIC ACID
1FUY 2001-01-17 2.25 CRYSTAL STRUCTURE OF BETAA169L/BETAC170W DOUBLE MUTANT OF TRYPTOPHAN SYNTHASE COMPLEXED WITH 5-FLUORO-INDOLE-PROPANOL PHOSPHATE
1C9D 1999-12-29 2.3 CRYSTAL STRUCTURE OF THE COMPLEX OF BACTERIAL TRYPTOPHAN SYNTHASE WITH THE TRANSITION STATE ANALOGUE INHIBITOR 4-(2-HYDROXY-4-FLUOROPHENYLTHIO)-BUTYLPHOSPHONIC ACID
1A50 1999-03-30 2.3 CRYSTAL STRUCTURE OF WILD-TYPE TRYPTOPHAN SYNTHASE COMPLEXED WITH 5-FLUOROINDOLE PROPANOL PHOSPHATE
1A5S 1999-03-30 2.3 CRYSTAL STRUCTURE OF WILD-TYPE TRYPTOPHAN SYNTHASE COMPLEXED WITH 5-FLUOROINDOLE PROPANOL PHOSPHATE AND L-SER BOUND AS AMINO ACRYLATE TO THE BETA SITE
1C29 2000-01-14 2.3 CRYSTAL STRUCTURE OF THE COMPLEX OF BACTERIAL TRYPTOPHAN SYNTHASE WITH THE TRANSITION STATE ANALOGUE INHIBITOR 4-(2-HYDROXYPHENYLTHIO)-1-BUTENYLPHOSPHONIC ACID
1TTP 1996-03-08 2.3 TRYPTOPHAN SYNTHASE (E.C.4.2.1.20) IN THE PRESENCE OF CESIUM, ROOM TEMPERATURE
1K7E 2002-07-10 2.3 CRYSTAL STRUCTURE OF WILD-TYPE TRYPTOPHAN SYNTHASE COMPLEXED WITH N-[1H-INDOL-3-YL-ACETYL]GLYCINE ACID
1CX9 1999-12-29 2.3 CRYSTAL STRUCTURE OF THE COMPLEX OF BACTERIAL TRYPTOPHAN SYNTHASE WITH THE TRANSITION STATE ANALOGUE INHIBITOR 4-(2-AMINOPHENYLTHIO)-BUTYLPHOSPHONIC ACID
1KFK 2003-10-21 2.4 Crystal structure of Tryptophan Synthase From Salmonella Typhimurium
2TSY 1997-04-01 2.5 CRYSTAL STRUCTURES OF MUTANT (BETAK87T) TRYPTOPHAN SYNTHASE ALPHA2 BETA2 COMPLEX WITH LIGANDS BOUND TO THE ACTIVE SITES OF THE ALPHA AND BETA SUBUNITS REVEAL LIGAND-INDUCED CONFORMATIONAL CHANGES
2DH5 2007-04-24 2.9 Crystal structure of E. coli Holo-TrpB
2DH6 2007-04-24 3.0 Crystal structure of E. coli Apo-TrpB
2WSY 1999-03-30 3.05 CRYSTAL STRUCTURE OF WILD-TYPE TRYPTOPHAN SYNTHASE

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
95.1 Tryptophan synthase alpha chain A9MPY6 TRPA_SALAR
98.9 Tryptophan synthase alpha chain B4TX39 TRPA_SALSV
99.3 Tryptophan synthase alpha chain B5BIC0 TRPA_SALPK
99.3 Tryptophan synthase alpha chain Q5PNM0 TRPA_SALPA
99.3 Tryptophan synthase alpha chain C0Q3N3 TRPA_SALPC
99.3 Tryptophan synthase alpha chain Q57NT2 TRPA_SALCH
99.6 Tryptophan synthase alpha chain Q8Z7E0 TRPA_SALTI
99.6 Tryptophan synthase alpha chain A9MWR3 TRPA_SALPB
99.6 Tryptophan synthase alpha chain B4T6X2 TRPA_SALNS
99.6 Tryptophan synthase alpha chain B4TJK9 TRPA_SALHS
99.6 Tryptophan synthase alpha chain B5R6M4 TRPA_SALG2
99.6 Tryptophan synthase alpha chain B5R3P3 TRPA_SALEP
99.6 Tryptophan synthase alpha chain B5FU65 TRPA_SALDC
99.6 Tryptophan synthase alpha chain B5F4M3 TRPA_SALA4
100.0 Tryptophan synthase alpha chain P00929 TRPA_SALTY
90.1 B Tryptophan synthase alpha chain B2VKT2 TRPB_ERWT9
90.9 B Tryptophan synthase alpha chain Q6D4U0 TRPB_PECAS
91.1 B Tryptophan synthase alpha chain A8GF82 TRPB_SERP5
91.6 B Tryptophan synthase alpha chain C6DGZ5 TRPB_PECCP
93.2 B Tryptophan synthase alpha chain A7MMG1 TRPB_CROS8
92.7 B Tryptophan synthase alpha chain A4WB02 TRPB_ENT38
93.7 B Tryptophan synthase alpha chain A6T7W6 TRPB_KLEP7
93.7 B Tryptophan synthase alpha chain B5XT02 TRPB_KLEP3
96.5 B Tryptophan synthase alpha chain Q31ZV4 TRPB_SHIBS
96.5 B Tryptophan synthase alpha chain B2U0F2 TRPB_SHIB3
96.5 B Tryptophan synthase alpha chain B7N474 TRPB_ECOLU
96.5 B Tryptophan synthase alpha chain Q1RCA6 TRPB_ECOUT
96.5 B Tryptophan synthase alpha chain A1AAN1 TRPB_ECOK1
96.5 B Tryptophan synthase alpha chain B7ML77 TRPB_ECO45
96.5 B Tryptophan synthase alpha chain P0A880 TRPB_SHIFL
96.5 B Tryptophan synthase alpha chain Q0T5D5 TRPB_SHIF8
96.5 B Tryptophan synthase alpha chain B6I9X5 TRPB_ECOSE
96.5 B Tryptophan synthase alpha chain P0A879 TRPB_ECOLI
96.5 B Tryptophan synthase alpha chain B1ITJ4 TRPB_ECOLC
96.5 B Tryptophan synthase alpha chain A7ZZJ7 TRPB_ECOHS
96.5 B Tryptophan synthase alpha chain B1XBL0 TRPB_ECODH
96.5 B Tryptophan synthase alpha chain B7LY17 TRPB_ECO8A
96.5 B Tryptophan synthase alpha chain B7L493 TRPB_ECO55
96.5 B Tryptophan synthase alpha chain A7ZL79 TRPB_ECO24
96.7 B Tryptophan synthase alpha chain Q32GS9 TRPB_SHIDS
96.7 B Tryptophan synthase alpha chain B1LH31 TRPB_ECOSM
96.7 B Tryptophan synthase alpha chain Q8FHV9 TRPB_ECOL6
96.7 B Tryptophan synthase alpha chain Q0TIA9 TRPB_ECOL5
96.7 B Tryptophan synthase alpha chain B7MUA0 TRPB_ECO81
96.7 B Tryptophan synthase alpha chain B7NVN1 TRPB_ECO7I
96.7 B Tryptophan synthase alpha chain B7UR67 TRPB_ECO27
96.7 B Tryptophan synthase alpha chain Q3Z109 TRPB_SHISS
96.7 B Tryptophan synthase alpha chain B5YZP1 TRPB_ECO5E
96.7 B Tryptophan synthase alpha chain Q8X7B6 TRPB_ECO57
97.0 B Tryptophan synthase alpha chain B7LS19 TRPB_ESCF3
96.7 B Tryptophan synthase alpha chain A8AG61 TRPB_CITK8
98.2 B Tryptophan synthase alpha chain A9MPY7 TRPB_SALAR
99.5 B Tryptophan synthase alpha chain Q57NT3 TRPB_SALCH
99.7 B Tryptophan synthase alpha chain P0A2K1 TRPB_SALTY
99.7 B Tryptophan synthase alpha chain P0A2K2 TRPB_SALTI
99.7 B Tryptophan synthase alpha chain B4TX38 TRPB_SALSV
99.7 B Tryptophan synthase alpha chain B5BIC1 TRPB_SALPK
99.7 B Tryptophan synthase alpha chain C0Q3N4 TRPB_SALPC
99.7 B Tryptophan synthase alpha chain A9MWR4 TRPB_SALPB
99.7 B Tryptophan synthase alpha chain Q5PD17 TRPB_SALPA
99.7 B Tryptophan synthase alpha chain B4T6X1 TRPB_SALNS
99.7 B Tryptophan synthase alpha chain B4TJK8 TRPB_SALHS
99.7 B Tryptophan synthase alpha chain B5R6M5 TRPB_SALG2
99.7 B Tryptophan synthase alpha chain B5R3P4 TRPB_SALEP
99.7 B Tryptophan synthase alpha chain B5FU66 TRPB_SALDC
99.7 B Tryptophan synthase alpha chain B5F4M4 TRPB_SALA4