Stepwise introduction of zinc binding site into porphobilinogen synthase of Pseudomonas aeruginosa (mutations A129C, D131C, D139C, P132E)

Tracking the Evolution of Porphobilinogen Synthase Metal Dependence in Vitro

PORPHOBILINOGEN SYNTHASE, ALAD, ALADHPORPHOBILINOGEN SYNTHASE

SITTING DROP. DROPS WERE MIXED OF 3 MICROLITER OF PROTEIN SOLUTION (12 MG/ML PROTEIN, 50 MM NA-HEPES PH 7.5, 10MM MGCL2, 1MM ZNCL2, 10 MM DTT) PLUS 3 MICROLITER OF RESERVOIR SOLUTION (1M LITHIUM SULFATE, 100MM TRIS-HCL PH 8.5, 10MM NICKEL CHLORIDE), 100 MICROLITER OF RESERVOIR SOLUTION, 96 WELL PLATES (NUNC) IN AN ANAEROBIC BOX

HIGH RESOLUTION CRYSTAL STRUCTURE OF A MG2- DEPENDENT 5- AMINOLEVULINIC ACID DEHYDRATASE

COMPLEX OF A MG2-DEPENDENT PORPHOBILINOGEN SYNTHASE (MUTANT D139N) WITH 5-FLUOROLEVULINIC ACID

STEPWISE INTRODUCTION OF A ZINC BINDING SITE INTO PORPHOBILINOGEN SYNTHASE FROM PSEUDOMONAS AERUGINOSA (MUTATION D139C)

STEPWISE INTRODUCTION OF ZINC BINDING SITE INTO PORPHOBILINOGEN SYNTHASE OF PSEUDOMONAS AERUGINOSA (MUTATIONS A129C AND D131C)

STEPWISE INTRODUCTION OF ZINC BINDING SITE INTO PORPHOBILINOGEN SYNTHASE OF PSEUDOMONAS AERUGINOSA (MUTATIONS A129C AND D139C)

STEPWISE INTRODUCTION OF ZINC BINDING SITE INTO PORPHOBILINOGEN SYNTHASE OF PSEUDOMONAS AERUGINOSA (MUTATIONS D131C AND D139C)

STEPWISE INTRODUCTION OF ZINC BINDING SITE INTO PORPHOBILINOGEN SYNTHASE OF PSEUDOMONAS AERUGINOSA (MUTATIONS A129C, D131C AND D139C)

STEPWISE INTRODUCTION OF ZINC BINDING SITE INTO PORPHOBILINOGEN SYNTHASE OF PSEUDOMONAS AERUGINOSA (MUTATIONS A129C, D131C, D139C, P132E, K229R)

SHEET
DETERMINATION METHOD: DSSP
THE SHEETS PRESENTED AS "AA BA" IN EACH CHAIN ON SHEET RECORDS
BELOW IS ACTUALLY AN 11-STRANDED BARREL THIS IS REPRESENTED BY
A 12-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS
ARE IDENTICAL.

CATALYTIC ACTIVITY: 2 5-AMINOLEVULINATE = PORPHOBILINOGEN + 2
H(2)O.
ENGINEERED MUTATION IN CHAINS A AND B, ALA 129 TO CYS
ENGINEERED MUTATION IN CHAINS A AND B, ASP 131 TO CYS
ENGINEERED MUTATION IN CHAINS A AND B, PRO 132 TO GLU
ENGINEERED MUTATION IN CHAINS A AND B, ASP 139 TO CYS
ACCIDENTALLY INTRODUCED MUTATION IN CHAINS A AND B,
ILE 199 TO VAL

ACCIDENTAL POINT MUTATION IN THE ORIGINAL GENE CAUSES I199V, FOUR ADDITIONAL ENGINEERED MUTATIONS A129C, D131C, D139C AND P132E

HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.

DELTA-AMINOLEVULINIC ACID DEHYDRATASE (E.C.4.2.1.24)

SYNTHASE, EVOLUTION, METALLOENZYME, PORPHOBILINOGEN SYNTHASE, PSEUDOMONAS AERUGINOSA, PROTEIN ENGINEERING