Stepwise introduction of a zinc binding site into Porphobilinogen synthase from Pseudomonas aeruginosa (mutation D139C)

Tracking the Evolution of Porphobilinogen Synthase Metal Dependence in Vitro

PORPHOBILINOGEN SYNTHASE, ALAD, ALADHPORPHOBILINOGEN SYNTHASE

MSFTPANRAYPYTRLRRNRRDDFSRRLVRENVLTVDDLILPVFVLDGVNQRESIPSMPGVERLSIDQLLIEAEEWVALGI
PALALFPVTPVEKKSLDAAEAYNPEGIAQRATRALRERFPELGIITDVALDPFTTHGQCGILDDDGYVLNDVSIDVLVRQ
ALSHAEAGAQVVAPSDMMDGRIGAIREALESAGHTNVRVMAYSAKYASAYYGPFRDAVGSASNLGKGNKATYQMDPANSD
EALHEVAADLAEGADMVMVKPGMPYLDIVRRVKDEFRAPTFVYQVSGEYAMHMGAIQNGWLAESVILESLTAFKRAGADG
ILTYFAKQAAEQLRRGR

HANGING DROP IN 24-WELL LIMBRO PLATES. DROPS MADE OF 5 MICROL PROTEIN SOLUTION (8,7 MG/ML PROTEIN, 50 MM NA-HEPES PH 7.5, 10 MM MGCL2, 10 ZNCL2, 10 MM DTT) PLUS 5 MICROL RESERVOIR SOLUTION (28,5 % (W/V) PEG-400, 100 MM NA-HEPES PH 7.5, 80MM MGCL2, 20MM DTT) ABOVE 500 MICROL OF RESERVOIR SOLUTION.

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 ZINC BINDING SITE INTO PORPHOBILINOGEN SYNTHASE OF PSEUDOMONAS AERUGINOSA (MUTANTIONS A129C AND D131C)

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

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

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

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

STEPWISE INTRODUCTION OF ZINC BINDING SITE INTO PORPHOBILINOGEN SYNTHASE OF PSEUDOMONAS AERUGINOSA (MUTANTIONS 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, ASP 139 TO CYS,
ILE 199 VAL

AMINO ACID EXCHANGE IN POSITION 139, ASP TURNED INTO CYS FORMIC ACID IS USED TO REPRESENT CARBOXYLIC GROUPS OF OTHERWISE DISORDERED MOLECULES BOUND TO THE ACTIVE SITE. THE SEQUENCE CONFLICT AT RESIDUE 199 IS DUE TO AN ACCIDENTAL POINT MUTATION IN THE GENE USED FOR THE MUTATIONAL STUDY.

HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.

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