TY - JOUR
T1 - Crystal structure of the pyocyanin biosynthetic protein PhzS
AU - Greenhagen, Bryan T.
AU - Shi, Katherine
AU - Robinson, Howard
AU - Gamage, Swarna
AU - Bera, Asim K.
AU - Ladner, Jane E.
AU - Parsons, James F.
PY - 2008/5/13
Y1 - 2008/5/13
N2 - The human pathogen Pseudomonas aeruginosa produces pyocyanin, a blue-pigmented phenazine derivative, which is known to play a role in virulence. Pyocyanin is produced from chorismic acid via the phenazine pathway, nine proteins encoded by a gene cluster. Phenazine-1-carboxylic acid, the initial phenazine formed, is converted to pyocyanin in two steps that are catalyzed by the enzymes PhzM and PhzS. PhzM is an adenosylmethionine dependent methyltransferase, and PhzS is a flavin dependent hydroxylase. It has been shown that PhzM is only active in the physical presence of PhzS, suggesting that a protein-protein interaction is involved in pyocyanin formation. Such a complex would prevent the release of 5-methyl-phenazine-1-carboxylate, the putative intermediate, and an apparently unstable compound. Here, we describe the three-dimensional structure of PhzS, solved by single anomalous dispersion, at a resolution of 2.4 Å. The structure reveals that PhzS is a member of the family of aromatic hydroxylases characterized by p-hydroxybenzoate hydroxylase. The flavin cofactor of PhzS is in the solvent exposed out orientation typically seen in unliganded aromatic hydroxylases. The PhzS flavin, however, appears to be held in a strained conformation by a combination of stacking interactions and hydrogen bonds. The structure suggests that access to the active site is gained via a tunnel on the opposite side of the protein from where the flavin is exposed. The C-terminal 23 residues are disordered as no electron density is present for these atoms. The probable location of the C-terminus, near the substrate access tunnel, suggests that it may be involved in substrate binding as has been shown for another structural homologue, RebC. This region also may be an element of a PhzM-PhzS interface. Aromatic hydroxylases have been shown to catalyze electrophilic substitution reactions on activated substrates. The putative PhzS substrate, however, is electron deficient and unlikely to act as a nucleophile, suggesting that PhzS may use a different mechanism than its structural relatives.
AB - The human pathogen Pseudomonas aeruginosa produces pyocyanin, a blue-pigmented phenazine derivative, which is known to play a role in virulence. Pyocyanin is produced from chorismic acid via the phenazine pathway, nine proteins encoded by a gene cluster. Phenazine-1-carboxylic acid, the initial phenazine formed, is converted to pyocyanin in two steps that are catalyzed by the enzymes PhzM and PhzS. PhzM is an adenosylmethionine dependent methyltransferase, and PhzS is a flavin dependent hydroxylase. It has been shown that PhzM is only active in the physical presence of PhzS, suggesting that a protein-protein interaction is involved in pyocyanin formation. Such a complex would prevent the release of 5-methyl-phenazine-1-carboxylate, the putative intermediate, and an apparently unstable compound. Here, we describe the three-dimensional structure of PhzS, solved by single anomalous dispersion, at a resolution of 2.4 Å. The structure reveals that PhzS is a member of the family of aromatic hydroxylases characterized by p-hydroxybenzoate hydroxylase. The flavin cofactor of PhzS is in the solvent exposed out orientation typically seen in unliganded aromatic hydroxylases. The PhzS flavin, however, appears to be held in a strained conformation by a combination of stacking interactions and hydrogen bonds. The structure suggests that access to the active site is gained via a tunnel on the opposite side of the protein from where the flavin is exposed. The C-terminal 23 residues are disordered as no electron density is present for these atoms. The probable location of the C-terminus, near the substrate access tunnel, suggests that it may be involved in substrate binding as has been shown for another structural homologue, RebC. This region also may be an element of a PhzM-PhzS interface. Aromatic hydroxylases have been shown to catalyze electrophilic substitution reactions on activated substrates. The putative PhzS substrate, however, is electron deficient and unlikely to act as a nucleophile, suggesting that PhzS may use a different mechanism than its structural relatives.
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U2 - 10.1021/bi702480t
DO - 10.1021/bi702480t
M3 - Article
C2 - 18416536
AN - SCOPUS:43249111574
VL - 47
SP - 5281
EP - 5289
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 19
ER -