CRYSTAL-STRUCTURES OF MUTANT PSEUDOMONAS-AERUGINOSA P-HYDROXYBENZOATEHYDROXYLASES - THE TYR201PHE, TYR385PHE, AND ASN300ASP VARIANTS

Structures of the mutant p-hydroxybenzoate hydroxylases, Tyr201Phe, Ty r385Phe, and Asn300Asp, each complexed with the substrate p-OHB have b een determined by X-ray crystallography. Crystals of these three mutan ts of the Pseudomonas aeruginosa enzyme, which differs from the wild-t ype Pseudomonas fluorescens enzyme at two surface positions (228 and 2 49), were isomorphous with crystals of the wild-type P. fluorescens en zyme, allowing the mutant structures to be determined by model buildin g and refinement, starting from the coordinates for the oxidized P. fl uorescens PHBH-3,4-diOHB complex [Schreuder, H. A., van der Laan, J. M ., Hol, W. G. J., and Drenth, J. (1988) J. Mel. Biol. 199, 637-648]. T he R factors for the structures described here are: Tyr385Phe, 0.178 f or data from 40.0 to 2.1 Angstrom; Tyr201Phe, 0.203 for data from 40.0 to 2.3 Angstrom; and Asn300Asp, 0.193 for data from 40.0 to 2.3 Angst rom. The functional effects of the Tyr201Phe and Tyr385Phe mutations, described earlier [Entsch, B., Palfey, B. A., Ballou, D. P., and Masse y, V. (1991) J. Biol. Chem. 266, 17341-17349], were rationalized with the assumption that the mutations perturbed the hydrogen-bonding inter actions of the tyrosine residues but caused no other changes in the en zyme structure. In agreement with these assumptions, the positions of the substrate, the flavin, and the modified residues are not altered i n the Tyr385Phe and Tyr201Phe structures. In contrast, substitution of Asp for Asn at residue 300 has more profound effects on the enzyme st ructure. The side chain of Asp300 moves away from the flavin, disrupti ng the interactions of the carboxamide group with the flavin O(2) atom , and the alpha-helix H10 that begins at residue 297 is displaced, alt ering its dipole interactions with the flavin ring. The functional con sequences of these changes in the enzyme structure and of the introduc tion of the carboxyl group at 300 are described and discussed in the a ccompanying paper (Palfey et al., 1994b).