Real-time enzyme dynamics illustrated with fluorescence spectroscopy of p-Hydroxybenzoate Hydroxylase.

We have used the flavoenzyme p-hydroxybenzoate hydroxylase (PHBH) to illustrate that a strongly fluorescent donor label can communicate with the flavin via single-pair Forster resonance energy transfer (spFRET). The accessible Cys-116 of PHBH was labeled with two different fluorescent maleimides with full preservation of enzymatic activity. One of these labels shows overlap between its fluorescence spectrum and the absorption spectrum of the FAD prosthetic group in the oxidized state, while the other fluorescent probe does not have this spectral overlap. The spectral overlap strongly diminished when the flavin becomes reduced during catalysis. The donor fluorescence properties can then be used as a sensitive antenna for the flavin redox state. Time-resolved fluorescence experiments on ensembles of labeled PHBH molecules were carried out in the absence and presence of enzymatic turnover. Distinct changes in fluorescence decays of spFRET-active PHBH can be observed when the enzyme is performing catalysis using both substrates p-hydroxybenzoate and NADPH. Single-molecule fluorescence correlation spectroscopy on spFRET-active PHBH showed the presence of a relaxation process (relaxation time of 23 mus) that is related to catalysis. In addition, in both labeled PHBH preparations the number of enzyme molecules reversibly increased during enzymatic turnover indicating that the dimer-monomer equilibrium is affected

Structure of an Engineered β-Lactamase Maltose Binding Protein Fusion Protein: Insights into Heterotropic Allosteric Regulation

Engineering novel allostery into existing proteins is a challenging endeavor to obtain novel sensors, therapeutic proteins, or modulate metabolic and cellular processes. The RG13 protein achieves such allostery by inserting a circularly permuted TEM-1 β-lactamase gene into the maltose binding protein (MBP). RG13 is positively regulated by maltose yet is, serendipitously, inhibited by Zn2+ at low µM concentration. To probe the structure and allostery of RG13, we crystallized RG13 in the presence of mM Zn2+ concentration and determined its structure. The structure reveals that the MBP and TEM-1 domains are in close proximity connected via two linkers and a zinc ion bridging both domains. By bridging both TEM-1 and MBP, Zn2+ acts to “twist tie” the linkers thereby partially dislodging a linker between the two domains from its original catalytically productive position in TEM-1. This linker 1 contains residues normally part of the TEM-1 active site including the critical β3 and β4 strands important for activity. Mutagenesis of residues comprising the crystallographically observed Zn2+ site only slightly affected Zn2+ inhibition 2- to 4-fold. Combined with previous mutagenesis results we therefore hypothesize the presence of two or more inter-domain mutually exclusive inhibitory Zn2+ sites. Mutagenesis and molecular modeling of an intact TEM-1 domain near MBP within the RG13 framework indicated a close surface proximity of the two domains with maltose switching being critically dependent on MBP linker anchoring residues and linker length. Structural analysis indicated that the linker attachment sites on MBP are at a site that, upon maltose binding, harbors both the largest local Cα distance changes and displays surface curvature changes, from concave to relatively flat becoming thus less sterically intrusive. Maltose activation and zinc inhibition of RG13 are hypothesized to have opposite effects on productive relaxation of the TEM-1 β3 linker region via steric and/or linker juxtapositioning mechanisms

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

Structures of the mutant p-hydroxybenzoate hydroxylases, Tyr201Phe, Tyr385Phe, and Asn300Asp, each complexed with the substrate p-OHB have been determined by X-ray crystallography. Crystals of these three mutants of the Pseudomonas aeruginosa enzyme, which differs from the wild-type Pseudomonas fluorescens enzyme at two surface positions (228 and 249), were isomorphous with crystals of the wild-type P. fluorescens enzyme, allowing the mutant structures to be determined by model building and refinement, starting from the coordinates for the oxidized P. fluorescens PHBH-3,4-diOHB complex [Schreuder, H. A., van der Laan, J. M., Hol, W. G. J., & Drenth, J. (1988) J. Mol. Biol. 199, 637-648]. The R factors for the structures described here are: Tyr385Phe, 0.178 for data from 40.0 to 2.1 A; Tyr201Phe, 0.203 for data from 40.0 to 2.3 A; and Asn300Asp, 0.193 for data from 40.0 to 2.3 A. The functional effects of the Tyr201Phe and Tyr385Phe mutations, described earlier [Entsch, B., Palfey, B. A., Ballou, D. P., & Massey, V. (1991) J. Biol. Chem. 266, 17341-17349], were rationalized with the assumption that the mutations perturbed the hydrogen-bonding interactions of the tyrosine residues but caused no other changes in the enzyme structure. In agreement with these assumptions, the positions of the substrate, the flavin, and the modified residues are not altered in the Tyr385Phe and Tyr201 Phe structures. In contrast, substitution of Asp for Asn at residue 300 has more profound effects on the enzyme structure. The side chain of Asp300 moves away from the flavin, disrupting the interactions of the carboxamide group with the flavin O(2) atom, and the ??-helix H10 that begins at residue 297 is displaced, altering its dipole interactions with the flavin ring. The functional consequences of these changes in the enzyme structure and of the introduction of the carboxyl group at 300 are described and discussed in the accompanying paper (Palfey et al., 1994b).close362