Phe161 and Arg166 variants of p-hydroxybenzoate hydroxylase Implications for NADPH recognition and structural stability

AbstractPhe161 and Arg166 of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens belong to a newly discovered sequence motif in flavoprotein hydroxylases with a putative dual function in FAD and NADPH binding [1]. To study their role in more detail, Phe161 and Arg166 were selectively changed by site-directed mutagenesis. F161A and F161G are catalytically competent enzymes having a rather poor affinity for NADPH. The catalytic properties of R166K are similar to those of the native enzyme. R166S and R166E show impaired NADPH binding and R166E has lost the ability to bind FAD. The crystal structure of substrate complexed F161A at 2.2 Å is indistinguishable from the native enzyme, except for small changes at the site of mutation. The crystal structure of substrate complexed R166S at 2.0 Å revealed that Arg166 is important for providing an intimate contact between the FAD binding domain and a long excursion of the substrate binding domain. It is proposed that this interaction is essential for structural stability and for the recognition of the pyrophosphate moiety of NADPH

The transfer of protein crystals from their original mother liquor to a solution with a completely different precipitant

A procedure is described for the transfer of protein crystals from an ammonium sulfate‐containing mother liquor to a solution with another precipitant, such as polyethylene glycol. The suitable concentration of the alternative precipitant is established via a novel protocol, using a hanging‐drop equilibration method. This crystal transfer procedure is illustrated by experiments with crystals of trypanosomal triosephosphate isomerase and bacterial p ‐hydroxybenzoate hydroxylase, but it might have more general applicability

Crystal structure of p-hydroxybenzoate hydroxylase complexed with its reaction product 3,4-dihydroxybenzoate

Crystals of the flavin-containing enzyme p-hydroxybenzoate hydroxylase (PHBHase) complexed with its reaction product were investigated in order to obtain insight into the catalytic cycle of this enzyme involving two substrates and two cofactors. PHBHase was crystallized initially with its substrate, p-hydroxybenzoate and the substrate was then converted into the product 3,4-dihydroxybenzoate by allowing the catalytic reaction to proceed in the crystals. In addition, crystals were soaked in mother liquor containing a high concentration of this product. Data up to 2.3 A (1 A = 0.1 nm) were collected by the oscillation method and the structure of the enzyme product complex was refined by alternate restrained least-squares procedures and model building by computer graphics techniques. A total of 273 solvent molecules could be located, four of them being presumably sulfate ions. The R-factor for 14,339 reflections between 6.0 A and 2.3 A is 19.3%. The 3-hydroxyl group of the product introduced by the enzyme is clearly visible in the electron density, showing unambiguously which carbon atom of the substrate is hydroxylated. A clear picture of the hydroxylation site is obtained. The plane of the product is rotated 21 degrees with respect to the plane of the substrate in the current model of enzyme-substrate complex. The 4-hydroxyl group of the product is hydrogen bonded to the hydroxyl group of Tyr201, its carboxyl group is interacting with the side-chains of Tyr222, Arg214 and Ser212, while the newly introduced 3-hydroxyl group makes a hydrogen bond with the backbone carbonyl oxygen of Pro293

Sulfamide as Zinc Binding Motif in Small Molecule Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa)

Previously disclosed TAFIa inhibitors having a urea zinc-binding motif were used as the starting point for the development of a novel class of highly potent inhibitors having a sulfamide zinc-binding motif. High-resolution X-ray cocrystal structures were used to optimize the structures and reveal a highly unusual sulfamide configuration. A selected sulfamide was profiled in vitro and in vivo and displayed a promising ADMET profile

Sulfamide as Zinc Binding Motif in Small Molecule Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa)

Previously disclosed TAFIa inhibitors having a urea zinc-binding motif were used as the starting point for the development of a novel class of highly potent inhibitors having a sulfamide zinc-binding motif. High-resolution X-ray cocrystal structures were used to optimize the structures and reveal a highly unusual sulfamide configuration. A selected sulfamide was profiled in vitro and in vivo and displayed a promising ADMET profile

Sulfamide as Zinc Binding Motif in Small Molecule Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa)

Previously disclosed TAFIa inhibitors having a urea zinc-binding motif were used as the starting point for the development of a novel class of highly potent inhibitors having a sulfamide zinc-binding motif. High-resolution X-ray cocrystal structures were used to optimize the structures and reveal a highly unusual sulfamide configuration. A selected sulfamide was profiled in vitro and in vivo and displayed a promising ADMET profile

Novel Small Molecule Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa) from Natural Product Anabaenopeptin

Anabaenopeptins isolated from cyanobacteria were identified as inhibitors of carboxypeptidase TAFIa. Cocrystal structures of these macrocyclic natural product inhibitors in a modified porcine carboxypeptidase B revealed their binding mode and provided the basis for the rational design of small molecule inhibitors with a previously unknown central urea motif. Optimization based on these design concepts allowed for a rapid evaluation of the SAR and delivered potent small molecule inhibitors of TAFIa with a promising overall profile