The Structural Basis for BVMO Substrate Profile and Stereospecificity

The Baeyer-Villiger monooxygenases (BVMOs) are a group of bacterial enzymes that are able to catalyze the synthetically useful Baeyer-Villiger oxidation reaction. As such, these enzymes have attracted considerable attention as potential industrial biocatalysts. The interest in these enzymes has led to a desire to be able to rationally design them for tailored biocatalytic applications. While recent years have seen the publication of a number of crystal structures (1-3), we have been lacking a structure of a BVMO that has its native substrate or product bound in a conformation that will allow the determination of substrate specificity and stereospecificity. Without such a structure, progress towards tailored BVMOs has been hampered. We have been able to solve two crystal structures of cyclohexanone monooxygenase (CHMO) with its lactone product, ε-caprolactone, bound. These structures place the lactone in an ideal position for the determination of its substrate specificity and stereospecificity. These structures have provided us with a better understanding of the structural basis for substrate binding, paving the way for the rational design of tailored BVMOs. At the same time, we have pursued small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) studies to better understand the dynamic nature of the enzyme. These studies have allowed us to explain the relationship between the various crystallized states of BVMOs and their complex, fourteen step enzyme mechanism.

"Structural characterization of aminoglycoside modifying enzyme ANT(2"")-Ia"

Aminoglycosides are a class of broad-spectrum antibiotics used in the treatment of serious Gram-negative bacterial infections, they target the 16S RNA subunit and upon binding cause errors in translation, eventually inducing a bactericidal effect [1]. Aminoglycoside nucleotidyltransferase (2")-Ia (ANT(2")-Ia) is an aminoglycoside modifying enzyme that prevents aminoglycosides from binding to the ribosomal subunit, making this enzyme a principle candidate structure-based drug design [2]. Characterization of ANT(2")-Ia has been proven to be difficult due to the low stability and solubility of overexpressed protein, where 95% of the protein being expressed is in the form of inclusion bodies [3]. We describe a protocol that has lead to successful expression and purification of ANT(2")-Ia. A successful enzymatic assay has also been adapted and the protein is active and stable under these conditions with a specific activity of 0.14 U/mg. Furthermore, nuclear magnetic resonance (NMR) studies have allowed for the assignment of 144 of the 176 non-proline backbone residues. Substrate binding NMR experiments have shown unique global chemical shift perturbations upon binding ATP and tobramycin, suggesting unique binding sites for each substrate. Structural determination of ANT(2")-Ia using NMR in conjunction with x-ray crystallography can be utilized in order to develop small molecules that will act as more effective aminoglycosides in order to inhibit ANT(2")-Ia from binding and modifying these antibiotics.

Study of 2'-macrolide phosphotransferase selectivity for different substrates

Macrolides are antibiotics that have been in use since the late 1950s to treat a wide range of bacterial infections (e.g. upper respiratory infections, skin and soft-tissue infections, stomach ulcers and some venereal diseases). The structure of these antibiotics contains a lactone ring of either 14, 15, or 16 members, with a variety of sugar moieties attached. Resistance to this class of antibiotics may result from the reaction carried out by macrolide phosphotransferases [MPHs]. MPHs belong to the family of antibiotic kinases which catalyzes the transfer of a phosphate group from a nucleoside triphosphate to a specific hydroxyl on the antibiotic. However, unlike most antibiotic kinases, MPHs utilize GTP as the phosphate donor. Specifically, 2'-macrolide phosphotransferase type I [MPH(2')-I] transfers the gamma-phosphate from GTP to the 2'-hydroxyl of 14- and 15-membered ring macrolides. Crystal structure of the ternary complexes of MPH(2')-I with both 14- and 15-membered lactone macrolides have been determined. To study the basis of substrate selectivity, we have generated mutations of several amino acid residues in the macrolide-binding pocket and examined the catalytic activities of these mutants on the different classes of macrolides, including those containing a 16-membered lactone. Furthermore, we will present kinetic studies of MPH(2')-I containing mutations in the nucleoside-binding pocket in order to study the mechanism for the enzyme's preference for GTP.

A catalytic switch uncovered in gentamicin resistance kinase APH(2'')-Ia

The APH(2'')-Ia domain of the bifunctional aminoglycoside resistance enzyme AAC(6')-Ie/APH(2'')-Ia confers high-level resistance to aminoglycoside antibiotics. Crystal structures of this kinase domain in complex with GTP analogues and acceptor substrates have uncovered a surprising conformational bistability of the GTP substrate, which may reduce futile hydrolysis of the cofactor by the enzyme. This conformational switch is influenced by the binding of aminoglycosides, and may represent an adaptive feature of the enzyme, improving its evolutionary fitness in bacterial populations. This mechanism combines with a remarkable flexibility observed in the binding of diverse aminoglycoside substrates to make this enzyme a formidable antibiotic resistance machine.

Determinants of nucleoside specificity of a macrolide phosphotransferase

2'-macrolide phosphotransferase type I [MPH(2')-I] is an antibiotic kinase that renders many macrolides, such as erythromycin, inactive by catalyzing the transfer of a phosphate group from a nucleoside triphosphate to the hydroxyl at the 2'-position of the antibiotic. MPH(2')-I is functionally and structurally analogous to the aminoglycoside kinases (APHs). However, it is distinct from most APHs in that it utilizes GTP exclusively as its phosphate donor. We will present the crystal structure of MPH(2')-I in its apo and ternary complex forms with guanosine nucleotide and different macrolide substrates. We will compare its nucleoside-binding pocket to that of the 2''-aminoglycoside phosphotransferases [APH(2'')], a subclass of aminoglycoside kinases that are capable of utilizing GTP as a phosphate donor. To further decipher the structural basis of the nucleoside specificity of MPH(2')-I, mutations of amino acid resides in the nucleoside-binding pocket have been carried out and their effects on the binding affinity of purine nucleotides were examined by isothermal titration calorimetry. Our preliminary results show that the "gatekeeper" residue plays a role in governing the nucleoside selectivity.

Crystal structures of hFPPS in complex with novel anticancer drug leads

Human farnesyl pyrophosphate synthase (hFPPS) produces farnesyl pyrophosphate, an isoprenoid required for a variety of essential cellular processes. Inhibition of hFPPS has been well established as the mechanism of action of the nitrogen-containing bisphosphonate (N-BP) drugs, currently best known for their anti-bone resorptive effects. Recent investigations indicate that hFPPS inhibition also produces potent anticancer effects both in vitro and vivo: N-BPs inhibit proliferation, motility, and viability of tumor cells, and act in synergy with other anticancer agents [1,2]. However, the physicochemical properties of the current N-BP drugs seriously compromise their full anticancer potential in non-skeletal tissues. They show poor membrane permeability and extreme affinity to bone, due mainly to their highly charged bisphosphonate moiety, which mimics the pyrophosphate of the substrates of hFPPS. Both the substrates and N-BPs bind to hFPPS via Mg ion-mediated interactions between their pyrophosphate/bisphosphonate moiety and two aspartate-rich surfaces of the enzyme's active site cavity. Recently, we took a structure-guided approach to develop bisphosphonates with higher lipophilicity for enhanced uptake into non-skeletal tissues. Surprisingly, some of the new compounds were found to bind to hFPPS even in the absence of Mg ions. Crystal structures of hFPPS in complex with a representative compound revealed that this bisphosphonate binds to the enzyme's active site in the presence of Mg ions, but also to a nearby allosteric inhibitory site in their absence. Furthermore, removal of a phosphonate group from the bisphosphonate moiety of this compound resulted in an inhibitor that binds exclusively to the allosteric site. Based on the crystal structures with these lead compounds, we generated of a novel class of non-bisphosphonate, allosteric inhibitors of hFPPS with superior physicochemical properties than those of the current N-BP drugs for broader tissue distribution.

Crystal structure of an alleged mannosylphosphate transferase Ktr6p at 3.06 Å

Ktr6p is an alleged mannosylphosphate transferase from yeast Golgi. It has been implicated in decorating both O-linked and N-lined glycans with mannosylphosphate in vivo. However, based on sequence similarity, Ktr6p belongs to GT15 family of α-1,2-mannosyltransferases. To address this disagreement, the soluble portion of Ktr6p was expressed in P. pastoris and purified by liquid chromatography. The purified protein, GDP-mannose and various acceptors were used in a number of direct and indirect activity assays, however, neither manosyltransferase nor mannosylphosphate transferase activity was detected. Ktr6p was crystallized in a number of PEG- containing conditions, but the crystals resisted all attempts at cryoprotection. Three crystals were used to collect a 3.06 Å resolution dataset on a home source at room temperature. The crystals belong to P 21 21 21 spacegroup with 2 molecules per asymmetric unit. The structure was solved by molecular replacement using a structure of Kre2p, a close homolog from GT15 family (40% sequence identity). The structure was refined to R/Rfree 16.1%/21.2% The overall structure of Ktr6p is very similar to the structure of Kre2p having less than 2 Å overall backbone RMSD. However even at 3 Å resolution the difference in the active site is striking. The guanine moiety binding pocked is occluded by a well-ordered loop making GDP-mannose binding impossible in this conformation. Several aminoacid substitutions in the Mn2+ coordinating environment suggest that Ktr6 does not depend on manganese for its postulated activity. These observations indicate that Ktr6p functions quite differently from Kre2p.