Identification and characterization of the first small molecule inhibitor of MDMX.

The p53 pathway is disrupted in virtually every human tumor. In approximately 50% of human cancers, the p53 gene is mutated, and in the remaining cancers, the pathway is dysregulated by genetic lesions in other genes that modulate the p53 pathway. One common mechanism for inactivation of the p53 pathway in tumors that express wild-type p53 is increased expression of MDM2 or MDMX. MDM2 and MDMX bind p53 and inhibit its function by distinct nonredundant mechanisms. Small molecule inhibitors and small peptides have been developed that bind MDM2 in the p53-binding pocket and displace the p53 protein, leading to p53-mediated cell cycle exit and apoptosis. To date, peptide inhibitors of MDMX have been developed, but no small molecule inhibitors have been reported. We have developed biochemical and cell-based assays for high throughput screening of chemical libraries to identify MDMX inhibitors and identified the first MDMX inhibitor SJ-172550. This compound binds reversibly to MDMX and effectively kills retinoblastoma cells in which the expression of MDMX is amplified. The effect of SJ-172550 is additive when combined with an MDM2 inhibitor. Results from a series of biochemical and structural modeling studies suggest that SJ-172550 binds the p53-binding pocket of MDMX, thereby displacing p53. This lead compound is a useful chemical scaffold for further optimization of MDMX inhibitors that may eventually be used to treat pediatric cancers and various adult tumors that overexpress MDMX or have similar genetic lesions. When combined with selective MDM2 inhibitors, SJ-172550 may also be useful for treating tumors that express wild-type p53.

How the MccB bacterial ancestor of ubiquitin E1 initiates biosynthesis of the microcin C7 antibiotic.

The 39-kDa Escherichia coli enzyme MccB catalyses a remarkable posttranslational modification of the MccA heptapeptide during the biosynthesis of microcin C7 (MccC7), a 'Trojan horse' antibiotic. The approximately 260-residue C-terminal region of MccB is homologous to ubiquitin-like protein (UBL) activating enzyme (E1) adenylation domains. Accordingly, MccB-catalysed C-terminal MccA-acyl-adenylation is reminiscent of the E1-catalysed activation reaction. However, unlike E1 substrates, which are UBLs with a C-terminal di-glycine sequence, MccB's substrate, MccA, is a short peptide with an essential C-terminal Asn. Furthermore, after an intramolecular rearrangement of MccA-acyl-adenylate, MccB catalyses a second, unique reaction, producing a stable phosphoramidate-linked analogue of acyl-adenylated aspartic acid. We report six-crystal structures of MccB in apo, substrate-, intermediate-, and inhibitor-bound forms. Structural and kinetic analyses reveal a novel-peptide clamping mechanism for MccB binding to heptapeptide substrates and a dynamic-active site for catalysing dual adenosine triphosphate-consuming reactions. The results provide insight into how a distinctive member of the E1 superfamily carries out two-step activation for generating the peptidyl-antibiotic MccC7.

Complexes of the enzyme phosphomannomutase/phosphoglucomutase with a slow substrate and an inhibitor.

Two complexes of the enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa with a slow substrate and with an inhibitor have been characterized by X-ray crystallography. Both ligands induce an interdomain rearrangement in the enzyme that creates a highly buried active site. Comparisons with enzyme-substrate complexes show that the inhibitor xylose 1-phosphate utilizes many of the previously observed enzyme-ligand interactions. In contrast, analysis of the ribose 1-phosphate complex reveals a combination of new and conserved enzyme-ligand interactions for binding. The ability of PMM/PGM to accommodate these two pentose phosphosugars in its active site may be relevant for future efforts towards inhibitor design.

The reaction of phosphohexomutase from Pseudomonas aeruginosa: structural insights into a simple processive enzyme.

The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa catalyzes the reversible conversion of 1-phospho to 6-phospho-sugars. The reaction entails two phosphoryl transfers, with an intervening 180 degrees reorientation of the reaction intermediate (e.g. glucose 1,6-bisphosphate) during catalysis. Reorientation of the intermediate occurs without dissociation from the active site of the enzyme and is, thus, a simple example of processivity, as defined by multiple rounds of catalysis without release of substrate. Structural characterization of two PMM/PGM-intermediate complexes with glucose 1,6-bisphosphate provides new insights into the reaction catalyzed by the enzyme, including the reorientation of the intermediate. Kinetic analyses of site-directed mutants prompted by the structural studies reveal active site residues critical for maintaining association with glucose 1,6-bisphosphate during its unique dynamic reorientation in the active site of PMM/PGM.

Evolutionary trace analysis of the alpha-D-phosphohexomutase superfamily.

The alpha-D-phosphohexomutase superfamily is composed of four related enzymes that catalyze a reversible, intramolecular phosphoryl transfer on their sugar substrates. The enzymes in this superfamily play important and diverse roles in carbohydrate metabolism in organisms from bacteria to humans. Recent structural and mechanistic studies of one member of this superfamily, phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa, have provided new insights into enzyme mechanism and substrate recognition. Here we use sequence-sequence and sequence-structure comparisons via evolutionary trace analysis to examine 71 members of the alpha-D-phosphohexomutase superfamily. These analyses show that key residues in the active site, including many of those involved in substrate contacts in the P. aeruginosa PMM/PGM complexes, are conserved throughout the enzyme family. Several important regions show class-specific differences in sequence that appear to be correlated with differences in substrate specificity exhibited by subgroups of the family. In addition, we describe the translocation of a 20-residue segment containing the catalytic phosphoserine of phosphoacetylglucosamine mutase, which uniquely identifies members of this subgroup.

Detailed kinetic studies of an aggregating inhibitor; inhibition of phosphomannomutase/phosphoglucomutase by disperse blue 56.

Phosphomannomutase/phosphoglucomutase occupies a central position in the pathways by which several virulence factors are synthesized in Pseudomonas aeruginosa. Virtual screening was used to identify potential inhibitors of phosphomannomutase/ phosphoglucomutase, and one compound, the anthraquinone-based dye Disperse Blue 56, showed potent inhibition in vitro. The kinetics of inhibition was complex; the time courses for reactions in the presence of the inhibitor were biphasic, suggestive of slow-binding inhibition. Quantitative analysis of the progress curves and preincubation experiments demonstrated that slow-binding inhibition was not occurring, however. Initial velocity kinetic studies indicated that Disperse Blue 56 was a parabolic, noncompetitve inhibitor. Progress curves for reactions in the presence of Disperse Blue 56 could be fitted very well by a model in which 2 equiv of the inhibitor bound to free enzyme or the enzyme-substrate complex. The inhibition was largely relieved by the inclusion of 0.01% Triton X-100 in the assay solutions, which has been suggested to be the hallmark for inhibition by compounds that exert their effect through aggregates [McGovern, S. L., Caselli, E., Grigorieff, N., and Shiochet, B. K. (2002) J. Med. Chem. 45, 1712-1722]. Our kinetic data appear to be consistent with either inhibition by a dimer of Disperse Blue 56 or inhibition by a Disperse Blue 56 aggregate, but the latter appears much more likely. We present a detailed analysis of the system to provide further information that may help in the recognition of inhibition through aggregation.

Structural basis of diverse substrate recognition by the enzyme PMM/PGM from P. aeruginosa.

Enzyme-substrate complexes of phosphomannomutase/phosphoglucomutase (PMM/PGM) reveal the structural basis of the enzyme's ability to use four different substrates in catalysis. High-resolution structures with glucose 1-phosphate, glucose 6-phosphate, mannose 1-phosphate, and mannose 6-phosphate show that the position of the phosphate group of each substrate is held constant by a conserved network of hydrogen bonds. This produces two distinct, and mutually exclusive, binding orientations for the sugar rings of the 1-phospho and 6-phospho sugars. Specific binding of both orientations is accomplished by key contacts with the O3 and O4 hydroxyls of the sugar, which must occupy equatorial positions. Dual recognition of glucose and mannose phosphosugars uses a combination of specific protein contacts and nonspecific solvent contacts. The ability of PMM/PGM to accommodate these four diverse substrates in a single active site is consistent with its highly reversible phosphoryl transfer reaction and allows it to function in multiple biosynthetic pathways in P. aeruginosa.

Roles of active site residues in Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase.

In Pseudomonas aeruginosa, the dual-specificity enzyme phosphomannomutase/phosphoglucomutase catalyzes the transfer of a phosphoryl group from serine 108 to the hydroxyl group at the 1-position of the substrate, either mannose 6-P or glucose 6-P. The enzyme must then catalyze transfer of the phosphoryl group on the 6-position of the substrate back to the enzyme. Each phosphoryl transfer is expected to require general acid-base catalysis, provided by amino acid residues at the enzyme active site. An extensive survey of the active site residues by site-directed mutagenesis failed to identify a single key residue that mediates the proton transfers. Mutagenesis of active site residues Arg20, Lys118, Arg247, His308, and His329 to residues that do not contain ionizable groups produced proteins for which V(max) was reduced to 4-12% of that of the wild type. The fact that no single residue decreased catalytic activity more significantly, and that several residues had similar effects on V(max), suggested that the ensemble of active site amino acids act by creating positive electrostatic potential, which serves to depress the pK of the substrate hydroxyl group so that it binds in ionized form at the active site. In this way, the necessity of positioning the reactive hydroxyl group near a specific amino acid residue is avoided, which may explain how the enzyme is able to promote catalysis of both phosphoryl transfers, even though the 1- and 6-positions do not occupy precisely the same position when the substrate binds in the two different orientations in the active site. When Ser108 is mutated, the enzyme retains a surprising amount of activity, which has led to the suggestion that an alternative residue becomes phosphorylated in the absence of Ser108. (31)P NMR spectra of the S108A protein confirm that it is phosphorylated. Although the S108A/H329N protein had no detectable catalytic activity, the (31)P NMR spectra were not consistent with a phosphohistidine residue.

Crystal structure of PMM/PGM: an enzyme in the biosynthetic pathway of P. aeruginosa virulence factors.

The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from P. aeruginosa is required for the biosynthesis of two bacterial exopolysaccharides: alginate and lipopolysaccharide (LPS). Both of these molecules play a role in the virulence of P. aeruginosa, an important human pathogen known for its ability to develop antibiotic resistance and cause chronic lung infections in cystic fibrosis patients. The crystal structure of PMM/PGM shows that the enzyme has four domains, three of which have a similar three-dimensional fold. Residues from all four domains of the protein contribute to the formation of a large active site cleft in the center of the molecule. Detailed information on the active site of PMM/PGM lays the foundation for structure-based inhibitor design. Inhibitors of sufficient potency and specificity should impair the biosynthesis of alginate and LPS, and may facilitate clearance of the bacteria by the host immune system and increase the efficacy of conventional antibiotic treatment against chronic P. aeruginosa infections.