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Eric A Althoff

Publications and source records attributed to Eric A Althoff.

5 recordsLinked to original sources
PubMed2006

New algorithms and an in silico benchmark for computational enzyme design.

The creation of novel enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Here we describe two new algorithms for enzyme design that employ hashing techniques to allow searching through large numbers of protein scaffolds for optimal catalytic site placement. We also describe an in silico benchmark, based on the recapitulation of the active sites of native enzymes, that allows rapid evaluation and testing of enzyme design methodologies. In the benchmark test, which consists of designing sites for each of 10 different chemical reactions in backbone scaffolds derived from 10 enzymes catalyzing the reactions, the new methods succeed in identifying the native site in the native scaffold and ranking it within the top five designs for six of the 10 reactions. The new methods can be directly applied to the design of new enzymes, and the benchmark provides a powerful in silico test for guiding improvements in computational enzyme design.

Algorithms↗
PubMed2005

Investigation of the mechanism of resistance to third-generation cephalosporins by class C beta-lactamases by using chemical complementation.

The widespread use of antibiotics to treat bacterial infections has led to the continuing challenge of antibiotic resistance. For beta-lactam antibiotics, the most common form of resistance is the expression of beta-lactamase enzymes, which inactivate the antibiotics by cleavage of the beta-lactam core. In this study, chemical complementation, which is a general method to link the formation or cleavage of a chemical bond to the transcription of a reporter gene in vivo, was employed in combination with combinatorial mutagenesis to study the mechanism by which the class C beta-lactamase P99 might evolve resistance to the commonly administered third-generation cephalosporin cefotaxime. The chemical complementation system was first shown to be able to distinguish between the wild-type (wt) class C beta-lactamase P99 and the clinically isolated extended-spectrum class C beta-lactamase GC1 in the presence of cefotaxime. The system was then employed to evaluate the activity of mutants of wt P99 towards cefotaxime. A number of single-point mutations at position 221 (Tyr in wt P99) were identified that conferred resistance towards inhibition by cefotaxime, with as much as a 2000-fold increase in k(cat) and a 100-fold increase in k(cat)/K(M) (k(cat)=the rate of catalysis; K(M)=the Michaelis constant), as compared to those of the wt enzyme. Finally, the chemical complementation system was employed in a high-throughput screen to identify a number of mutants of P99 that have multiple mutations around the substrate-binding pocket that increase resistance towards cefotaxime inhibition. The catalytic turnover of cefotaxime by the most active mutant identified was 5500 times higher than that of the wt P99. The resistant mutants suggest a mechanism by which a number of mutations can confer resistance by increasing the flexibility of the Omega loop and altering the positioning of residue 221. Thus, as illustrated in this study, chemical complementation has the potential to be used as a high-throughput screen to study a wide range of enzyme-drug interactions.

Anti-Bacterial Agents↗
PubMed2004

Correlation between ligand-receptor affinity and the transcription readout in a yeast three-hybrid system.

The yeast two-hybrid assay has proven to be a powerful method to detect protein-protein interactions as well as to derive genome-wide protein interaction maps. More recently, three-hybrid assays have emerged as a means to detect both protein-RNA and protein-small molecule interactions. Despite the routine use of the two-hybrid assay and the potential of three-hybrid systems, there has been little quantitative characterization to understand how the strength of the protein interaction correlates with transcription activation. It is not known if the additional interaction in three-hybrid systems compromises the sensitivity of the system. Thus, here, we set out to determine the K(D) cutoff of a small molecule three-hybrid system and to determine if there is a correlation between the K(D) and the levels of transcription activation. A series of mutations to FK506-binding protein 12 (FKBP12) were designed to vary the affinity of this protein for the small molecule synthetic ligand for FK506-binding protein 12 (SLF). These FKBP12 variants were overexpressed and purified, and their K(D)'s for SLF were measured using a fluorescence polarization assay. Then the levels of transcription activation in a Mtx-DHFR yeast three-hybrid system were determined for these variants using a lacZ reporter gene. The K(D) cutoff of the Mtx yeast three-hybrid system is found to be ca. 50 nM. Further, the levels of transcription activation correlate with the strength of the binding interaction, though the dynamic range is only 1 order of magnitude. These results establish that the three-hybrid assay has the requisite sensitivity for drug discovery. However, the small dynamic range highlights a limitation to equilibrium-based assays for discriminating interactions based on affinity.

Escherichia coli↗
PubMed2002

Receptor-dependence of the transcription read-out in a small-molecule three-hybrid system.

Small-molecule three-hybrid systems show promise as an in vivo alternative to affinity chromatography for detecting small-molecule-protein interactions. While several three-hybrid systems have been reported, little has been done to characterize these systems and, in particular, to test the assumption that the protein-small-molecule interaction can be varied without disrupting the transcription read-out. Recently we reported a dexamethasone-methotrexate chemical inducer of dimerization (CID) for use in the yeast three-hybrid system, based on the well-studied ligand-receptor pairs dexamethasone (Dex)-glucocorticoid receptor (GR) and methotrexate (Mtx)-dihydrofolate reductase (DHFR). Here we describe our first efforts to characterize this system, by focusing on a comparison of the activity of a bacterial and a mammalian DHFR as a test case of the influence of the ligand-receptor pair on the transcription read-out. By using a lacZ reporter gene, the activity of several GR and DHFR protein chimeras with different orientations and linker sequences and Dex-Mtx CIDs with different chemical linkers have been compared. In addition, Western analyses and in vivo biochemical assays have been carried out to confirm the integrity of the GR and DHFR protein chimeras. The transcription read-out is found to be much more sensitive to the structure of the protein chimeras than the CID. The most surprising result is that the levels of transcription activation are consistently higher with the bacterial than the mammalian DHFR, despite the fact that both proteins bind Mtx with an inhibition constant (K(I)) in the low pM range. These results set the stage for understanding three-hybrid systems at the biochemical level so that they can be used to detect ligand-receptor pairs with a range of structures and dissociation constants.

Alcohol Oxidoreductases↗