The structure of L-aspartate-alpha-decarboxylase from E. coli has been determined at 2.2 A resolution. The enzyme is a tetramer with pseudofour-fold rotational symmetry. The subunits are six-stranded beta-barrels capped by small alpha-helices at each end. The active sites are located between adjacent subunits. The electron density provides evidence for catalytic pyruvoyl groups at three active sites and an ester at the fourth. The ester is an intermediate in the autocatalytic self-processing leading to formation of the pyruvoyl group. This unprecedented structure provides novel insights into the general phenomenon of protein processing.
Drug discovery faces economic and scientific imperatives to deliver lead molecules rapidly and efficiently. Using traditional paradigms the molecular design, synthesis, and screening loops enforce a significant time delay leading to inefficient use of data in the iterative molecular design process. Here, we report the application of a flow technology platform integrating the key elements of structure-activity relationship (SAR) generation to the discovery of novel Abl kinase inhibitors. The platform utilizes flow chemistry for rapid in-line synthesis, automated purification, and analysis coupled with bioassay. The combination of activity prediction using Random-Forest regression with chemical space sampling algorithms allows the construction of an activity model that refines itself after every iteration of synthesis and biological result. Within just 21 compounds, the automated process identified a novel template and hinge binding motif with pIC50 > 8 against Abl kinase--both wild type and clinically relevant mutants. Integrated microfluidic synthesis and screening coupled with machine learning design have the potential to greatly reduce the time and cost of drug discovery within the hit-to-lead and lead optimization phases.
The Escherichia coli panD gene, encoding l-aspartate-alpha-decarboxylase, was cloned by PCR, and shown to complement a panD mutant defective in beta-alanine biosynthesis. Aspartate decarboxylase is a pyruvoyl-dependent enzyme, and is synthesized initially as an inactive proenzyme (the pi-protein), which is proteolytically cleaved at a specific X-Ser bond to produce a beta-subunit with XOH at its C-terminus and an alpha-subunit with a pyruvoyl group at its N-terminus, derived from the serine. The recombinant enzyme, as purified, is a tetramer, and comprises principally the unprocessed pi-subunit (of 13.8 kDa), with a small proportion of the alpha- and beta-subunits (11 kDa and 2.8 kDa respectively). Incubation of the purified enzyme at elevated temperatures for several hours results in further processing. Using fluorescein thiosemicarbazide, the completely processed enzyme was shown to contain three pyruvoyl groups per tetrameric enzyme. The presence of unchanged serine at the N-terminus of some of the alpha-subunits was confirmed by electrospray mass spectrometry (ESMS) and N-terminal amino acid sequencing. A novel HPLC assay for aspartate decarboxylase was established and used to determine the Km and kcat for l-aspartate as 151+/-16 microM and 0.57 s-1 respectively. ESMS was also used to observe substrate and product adducts trapped on the pyruvoyl group by sodium cyanoborohydride treatment.
Chorismate synthase (EC 4.6.1.4) is the seventh enzyme of the shikimate pathway (1, 2) and catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate 1 (EPSP) 1 to chorismate 2 (Scheme 1). Chorismate is a common intermediate in the biosynthesis of aromatic metabolites such as aromatic amino acids and folate cofactors. The chorismate synthase reaction involves the 1,4-elimination of phosphate and the C-(6proR) hydrogen with overall anti-stereochemistry (3-5). A concerted elimination appears to be unlikely since studies with model systems (6, 7) and molecular orbital considerations (8, 9) suggest that concerted 1,4-eliminations with syn-stereochemistry are favored. For these reasons, several non-concerted mechanisms have been suggested for the chorismate synthase reaction (10). Proposed mechanisms include an X-group mechanism, involving the nucleophilic attack of the C-1 position of the substrate (5) (Scheme 2d), and a cationic mechanism, involving the step-wise loss of phosphate and a proton (11, 12) (Scheme 2a). An allylic rearrangement of phosphate followed by a 1,2-elimination (13) has been discounted because the intermediate iso-EPSP is a competitive inhibitor rather than a substrate of the Neurospora crassa enzyme (14).Reduced FMN is required for enzyme activity despite there being no overall reduction or oxidation in the conversion of EPSP to chorismate (15)(16)(17)(18)(19). The role of flavin is not yet clear but it appears to be directly involved in catalysis. A transient flavin intermediate has been characterized by uv/visible spectroscopy during single and multiple turnover experiments using the enzyme from Escherichia coli (20,21). It has been suggested that this spectrum is consistent with a charge transfer complex or a C-4a-flavin adduct (20). However, such a perturbation of the reduced flavin spectrum could, at least in part, be due to noncovalent interactions between the substrate and the flavin. The absence of detectable activity of the N. crassa (22) and E. coli (23) enzymes reconstituted with reduced 5-deaza-FMN provides additional evidence that reduced flavin is chemically, and not just structurally, involved in turnover. In the presence of the inhibitor (6R)-6-fluoro-EPSP the E. coli enzyme forms a protein-bound flavin semiquinone, suggesting the possibility of radical intermediates during normal turnover (24). A radical mechanism involving the initial abstraction of a hydrogen atom from the C-6 position of the substrate (11, 22, 25) seems unlikely since there are no obvious candidates for a catalytic center that can accept single electrons, such as transition metal ions (24), particularly given the requirement for fully reduced flavin for activity.
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