Background: A hierarchy of catalytic steps characterizes multifunctional cytochrome P450 enzymes. Results: In the post-polyketide oxidative tailoring of mycinamicins by MycG, the two methoxy groups of mycinose are sensors that mediate initial recognition and discriminate between closely related molecules. Conclusion: Bulky and conformationally restrained macrolide substrates advance to the catalytically productive mode through multiple steps. Significance: Protein engineering facilitating substrate progression may enhance catalysis.
Cytochrome P450 monooxygenases typically catalyze the insertion of one atom of oxygen from O2 into unactivated carbon-hydrogen and carbon-carbon bonds, with concomitant reduction of the other oxygen atom to H2O by NAD(P)H. Comparison of the average structures of the camphor hydroxylase cytochrome P450cam (CYP101) obtained from residual dipolar coupling (RDC)-restrained molecular dynamics (MD) in the presence and absence of substrate camphor shows structural displacements resulting from the essential collapse of the active site upon substrate removal. This collapse has conformational consequences that extend across the protein structure, none of which were observed in analogous crystallographic structures. Mutations were made to test the involvement of the observed conformational changes in substrate binding and recognition. All of the mutations performed based upon the NMR-detected perturbations, even those remote from the active site, resulted in modified substrate selectivity, enzyme efficiency and/or haem iron spin state. The results demonstrate that solution NMR can provide insights into enzyme structure-function relationships that are difficult to obtain by other methods.
Neuronopathic glycosphingolipidoses are a sub-group of lysosomal storage disorders for which there are presently no effective therapies. Here, we evaluated the potential of substrate reduction therapy (SRT) using an inhibitor of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide (GL1) and related glycosphingolipids. The substrates that accumulate in Sandhoff disease (e.g., ganglioside GM2 and its nonacylated derivative, lyso-GM2) are distal to the drug target, GCS. Treatment of Sandhoff mice with a GCS inhibitor that has demonstrated CNS access (Genz-682452) reduced the accumulation of GL1 and GM2, as well as a variety of diseaseassociated substrates in the liver and brain. Concomitant with these effects was a significant decrease in the expression of CD68 and glycoprotein non-metastatic melanoma B protein (Gpnmb) in the brain, indicating a reduction in microgliosis in the treated mice. Moreover, using in vivo imaging, we showed that the monocytic biomarker translocator protein (TSPO), which was elevated in Sandhoff mice, was normalized following Genz-682452 treatment. These positive effects translated in turn into a delay ($28 days) in loss of motor function and coordination, as measured by rotarod latency, and a significant increase in longevity ($17.5%). Together, these results support the development of SRT for the treatment of gangliosidoses, particularly in patients with residual enzyme activity.
MycG is a P450 monoxygenase that catalyzes the sequential hydroxylation and epoxidation of mycinamicin IV (M-IV), the last two steps in the biosynthesis of mycinamicin II, a macrolide antibiotic isolated from M. griseorubida. The crystal structure of MycG with M-IV bound was previously determined, but showed the bound substrate in an orientation that did not rationalize the observed regiochemistry of M-IV hydroxylation. NMR paramagnetic relaxation enhancements (PRE) gave evidence for an orientation of M-IV in the MycG active site more compatible with the observed chemistry, but substrate-induced changes in the enzyme structure were not characterized. We now describe the use of amide 1H-15N residual dipolar couplings (RDCs) as experimental restraints in solvated “soft annealing” molecular dynamics simulations to generate solution structural ensembles of M-IV-bound MycG. Chemical shift perturbations, hydrogen-deuterium exchange and 15N relaxation behavior provide insight into dynamic and electronic perturbations in the MycG structure in response to M-IV binding. The solution and crystallographic structures are compared, and the possibility that the crystallographic orientation of bound M-IV represents an inhibitory mode is discussed.
Cytochrome P450 monooxygenases CYP101A1 and MycG catalyze regio- and stereospecific oxidations of their respective substrates, d-camphor and mycinamicin IV. Despite the low sequence homology between the two enzymes (29% identity) and differences in size and hydrophobicity of their substrates, the conformational changes that occur upon substrate binding in both enzymes as determined by solution NMR methods show some striking similarities. Many of the same secondary structural features in both enzymes are perturbed, suggesting the existence of a common mechanism for substrate binding and recognition in the P450 superfamily.
SLC6A19 (B0AT1) is a neutral amino acid transporter, the loss of function of which results in Hartnup disease. SLC6A19 is also believed to have an important role in amino acid homeostasis, diabetes, and weight control. A small-molecule inhibitor of human SLC6A19 (hSLC6A19) was identified using two functional cell-based assays: a fluorescence imaging plate reader (FLIPR) membrane potential (FMP) assay and a stable isotope-labeled neutral amino acid uptake assay. A diverse collection of 3440 pharmacologically active compounds from the Microsource Spectrum and Tocriscreen collections were tested at 10 µM in both assays using MDCK cells stably expressing hSLC6A19 and its obligatory subunit, TMEM27. Compounds that inhibited SLC6A19 activity in both assays were further confirmed for activity and selectivity and characterized for potency in functional assays against hSLC6A19 and related transporters. A single compound, cinromide, was found to robustly, selectively, and reproducibly inhibit SLC6A19 in all functional assays. Structurally related analogs of cinromide were tested to demonstrate structure–activity relationship (SAR). The assays described here are suitable for carrying out high-throughput screening campaigns to identify modulators of SLC6A19.
Certain recessively inherited diseases result from an enzyme deficiency within lysosomes. In mucopolysaccharidoses (MPS), a defect in glycosaminoglycan (GAG) degradation leads to GAG accumulation followed by progressive organ and multiple system dysfunctions. Current methods of GAG analysis used to diagnose and monitor the diseases lack sensitivity and throughput. Here we report a LC-MS method with accurate metabolite mass analysis for identifying and quantifying biomarkers for MPS type I without the need for extensive sample preparation. The method revealed 225 LC-MS features that were >1000-fold enriched in urine, plasma and tissue extracts from untreated MPS I mice compared to MPS I mice treated with iduronidase to correct the disorder. Levels of several trisaccharides were elevated >10000-fold. To validate the clinical relevance of our method, we confirmed the presence of these biomarkers in urine, plasma and cerebrospinal fluid from MPS I patients and assessed changes in their levels after treatment.
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