The Allen Lab employs biochemical, biophysical, and bioinformatic/computational techniques towards projects including: enzyme evolution, enzyme promiscuity vs. specificity, and drug discovery. We study superfamilies of enzymes involved in phosphoryl (haloalkanoate dehalogenase superfamily (HADSF)) and phosphoglycosyl (phosphoglycosyl transferase (PGT)) superfamily) transfer. We probe how members have evolved their architecture to use the same chemistry to act upon diverse substrates (HADSF) or evolved high specificity for very similar substrates (PGTs). Within drug discovery, we research enzymes involved in metabolic disorders including hereditary fructose intolerance (KHK and AMPD), congenital defects of glycosylation, and galactosemia (PMM2), and diseases modulated by inhibition of protein-protein interactions involved in transcriptional regulation pathways, including oxidative stress pathways (KEAP1) and NF-κB pathway (NEMO) involved in inflammatory response. Through these diverse research projects, we center around the common theme of elucidating mechanistic and structural foundations and implications of these systems.
Human AMP deaminase is an enzyme involved in the fructose metabolism pathway. During fructose consumption, ATP gets depleted and metabolized into AMP. This pathway ultimately leads to uric acid, which is involved in metabolic syndrome, renal and liver failure.
This enzyme can be used as a target for individuals with fructose-consumption related diseases such as hereditary fructose intolerance and metabolic syndrome. Using X-ray crystallography and kinetic analysis, we aim to determine the structure of this enzyme for structure-activity relationship studies to find specific potent inhibitors.
in collaboration with Dean Tolan at Boston University
Phosphomannomutases (PMMs) catalyze the conversion of mannose 6-phosphate to mannose 1-phosphate in the N-linked glycosylation pathway. Mutations or inhibition of PMM2 cause congenital defects of glycosylation, which leads decreased glycoconjugates, as well as developmental abnormalities.
We are interested in questions regarding specificity and divergent physiological roles of the two human PMM isoforms. We are using in vitro kinetic assays, X-ray crystallography, and bioinformatic techniques to answer these questions.
KEAP1 regulates oxidative stress response by sequestering transcription factor Nrf2 under basal conditions.
A series of cyclic peptide inhibitors have been designed based on the binding loop of Nrf2. The binding affinity of each peptide was tested using fluorescence anisotropy, and structures of each bound to KEAP1 were obtained using X-ray crystallography. Despite large variances in binding affinity, we observed few changes in the bound conformation.
in collaboration with Adrian Whitty at Boston University
X-ray crystal structure of the Kelch domain of KEAP1 (wheat) bound to the binding loop of Nrf2 (gray).
NF-κB Essential Modulator (NEMO) activates the NF-κB pathway responsible for immune and inflammatory response. IKKβ binding to NEMO triggers a conformational change and compaction of NEMO by 50 Å. We are currently using Small-Angle X-ray Scattering (SAXS) and X-ray crystallography to further understand this conformational change and role of intervening domain (IVD) on downstream processes.
in collaboration with Adrian Whitty and Tom Gilmore at Boston University
Bacterial glycosylation pathways proceed in an en bloc mechanism, sequentially building a glycan chain on a membrane resonant prenyl diphosphate carrier. Within this pathway, we are interested in questions about sugar and glycan specificity with respects to individual enzymes in the pathway, and pathway throughput. We also aim to elucidate the binding determinants and specificity for the prenyl phosphate carriers.
in collaboration with Barbara Imperiali at MIT
PDB: 5W7L
C. consisus PglC
Positive funnel for phosphate-rich substrates
Glycan length preference of glycosyltransferases
Coming soon...
Application of various machine learning techniques (including PLSR, VIP, PCA, and PLSDA) has facilitated deeper analysis of structures obtained by X-ray crystallography. Partial least squares regression (PLSR) analysis of our bound structures of KEAP1 elucidated the effects of strain and pre-organization on the binding of our cyclic peptide inhibitors.
C. jejuni
