Terentis, Andrew C.

Methionine-γ-lyase (MGL) is a pyridoxal-5′-phosphate (PLP) dependent enzyme found in bacteria and protozoa that catalyzes a variety of reactions, including the γ-elimination of L-methionine (L-Met). Besides its physiological roles in organisms, MGL is also a therapeutic target for pathogenic diseases and cancer. Since MGL’s catalytic mechanism remains uncertain, a new spectrophotometric assay was validated for measuring the kinetics of MGL catalyzed reactions. Kinetic data and density functional theory (DFT) data for the γ-elimination reaction of L-Met and several other substrate analogues by MGL from P. gingivalis is reported. A direct correlation between experimental kcat values and DFT-calculated activation energies of the γ-cleavage reaction of the enamine intermediate was identified for various substrates. Considering these data, we propose a catalytic mechanism for MGL catalysis, whereby the γ-cleavage step is rate-limiting. This conclusion has direct implications for the rational design of substrates or inhibitors aimed at regulating MGL activity.
PLP-dependent enzymes are present in all organisms and account for ~4% of all discovered enzyme activities. They all possess a common external aldimine and quinonoid intermediate at the start of their reaction pathway but likely diverge thereafter into different reaction pathways based on their substrate(s) and active site environment. To yield insight into the divergent reactivity of the quinonoid intermediate, DFT calculations of several PLP-dependent enzymes with unique reactivity revealed that the charge distribution is modulated on Cα and C4’, which allows for the regioselectivity of the quinonoid intermediate.
The mammalian heme enzyme indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes the first reaction of L-tryptophan (L-Trp) oxidation along the kynurenine pathway. IDO1 is a central immunoregulatory enzyme with important implications for inflammation, infectious disease, autoimmune disorders, and cancer. Since IDO1’s catalytic mechanism remains uncertain, kinetic data and DFT data for the dioxygenation reaction of L-Trp and several other substrate analogues by human IDO1 is reported. A direct correlation between experimental kcat values and DFT-calculated activation energies of the C2-O cleavage reaction of the L-Trp-epoxide intermediate was identified for various substrates. This conclusion has direct implications for the rational design of substrates or inhibitors aimed at regulating IDO1 activity and yields insight into heme-dependent dioxygenation chemistry.

FAU's Office of Undergraduate Research and Inquiry hosts an annual symposium where students engaged in undergraduate research may present their findings either through a poster presentation or an oral presentation.

Cell penetrating peptides (CPPs) are short sequences of amino acids that excel in
crossing the cellular membrane without inducing cytotoxicity Interest in these peptides
stem from their ability to be attached, and grant their penetrating properties to, a variety
of cargo In this work we have combined the application of Confocal Raman Microscopy
(CRM) and Atomic Force Microscopy for the first time to examine the interactions of
unlabeled Transportan (TP), one of the most well studied CPPs, with mammalian cells
CRM’s capability to discriminate control and treated cell groups was verified by principal
component analysis (PCA) and linear discriminant analysis (LDA) and was 93-100%
accurate We’ve determined that at a concentration of 20 μM TP enters cells through a
non-endocytotic mechanism, has a high affinity for the cytoplasm and membranes, and
results in a significant increase in cellular stiffness Our work provides the first direct
evidence of this cell-stiffening phenomenon SFTI-1, the smallest member of a bicyclic, cysteine rich class of CPPs, was
examined by CRM to determine the potential role of cyclic structure on cellular uptake
The peptide, along with monocyclic and linear analogs was heavy isotope labeled and
incubated with mammalian cells at numerous concentrations and timespans Our work is
the first SFTI-1 uptake study forgoing the use of fluorophore conjugates, which have
been linked to artificial cellular uptake We demonstrate herein the absence of any CRM
detectable uptake, providing the first evidence that SFTI-1 may not be a CPP
Finally, CRM was applied to the discrimination of normal and basal cell
carcinoma cells obtained from the same donor The use of patient matched cells avoids
the normal biochemical variations that exist among individuals, ensuring that
discrimination is based solely on the cell’s diseased state CRM spectra, analyzed by
PCA and LDA, were capable of spectral discrimination with 100% accuracy Major
differences in the cancerous cells were an increase in lipids and nucleic acids, and an
overall decrease in protein We also demonstrate an enhancement in Raman signal
through the use of an aluminum foil substrate, providing a practical approach for
measuring cells with thin morphologies

G-quadruplexes (G4s) are nucleic acid structures formed from π-stacked planar sets of four Hoogsteen hydrogen bonded guanine bases. G4s emerged as potential therapeutic targets based on their ability to modulate gene expression and inhibit the ability of telomerase to elongate chromosomal telomeres. Raman spectroscopy, polarized Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), and other optical spectroscopic techniques were used to characterize the G4s formed by four different DNA sequences: human telomeric (HT), thrombin-binding aptamer (TBA), nuclease hypersensitive element III1 region of the c- Myc gene promoter (Myc), and a single loop-isomer of Myc (MycL1).

The heme enzyme indoleamine 2,3-dioxygenase (IDO) catalyses L-tryptophan (LTrp) oxidation along the kynurenine pathway and is a key regulator of the mammalian immune system. It’s unknown if the enzyme is under redox control and the use of new potent inhibitors of IDO represents a novel therapeutic approach.

Skin cancer is a growing health concern worldwide. In this work, confocal Raman micro-spectroscopy (CRM) was combined with two types of multivariate analysis, principal component analysis (PCA) and linear discriminant analysis (LDA), to accurately differentiate between skin cancer and normal skin. CRM was employed to study three distinct intracellular regions – cytoplasm, nucleoplasm, and nucleolus – within human metastatic melanoma (SK) and skin fibroblast (BJ) cells. PCA/LDA was 92-98% successful in discriminating BJ from SK cells, with higher RNA identified in the nucleoli of BJ cells and higher lipids and collagen identified in the cytoplasm of SK cells. CRM measurements were also done on SCC, BCC, and normal skin tissue samples to determine the feasibility of combining Raman spectroscopy with CO2 ablation. Differentiation with PCA was possible between normal and SCC tissue that had been ablated, with 78% correct identification when non-ablated and 92% when ablated.