Department of Biochemistry

Alzheimer's disease (AD) is characterized by metabolic dysfunctions, making cerebrospinal fluid (CSF) an ideal biological matrix for characterizing brain metabolism. Very high-field 900 MHz nuclear magnetic resonance (NMR) spectroscopy was used to profile CSF metabolites from 47 patients (n = 27 AD, n = 20 non-AD). A total of forty-eight metabolites were identified and quantified. CSF concentrations of four nonpolar amino acids were reduced in AD: l-alanine (p = 0.001), l-valine (p = 0.02), l-leucine (p = 0.04), and l-phenylalanine (p = 0.04) (two-way ANOVA, pathology and sex as factors). No significant differences were observed for the other identified metabolites. A nonpolar amino acid factor (NAAF) was computed as the sum of these four metabolites (p < 0.001). Partial least-squares discriminant analysis (PLS-DA) based on the NAAF yielded moderate group separation (accuracy = 72%, Q² = 0.22, p = 0.006). Receiver operating characteristic (ROC) analysis demonstrated that the NAAF achieved the highest discriminatory performance over individual nonpolar amino acids (AUC = 0.83). l-Alanine correlated negatively with CSF tau markers (p-tau: r = -0.40, p = 0.005; t-tau: r = -0.42, p = 0.003); the NAAF displayed similar patterns (p-tau: r = -0.38, p = 0.009; t-tau: r = -0.40, p = 0.006). Very high-field NMR spectroscopy revealed a depletion of nonpolar amino acids in AD CSF, reflecting metabolic dysregulation in this neurodegenerative disease.

The ADP-ribosylation of proteins is a versatile, reversible, posttranslational modification involved in the dynamic regulation of numerous cellular processes. Human ADP-ribosyltransferase 1 ( hs ART1, EC: 2.4.2.31) is a membrane-associated, GPI-anchored, mono-ADP-ribosyltransferase selective for mono-ADP ribosylation (MARylation) of L-arginine residues. Dysregulation of hs ART1 activity has been shown to permit immune cell evasion in non–small cell lung cancer (NSCLC) through elevated MARylation at Arg125 of the purinergic type 2 receptor (P2X7) in P2X7-positive T cells, resulting in NAD + -induced cell death (NICD) of tumor-penetrating immune cells. With hs ART1 emerging as an immunotherapy target in select cancers, there is a need to develop small-molecule inhibitors. The transition state (TS) for the MARylation of P2X7 peptide was determined from kinetic isotope effect (KIE) measurements of 3 H-, 14 C-, 18 O-, and 15 N-labeled NAD + substrates. Quantum mechanical (QM) calculations of the reaction coordinate, mapped with experimental KIEs, identify a TS geometry consistent with a highly dissociative, asymmetric, concerted mechanism with minimal contributions from the leaving group nicotinamide (NAM) and minor contributions from the incoming L-arginine guanidinium. The absence of a normal deuterium solvent isotope effect identifies a positively charged guanidinium nucleophile, leading to a dication N-ribosyltransferase mechanism. Together with the unusual, normal 18 O–O4 ′ KIE, we identified unique charge accumulation across the oxocarbenium at the TS caused by an increased bond order between the C1 ′ –C2 ′ , and decreased bond order between the C4 ′ –O4 ′ of the nicotinamide mononucleotide ribose. This is the first L-arginine-specific ADP-ribosylation TS to be characterized, a step toward the design of TS analogs.

Flavin-dependent halogenases (FDHs) provide a biocatalytic approach for the site-selective halogenation of aromatic compounds, but their use in late-stage functionalization of peptides has remained limited. Here, we show that tryptophan (Trp) 7-halogenase RebH and an engineered variant (4V) originally optimized for larger small-molecule scaffolds can brominate peptidyl-Trp residues across a broad range of sequences and positional contexts. Through extensive analysis of diverse substrate sequences, we define features that enable RebH activity and reveal 4V's expanded sequence tolerance. We applied 4V for enzymatic bromination of diverse bioactive peptide scaffolds, including an antimicrobial peptide, a cell-penetrating peptide, and a G protein-coupled receptor agonist, without the need for sequence modification. These brominated peptides served as substrates for Suzuki-Miyaura coupling, enabling the installation of modifications that conferred new functional properties or tuned the biological activity of these peptides. Our results expand the substrate landscape of FDHs and establish bromination-enabled cross-coupling as a general approach for late-stage diversification of bioactive peptides.