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.

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.

Phosphonate natural products have proven value to society as antibiotics and herbicides. They inhibit a range of enzyme targets usually by mimicking the enzyme substrates. In this study, we investigate a family of phosphonate biosynthetic gene clusters (BGCs) found in Burkholderia. Heterologous expression in Escherichia coli resulted in production of an antimicrobial compound. Spectroscopic characterization and chemical synthesis assigned its structure as 2,4-dioxopentylphosphonic acid. One of the biosynthetic enzymes is a member of the domain of unknown function (DUF) 849 family with homology to β-keto acid cleavage enzymes (BKACEs). In vitro characterization shows that this enzyme catalyzes chemistry that is divergent from previously characterized BKACEs. The observed catalytic activity is explained by a series of cocrystal structures with substrates and intermediates. The BGC also contains a gene encoding lumazine synthase (LS), an essential enzyme in flavin biosynthesis. Biochemical experiments revealed that 2,4-dioxopentylphosphonic acid inhibits LS. In addition, expression of the LS encoded in the BGC, or LS orthologs from a range of organisms, in E. coli conferred resistance to the new phosphonate, which we therefore name flavophos.