Discovery and biosynthesis of karnamycins as angiotensin-converting enzyme inhibitors

Cardiovascular diseases are the leading global cause of death, with a large burden in developing countries and a strong association with hypertension. ACE, a zinc-dependent dipeptide carboxypeptidase, increases blood pressure by producing angiotensin II and degrading bradykinin; thus ACE inhibition is an established therapeutic strategy. Current ACE inhibitors such as captopril and enalapril can cause side effects, motivating the search for new inhibitors from microbial natural products. Pyridine and thiazole motifs are prevalent in drugs and form the core of karnamycins, a class first isolated as weak antibiotics. This study aims to discover new karnamycins from Lechevalieria rhizospherae NEAU-A2, elucidate their biosynthesis—particularly the fully substituted hydroxypyridine and thiazole features—and evaluate their ACE inhibitory activities as potential antihypertensive leads.

Prior work established the clinical importance and side effects of existing ACE inhibitors. Pyridine, thiazole, and hybrid pyridine–thiazole structures are common in bioactive compounds and approved drugs. Karnamycins were originally isolated from Saccharothrix aerocolonigenes N800-6 (1989) and karnamycin B was totally synthesized in 1997, but little biosynthetic or pharmacological follow-up occurred. Related 2,2′-bipyridine pathways (e.g., caerulomycin, collismycin) use hybrid PKS–NRPS logic with flavoprotein monooxygenases. These precedents informed genome mining and functional hypotheses for karnamycin assembly.

- Isolation and structural elucidation: Fermentation of L. rhizophorae NEAU-A2 from wheat rhizosphere soil, extraction (EtOAc, MeOH), chromatographic purification (silica gel, semi-preparative HPLC-DAD). Structures characterized by HR-ESI-MS, 1H/13C NMR, HSQC/HMBC/COSY, and X-ray crystallography (for compound 7). - Isotopic labeling: Feeding experiments with [1-13C] and [2-13C] acetate to trace incorporation into pyridine and side-chain moieties; analysis by NMR to assign acetate units and support PKS involvement. - Genome sequencing and mining: Genome of L. rhizophorae NEAU-A2 annotated (GeneMarkS). antiSMASH and PRISM3 identified a candidate karnamycin BGC (knm) with 16 ORFs encoding PKS–NRPS components, FAD-dependent monooxygenases, amidohydrolase, methyltransferase, transporter/regulators, and dehydrogenase; similarity to known 2,2′-bipyridine clusters noted. - Genetics: Targeted gene disruptions (e.g., knm2 PKS–NRPS; knmB1, knmB2 FPMOs; knmC, knmE, knmF) via homologous recombination and Red-mediated mutagenesis in L. rhizophorae NEAU-A2 and heterologous host Streptomyces albus 5C1; verification by PCR. HPLC profiling of mutants to assign gene functions in the pathway. - Enzymology: Heterologous expression and purification of FAD-dependent monooxygenases (KmnlB/KmnB1 and KmnlB2) and methyltransferase (KmnF) in E. coli BL21(DE3). Cofactor analysis confirmed FAD binding. In vitro assays assessed regioselective hydroxylation and O-methylation on isolated intermediates (e.g., 24–27, 32–38) with NADPH and SAM as cofactors, analyzed by HPLC. - Computational studies: Multiple sequence alignments, phylogenetics, AlphaFold structure predictions of KmnB1/B2, and molecular docking of substrates/intermediates to propose active-site residues and mechanisms of regiospecific hydroxylation. - ACE inhibition assay: Compounds dissolved in DMSO, serially diluted in HEPES–NaCl buffer; incubated with ACE (0.02 U/mL) followed by substrate; absorbance at 340 nm measured. Percent inhibition plotted versus log concentration; IC50 values determined using GraphPad Prism. Captopril used as positive control.

- Discovery: Six new karnamycins E1–E6 (1–6) and several known analogs (7–11) were isolated from L. rhizophorae NEAU-A2. Structures feature fully substituted hydroxypyridine and thiazole moieties. - Biosynthetic origin: 13C acetate feeding showed intact acetate incorporation into the pyridine ring and polyketide-derived segments, implicating PKS involvement. - BGC identification: A 16-ORF knm cluster encodes a hybrid PKS–NRPS (for assembly of the core), two FAD-dependent monooxygenases, a methyltransferase, an amidohydrolase, and accessory enzymes; a similar cluster was found in Actinothelodes cygnaricus. - Functional genetics: Deletion of knm2 (PKS–NRPS) abolished karnamycin production; deletions of knmB1 and knmB2 eliminated final products and led to accumulation of hydroxylation-state-specific intermediates, enabling assignment of enzyme functions. - Enzyme functions: KmnB2 catalyzes C4- (or first) hydroxylation of the pyridine core requiring a pre-existing C5-OH; KmnB1 catalyzes C6-hydroxylation to form a trihydroxypyridine intermediate (28). KmnF performs O-methylation (notably C5-O-methylation) to stabilize intermediates. Methylation shows some substrate promiscuity leading to observed methylated intermediates (e.g., 32–33, 35–37). - Mechanism/structure: AlphaFold-based models, docking, and mutagenesis revealed that KmnB1/B2, though FPMOs, utilize distinct active-site residues compared to known aromatic hydroxylases to direct chemo- and regiospecific pyridine hydroxylation. - ACE inhibition: Multiple karnamycins and intermediates showed significant ACE inhibitory activity. Reported IC50 values include 1 (1.22 ± 0.02 μM), 2 (1.10 ± 0.09 μM), 3 (0.30 ± 0.05 μM), 4 (0.24 ± 0.01 μM), 5 (0.71 ± 0.05 μM), 6 (0.50 ± 0.03 μM), 11 (5.36 ± 2.97 μM), 25 (5.81 ± 0.47 μM), 26 (2.94 ± 0.38 μM), 34 (5.42 ± 0.10 μM), 36 (0.62 ± 0.07 μM), 37 (2.58 ± 0.30 μM), and 20 (0.14 ± 0.51 μM). Overall, activities commonly span submicromolar to low micromolar, supporting potential as antihypertensive leads.

The study addresses the need for new ACE inhibitors by discovering and characterizing karnamycins with robust in vitro ACE inhibition. Elucidation of the biosynthetic logic, including a hybrid PKS–NRPS assembly and two regiospecific FPMOs coupled with a methyltransferase, clarifies how the fully substituted hydroxypyridine core is constructed. Understanding the regiospecific hydroxylation steps and methylation provides targets for pathway engineering to tailor substitution patterns that influence ACE inhibitory potency. Structural insights from AlphaFold models and docking suggest unique active-site determinants in KmnB1/B2 that expand the known catalytic repertoire of FPMOs for pyridine hydroxylation, informing future enzyme engineering for selective oxyfunctionalization. The observed structure–activity trends (e.g., effects of side-chain hydroxylation and methylation) guide medicinal chemistry optimization of karnamycin derivatives.

This work discovers six new karnamycins from L. rhizophorae NEAU-A2, defines their biosynthetic gene cluster and assembly pathway, and identifies two unusual FAD-dependent monooxygenases (KmnB1/B2) and a methyltransferase (KmnF) that build a fully substituted hydroxypyridine core with defined regiochemistry. Several karnamycins and intermediates exhibit submicromolar to low micromolar ACE inhibitory activities, highlighting their promise as antihypertensive leads. Future research should include enzyme engineering to modulate hydroxylation/methylation patterns, heterologous production and pathway refactoring to increase yields and expand analog diversity, systematic SAR-guided medicinal chemistry, and in vivo efficacy and pharmacokinetic evaluations.

- Some intermediates (e.g., trihydroxypyridine intermediate 28) were unstable, complicating detection and in vitro characterization; lack of expected demethylated products upon kmmK deletion was attributed to instability. - In vitro assays showed limited activity for certain enzyme–substrate combinations, indicating potential substrate or cofactor context requirements not fully replicated in vitro. - Biological evaluation focused on in vitro ACE inhibition; antibacterial activity was weak and no in vivo antihypertensive studies were performed, limiting translational conclusions. - Affiliation details and comprehensive biochemical kinetics for enzymes were not provided in the excerpt; quantitative enzyme parameters remain to be established.