Brian O. Bachmann

A genomics-guided approach for discovering and expressing cryptic metabolic pathways

Genome analysis of actinomycetes has revealed the presence of numerous cryptic gene clusters encoding putative natural products. These loci remain dormant until appropriate chemical or physical signals induce their expression. Here we demonstrate the use of a high-throughput genome scanning method to detect and analyze gene clusters involved in natural-product biosynthesis. This method was applied to uncover biosynthetic pathways encoding enediyne antitumor antibiotics in a variety of actinomycetes. Comparative analysis of five biosynthetic loci representative of the major structural classes of enediynes reveals the presence of a conserved cassette of five genes that includes a novel family of polyketide synthase (PKS). The enediyne PKS (PKSE) is proposed to be involved in the formation of the highly reactive chromophore ring structure (or “warhead”) found in all enediynes. Genome scanning analysis indicates that the enediyne warhead cassette is widely dispersed among actinomycetes. We show that selective growth conditions can induce the expression of these loci, suggesting that the range of enediyne natural products may be much greater than previously thought. This technology can be used to increase the scope and diversity of natural-product discovery.

Structure of beta-lactam synthetase reveals how to synthesize antibiotics instead of asparagine.

The enzyme β-lactam synthetase (β-LS) catalyzes the formation of the β-lactam ring in clavulanic acid, a clinically important β-lactamase inhibitor. Whereas the penicillin β-lactam ring is generated by isopenicillin N synthase (IPNS) in the presence of ferrous ion and dioxygen, β-LS uses ATP and Mg2+ as cofactors. According to sequence alignments, β-LS is homologous to class B asparagine synthetases (AS-Bs), ATP/Mg2+-dependent enzymes that convert aspartic acid to asparagine. Here we report the first crystal structure of a β-LS. The 1.95 Å resolution structure of Streptomyces clavuligerus β-LS provides a fully resolved view of the active site in which substrate, closely related ATP analog α,β-methyleneadenosine 5′-triphosphate (AMP-CPP) and a single Mg2+ ion are present. A high degree of substrate preorganization is observed. Comparison to Escherichia coli AS-B reveals the evolutionary changes that have taken place in β-LS that impede interdomain reaction, which is essential in AS-B, and that accommodate β-lactam formation. The structural data provide the opportunity to alter the synthetic potential of β-LS, perhaps leading to the creation of new β-lactamase inhibitors and β-lactam antibiotics.

The catalytic cycle of β-lactam synthetase observed by x-ray crystallographic snapshots

The catalytic cycle of the ATP/Mg2+-dependent enzyme β-lactam synthetase (β-LS) from Streptomyces clavuligerus has been observed through a series of x-ray crystallographic snapshots. Chemistry is initiated by the ordered binding of ATP/Mg2+ and N2-(carboxyethyl)-l-arginine (CEA) to the apoenzyme. The apo and ATP/Mg2+ structures described here, along with the previously described CEA⋅α,β-methyleneadenosine 5′-triphosphate (CEA⋅AMP-CPP)/Mg2+ structure, illuminate changes in active site geometry that favor adenylation. In addition, an acyladenylate intermediate has been trapped. The substrate analog N2-(carboxymethyl)-l-arginine (CMA) was adenylated by ATP in the crystal and represents a close structural analog of the previously proposed CEA-adenylate intermediate. Finally, the structure of the ternary product complex deoxyguanidinoproclavaminic acid (DGPC)⋅AMP/PPi/Mg2+ has been determined. The CMA-AMP/PPi/Mg2+ and DGPC⋅AMP/PPi/Mg2+ structures reveal interactions in the active site that facilitate β-lactam formation. All of the ATP-bound structures differ from the previously described CEA⋅AMP-CPP/Mg2+ structure in that two Mg2+ ions are found in the active sites. These Mg2+ ions play critical roles in both the adenylation and β-lactamization reactions.

Phosphonopeptide K-26 biosynthetic intermediates in Astrosporangium hypotensionis

Precursors and advanced intermediates for phosphonopeptide K-26 biosynthesis were synthesized and incorporation studies in Astrosporangium hypotensionis suggest a new mechanism of C-P bond formation in aromatic phosphonates.