Andrew C. Eliot

Cloning, Expression, and Biochemical Characterization of Streptomyces rubellomurinus Genes Required for Biosynthesis of Antimalarial Compound FR900098

The antibiotics fosmidomycin and FR900098 are members of a unique class of phosphonic acid natural products that inhibit the nonmevalonate pathway for isoprenoid biosynthesis. Both are potent antibacterial and antimalarial compounds, but despite their efficacy, little is known regarding their biosynthesis. Here we report the identification of the Streptomyces rubellomurinus genes required for the biosynthesis of FR900098. Expression of these genes in Streptomyces lividans results in production of FR900098, demonstrating their role in synthesis of the antibiotic. Analysis of the putative gene products suggests that FR900098 is synthesized by metabolic reactions analogous to portions of the tricarboxylic acid cycle. These data greatly expand our knowledge of phosphonate biosynthesis and enable efforts to overproduce this highly useful therapeutic agent.

Biosynthesis of 2-hydroxyethylphosphonate, an unexpected intermediate common to multiple phosphonate biosynthetic pathways.

Phosphonic acids encompass a common yet chemically diverse class of natural products that often possess potent biological activities. Here we report that, despite the significant structural differences among many of these compounds, their biosynthetic routes contain an unexpected common intermediate, 2-hydroxyethyl-phosphonate, which is synthesized from phosphonoacetaldehyde by a distinct family of metal-dependent alcohol dehydrogenases (ADHs). Although the sequence identity of the ADH family members is relatively low (34–37%), in vitro biochemical characterization of the homologs involved in biosynthesis of the antibiotics fosfomycin, phosphinothricin tripeptide, and dehydrophos (formerly A53868) unequivocally confirms their enzymatic activities. These unique ADHs have exquisite substrate specificity, unusual metal requirements, and an unprecedented monomeric quaternary structure. Further, sequence analysis shows that these ADHs form a monophyletic group along with additional family members encoded by putative phosphonate biosynthetic gene clusters. Thus, the reduction of phosphonoacetaldehyde to hydroxyethyl-phosphonate may represent a common step in the biosynthesis of many phosphonate natural products, a finding that lends insight into the evolution of phosphonate biosynthetic pathways and the chemical structures of new C–P containing secondary metabolites.

Molecular Cloning and Heterologous Expression of the Dehydrophos Biosynthetic Gene Cluster

Dehydrophos is a vinyl phosphonate tripeptide produced by Streptomyces luridus with demonstrated broad-spectrum antibiotic activity. To identify genes necessary for biosynthesis of this unusual compound we screened a fosmid library of S. luridus for the presence of the phosphoenolpyruvate mutase gene, which is required for biosynthesis of most phosphonates. Integration of one such fosmid clone into the chromosome of S. lividans led to heterologous production of dehydrophos. Deletion analysis of this clone allowed identification of the minimal contiguous dehydrophos cluster, which contained 17 open reading frames (ORFs). Bioinformatic analyses of these ORFs are consistent with a proposed biosynthetic pathway that generates dehydrophos from phosphoenolpyruvate. The early steps of this pathway are supported by analysis of intermediates accumulated by blocked mutants and in vitro biochemical experiments.

Structure of ACC synthase inactivated by the mechanism-based inhibitor l-vinylglycine

l‐Vinylglycine (l‐VG) is both a substrate for and a mechanism‐based inhibitor of 1‐aminocyclopropane‐1‐carboxylate (ACC) synthase. The ratio of the rate constants for catalytic conversion to α‐ketobutyrate and ammonia to inactivation is 500/1. The crystal structure of the covalent adduct of the inactivated enzyme was determined at 2.25 Å resolution. The active site contains an external aldimine of the adduct of l‐VG with the pyridoxal 5′‐phosphate cofactor. The side chain γ‐carbon of l‐VG is covalently bound to the ε‐amino group of Lys273. This species corresponds to one of the two alternatives proposed by Feng and Kirsch [Feng, L. and Kirsch, J.F. (2000) l‐Vinylglycine is an alternative substrate as well as a mechanism‐based inhibitor of 1‐aminocyclopropane‐1‐carboxylate synthase. Biochemistry 39, 2436–2444] and presumably results from Michael addition to a vinylglycine ketimine intermediate.

Structural basis for reduced activity of 1-aminocyclopropane-1-carboxylate synthase affected by a mutation linked to andromonoecy.

1‐aminocyclopropane‐1‐carboxylate synthase (ACS) is a key enzyme in the biosynthesis of the plant hormone ethylene. Recently, a new biological role for ACS has been found in Cucumis melo where a single point mutation (A57V) of one isoform of the enzyme, causing reduced activity, results in andromonoecious plants. We present here a straightforward structural basis for the reduced activity of the A57V mutant, based on our work on Malus domestica ACS, including a new structure of the unliganded apple enzyme at 1.35 Å resolution.