Molecular Basis of Bacillus subtilis ATCC 6633 Self-Resistance to the Phosphono-oligopeptide Antibiotic Rhizocticin.

Rhizocticins are phosphono-oligopeptide antibiotics that contain a toxic C-terminal ( Z) -l -2-amino-5-phosphono-3-pentenoic acid (APPA) moiety. APPA is an irreversible inhibitor of threonine synthase (ThrC), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-phospho-l-homoserine to l-threonine. ThrCs are essential for the viability of bacteria, plants, and fungi and are a target for antibiotic development, as de novo threonine biosynthetic pathway is not found in humans. Given the ability of APPA to interfere in threonine metabolism, it is unclear how the producing strain B. subtilis ATCC 6633 circumvents APPA toxicity. Notably, in addition to the housekeeping APPA-sensitive ThrC ( BsThrC), B. subtilis encodes a second threonine synthase (RhiB) encoded within the rhizocticin biosynthetic gene cluster. Kinetic and spectroscopic analyses show that PLP-dependent RhiB is an authentic threonine synthase, converting O-phospho-l-homoserine to threonine with a catalytic efficiency comparable to BsThrC. To understand the structural basis of inhibition, we determined the crystal structure of APPA bound to the housekeeping BsThrC, revealing a covalent complex between the inhibitor and PLP. Structure-based sequence analyses reveal structural determinants within the RhiB active site that contribute to rendering this ThrC homologue resistant to APPA. Together, this work establishes the self-resistance mechanism utilized by B. subtilis ATCC 6633 against APPA exemplifying one of many ways by which bacteria can overcome phosphonate toxicity.

Structure and function of phosphonoacetaldehyde dehydrogenase: the missing link in phosphonoacetate formation.

Phosphonates (C-PO3²â») have applications as antibiotics, herbicides, and detergents. In some environments, these molecules represent the predominant source of phosphorus, and several microbes have evolved dedicated enzymatic machineries for phosphonate degradation. For example, most common naturally occurring phosphonates can be catabolized to either phosphonoacetaldehyde or phosphonoacetate, which can then be hydrolyzed to generate inorganic phosphate and acetaldehyde or acetate, respectively. The phosphonoacetaldehyde oxidase gene (phnY) links these two hydrolytic processes and provides a previously unknown catabolic mechanism for phosphonoacetate production in the microbial metabolome. Here, we present biochemical characterization of PhnY and high-resolution crystal structures of the apo state, as well as complexes with substrate, cofactor, and product. Kinetic analysis of active site mutants demonstrates how a highly conserved aldehyde dehydrogenase active site has been modified in nature to generate activity with a phosphonate substrate.

Structural and mechanistic insights into C-P bond hydrolysis by phosphonoacetate hydrolase.

Bacteria have evolved pathways to metabolize phosphonates as a nutrient source for phosphorus. In Sinorhizobium meliloti 1021, 2-aminoethylphosphonate is catabolized to phosphonoacetate, which is converted to acetate and inorganic phosphate by phosphonoacetate hydrolase (PhnA). Here we present detailed biochemical and structural characterization of PhnA that provides insights into the mechanism of C-P bond cleavage. The 1.35 Å resolution crystal structure reveals a catalytic core similar to those of alkaline phosphatases and nucleotide pyrophosphatases but with notable differences, such as a longer metal-metal distance. Detailed structure-guided analysis of active site residues and four additional cocrystal structures with phosphonoacetate substrate, acetate, phosphonoformate inhibitor, and a covalently bound transition state mimic provide insight into active site features that may facilitate cleavage of the C-P bond. These studies expand upon the array of reactions that can be catalyzed by enzymes of the alkaline phosphatase superfamily.

Genetic and biochemical characterization of a pathway for the degradation of 2-aminoethylphosphonate in Sinorhizobium meliloti 1021.

A variety of microorganisms have the ability to use phosphonic acids as sole sources of phosphorus. Here, a novel pathway for degradation of 2-aminoethylphosphonate in the bacterium Sinorhizobium meliloti 1021 is proposed based on the analysis of the genome sequence. Gene deletion experiments confirmed the involvement of the locus containing phnW, phnA, and phnY genes in the conversion of 2-aminoethylphosphonate to inorganic phosphate. Biochemical studies of the recombinant PhnY and PhnA proteins verified their roles as phosphonoacetaldehyde dehydrogenase and phosphonoacetate hydrolase, respectively. This pathway is likely not limited to S. meliloti as suggested by the presence of homologous gene clusters in other bacterial genomes.

A de novo approach to the synthesis of glycosylated methymycin analogues with structural and stereochemical diversity.

A divergent and highly stereoselective route to 11 glycosylated methymycin analogues has been developed. The key to the success of this method was the iterative use of the Pd-catalyzed glycosylation reaction and postglycosylation transformation. This unique application of Pd-catalyzed glycosylation demonstrates the breath of α/ß- and d/l-glycosylation of macrolides that can be efficiently prepared using a de novo asymmetric approach to the carbohydrate portion.

Characterization of glycosyltransferase DesVII and its auxiliary partner protein DesVIII in the methymycin/picromycin biosynthetic pathway.

The in vitro characterization of the catalytic activity of DesVII, the glycosyltransferase involved in the biosynthesis of the macrolide antibiotics methymycin, neomethymycin, narbomycin, and pikromycin in Streptomyces venezuelae, is described. DesVII is unique among glycosyltransferases in that it requires an additional protein component, DesVIII, for activity. Characterization of the metabolites produced by a S. venezuelae mutant lacking the desVIII gene confirmed that desVIII is important for the biosynthesis of glycosylated macrolides but can be replaced by at least one of the homologous genes from other pathways. The addition of recombinant DesVIII protein significantly improves the glycosylation efficiency of DesVII in the in vitro assay. When affinity-tagged DesVII and DesVIII proteins were coproduced in Escherichia coli, they formed a tight (αß)(3) complex that is at least 10(3)-fold more active than DesVII alone. The formation of the DesVII/DesVIII complex requires coexpression of both genes in vivo and cannot be fully achieved by mixing the individual protein components in vitro. The ability of the DesVII/DesVIII system to catalyze the reverse reaction with the formation of TDP-desosamine was also demonstrated in a transglycosylation experiment. Taken together, our data suggest that DesVIII assists the folding of DesVII during protein production and remains tightly bound during catalysis. This requirement must be taken into consideration in the design of combinatorial biosynthetic experiments with new glycosylated macrolides.

Biosynthesis of rhizocticins, antifungal phosphonate oligopeptides produced by Bacillus subtilis ATCC6633.

Rhizocticins are phosphonate oligopeptide antibiotics containing the C-terminal nonproteinogenic amino acid (Z)-l-2-amino-5-phosphono-3-pentenoic acid (APPA). Here we report the identification and characterization of the rhizocticin biosynthetic gene cluster (rhi) in Bacillus subtilis ATCC6633. Rhizocticin B was heterologously produced in the nonproducer strain Bacillus subtilis 168. A biosynthetic pathway is proposed on the basis of bioinformatics analysis of the rhi genes. One of the steps during the biosynthesis of APPA is an unusual aldol reaction between phosphonoacetaldehyde and oxaloacetate catalyzed by an aldolase homolog RhiG. Recombinant RhiG was prepared, and the product of an in vitro enzymatic conversion was characterized. Access to this intermediate allows for biochemical characterization of subsequent steps in the pathway.

Linear aglycones are the substrates for glycosyltransferase DesVII in methymycin biosynthesis: analysis and implications.

The two essential structural components of macrolide antibiotics are the polyketide aglycone and the appended sugars. The aglycone formation is catalyzed by polyketide synthase (PKS), and glycosylation is catalyzed by an appropriate glycosyltransferase. Although it has been shown that glycosylation occurs after the cyclic aglycone is released from PKS, it is not known whether the acyl carrier protein (ACP)-bound linear polyketide chain can also be processed by the corresponding glycosyltransferase. To explore this possibility, the aglycone, 10-deoxymethynolide, which is the precursor of methymycin and neomethymycin, was chemically synthesized in the linear form as a N-acetylcysteamine (NAC) thioester. Subsequent incubation with TDP-d-desosamine in the presence of the dedicated glycosyltransferase, DesVII, and activator, DesVIII, produces a more polar product whose high-resolution mass is consistent with the anticipated glycosylated product. This study demonstrated for the first time that a macrolide glycosyltransferase can also recognize and process the linear precursor of its macrolactone substrate with a reduced but measurable activity.

Characterization of the glycosyltransferase activity of desVII: analysis of and implications for the biosynthesis of macrolide antibiotics.

In vitro catalytic activity of DesVII, the glycosyltransferase involved in the biosynthesis of methymycin, neomethymycin, narbomycin, and pikromycin in Streptomyces venezuelae, is described. This is the first report of demonstrated in vitro activity of a glycosyltransferase involved in the biosynthesis of macrolide antibiotics. DesVII is unique among glycosyltransferases in that it requires an additional protein component, DesVIII, as well as basic pH for its full activity.

Beta-glucosylation as a part of self-resistance mechanism in methymycin/pikromycin producing strain Streptomyces venezuelae.

In our study of the biosynthesis of D-desosamine in Streptomyces venezuelae, we have cloned and sequenced the entire desosamine biosynthetic cluster. The deduced product of one of the genes, desR, in this cluster shows high sequence homology to beta-glucosidases, which catalyze the hydrolysis of the glycosidic linkages, a function not required for the biosynthesis of desosamine. Disruption of the desR gene led to the accumulation of glucosylated methymycin/neomethymycin products, all of which are biologically inactive. It is thus conceivable that methymycin/neomethymycin may be produced as inert diglycosides, and the DesR protein is responsible for transforming these antibiotics from their dormant to their active forms. This hypothesis is supported by the fact that the translated desR gene has a leader sequence characteristic of secretory proteins, allowing it to be transported through the cell membrane and hydrolyze the modified antibiotics extracellularly to activate them. Expression of desR and biochemical characterization of the purified protein confirmed the catalytic function of this enzyme as a beta-glycosidase capable of catalyzing the hydrolysis of glucosylated methymycin/neomethymycin produced by S. venezuelae. These results provide strong evidence substantiating glycosylation/deglycosylation as a likely self-resistance mechanism of S. venezuelae. However, further experiments have suggested that such a glycosylation/deglycosylation is only a secondary self-defense mechanism in S. venezuelae, whereas modification of 23S rRNA, which is the target site for methymycin and its derivatives, by PikR1 and PikR2 is a primary self-resistance mechanism. Considering that postsynthetic glycosylation is an effective means to control the biological activity of macrolide antibiotics, the availability of macrolide glycosidases, which can be used for the activation of newly formed antibiotics that have been deliberately deactivated by engineered glycosyltransferases, may be a valuable part of an overall strategy for the development of novel antibiotics using the combinatorial biosynthetic approach.