Glycosylation of acyl carrier protein-bound polyketides during pactamycin biosynthesis.

Glycosylation is a common modification reaction in natural product biosynthesis and has been known to be a post-assembly line tailoring process in glycosylated polyketide biosynthesis. Here, we show that in pactamycin biosynthesis, glycosylation can take place on an acyl carrier protein (ACP)-bound polyketide intermediate. Using in vivo gene inactivation, chemical complementation and in vitro pathway reconstitution, we demonstrate that the 3-aminoacetophenone moiety of pactamycin is derived from 3-aminobenzoic acid by a set of discrete polyketide synthase proteins via a 3-(3-aminophenyl)3-oxopropionyl-ACP intermediate. This ACP-bound intermediate is then glycosylated by an N-glycosyltransferase, PtmJ, providing a sugar precursor for the formation of the aminocyclopentitol core structure of pactamycin. This is the first example of glycosylation of a small molecule while tethered to a carrier protein. Additionally, we demonstrate that PtmO is a hydrolase that is responsible for the release of the ACP-bound product to a free ß-ketoacid that subsequently undergoes decarboxylation.

Functional Characterization of 3-Aminobenzoic Acid Adenylation Enzyme PctU and UDP-N-Acetyl-d-Glucosamine: 3-Aminobenzoyl-ACP Glycosyltransferase PctL in Pactamycin Biosynthesis.

Pactamycin is an antibiotic produced by Streptomyces pactum with antitumor and antimalarial properties. Pactamycin has a unique aminocyclitol core that is decorated with 3-aminoacetophenone, 6-methylsaliciate, and an N,N-dimethylcarbamoyl group. Herein, we show that the adenylation enzyme PctU activates 3-aminobenzoic acid (3ABA) with adenosine triphosphate and ligates it to the holo form of the discrete acyl carrier protein PctK to yield 3ABA-PctK. Then, 3ABA-PctK is N-glycosylated with uridine diphosphate-N-acetyl-d-glucosamine (UDP-GlcNAc) by the glycosyltransferase PctL to yield GlcNAc-3ABA-PctK. Because 3ABA is known to be a precursor of the 3-aminoacetophenone moiety, PctU appears to be a gatekeeper that selects the appropriate 3-aminobenzoate starter unit. Overall, we propose that acyl carrier protein-bound glycosylated 3ABA derivatives are biosynthetic intermediates of pactamycin biosynthesis.

The secondary metabolite pactamycin with potential for pharmaceutical applications: biosynthesis and regulation.

The antitumor antibiotic pactamycin is a highly substituted aminocyclopentitol-derived secondary metabolite produced by the soil bacterium Streptomyces pactum. It has exhibited potent antibacterial, antitumor, antiviral, and antiprotozoal activities. Despite its outstanding biological activities, the complex chemical structure and broad-spectrum toxicity have hampered its development as a therapeutic, limiting its contribution to biomedical science to a role as a molecular probe for ribosomal function. However, a detailed understanding of its biosynthesis and how the biosynthesis is regulated has made it possible to tactically design and produce new pactamycin analogues, some of which have shown improved pharmacological properties. This mini-review describes the biosynthesis, regulation, engineered production, and biological activities of pactamycin and its congeners. It also highlights the suitability of biosynthetic methods as a feasible approach to generate new analogues of complex natural products and underscores the importance of utilizing biosynthetic enzymes as tools for chemoenzymatic production of structurally diverse bioactive compounds.

Pyridoxal phosphate-dependent reactions in the biosynthesis of natural products.

Covering: up to mid-2018 Pyridoxal 5'-phosphate (PLP) is a versatile organic cofactor used to catalyze diverse reactions on amino acid, oxoacid, and amine substrates. Here we review the reactions catalyzed by PLP-dependent enzymes, highlighting enzymes reported in the natural product biosynthetic literature. We describe enzymes that catalyze transaminations, Claisen-like condensations, and ß- and γ-eliminations and substitutions, along with epimerizations, decarboxylations, and transaldolations. Finally, we describe a newly reported group of O2-, PLP-dependent enzymes. Altogether, natural product biosynthesis showcases the incredible versatility of PLP-dependent transformations for building chemical complexity.

Global and pathway-specific transcriptional regulations of pactamycin biosynthesis in Streptomyces pactum.

Pactamycin, a structurally unique aminocyclitol natural product isolated from Streptomyces pactum, has potent antibacterial, antitumor, and anti-protozoa activities. However, its production yields under currently used culture conditions are generally low. To understand how pactamycin biosynthesis is regulated and explore the possibility of improving pactamycin production in S. pactum, we investigated the transcription regulations of pactamycin biosynthesis. In vivo inactivation of two putative pathway-specific regulatory genes, ptmE and ptmF, resulted in mutant strains that are not able to produce pactamycin. Genetic complementation using a cassette containing ptmE and ptmF integrated into the S. pactum chromosome rescued the production of pactamycin. Transcriptional analysis of the ΔptmE and ΔptmF strains suggests that both genes control the expression of the whole pactamycin biosynthetic gene cluster. However, attempts to overexpress these regulatory genes by introducing a second copy of the genes in S. pactum did not improve the production yield of pactamycin. We discovered that pactamycin biosynthesis is sensitive to phosphate regulation. Concentration of inorganic phosphate higher than 2 mM abolished both the transcription of the biosynthetic genes and the production of the antibiotic. Draft genome sequencing of S. pactum and bioinformatics studies revealed the existence of global regulatory genes, e.g., genes that encode a two-component PhoR-PhoP system, which are commonly involved in secondary metabolism. Inactivation of phoP did not show any significant effect to pactamycin production. However, in the phoP::aac(3)IV mutant, pactamycin biosynthesis is not affected by external inorganic phosphate concentration.

NAD+ -Dependent Dehydrogenase PctP and Pyridoxal 5'-Phosphate Dependent Aminotransferase PctC Catalyze the First Postglycosylation Modification of the Sugar Intermediate in Pactamycin Biosynthesis.

The unique five-membered aminocyclitol core of the antitumor antibiotic pactamycin originates from d-glucose, so unprecedented enzymatic modifications of the sugar intermediate are involved in the biosynthesis. However, the order of the modification reactions remains elusive. Herein, we examined the timing of introduction of an amino group into certain sugar-derived intermediates by using recombinant enzymes that were encoded in the pactamycin biosynthesis gene cluster. We found that the NAD+ -dependent alcohol dehydrogenase PctP and pyridoxal 5'-phosphate dependent aminotransferase PctC converted N-acetyl-d-glucosaminyl-3-aminoacetophonone into 3'-amino-3'-deoxy-N-acetyl-d-glucosaminyl-3-aminoacetophenone. Further, N-acetyl-d-glucosaminyl-3-aminophenyl-ß-oxopropanoic acid ethyl ester was converted into the corresponding 3'-amino derivative. However, PctP did not oxidize most of the tested d-glucose derivatives, including UDP-GlcNAc. Thus, modification of the GlcNAc moiety in pactamycin biosynthesis appears to occur after the glycosylation of aniline derivatives.

A Highly Promiscuous ß-Ketoacyl-ACP Synthase (KAS) III-like Protein Is Involved in Pactamycin Biosynthesis.

ß-Ketoacyl-acyl carrier protein (ß-Ketoacyl-ACP) synthase (KAS) III catalyzes the first step in fatty acid biosynthesis, involving a Claisen condensation of the acetyl-CoA starter unit with the first extender unit, malonyl-ACP, to form acetoacetyl-ACP. KAS III-like proteins have also been reported to catalyze acyltransferase reactions using coenzyme A esters or discrete ACP-bound substrates. Here, we report the in vivo and in vitro characterizations of a KAS III-like protein (PtmR), which directly transfers a 6-methylsalicylyl moiety from an iterative type I polyketide synthase to an aminocyclopentitol unit in pactamycin biosynthesis. PtmR is highly promiscuous, recognizing a wide array of S-acyl-N-acetylcysteamines as substrates to produce a suite of pactamycin derivatives with diverse alkyl and aromatic features. The results suggest that KAS III-like proteins may be used as versatile tools for modifications of complex natural products.

Interrogating the Tailoring Steps of Pactamycin Biosynthesis and Accessing New Pactamycin Analogues.

Pactamycin is a bacteria-derived aminocyclitol antibiotic with a wide-range of biological activity. Its chemical structure and potent biological activities have made it an interesting lead compound for drug discovery and development. Despite its unusual chemical structure, many aspects of its formation in nature remain elusive. Using a combination of genetic inactivation and metabolic analysis, we investigated the tailoring processes of pactamycin biosynthesis in Streptomyces pactum. The results provide insights into the sequence of events during the tailoring steps of pactamycin biosynthesis and explain the unusual production of various pactamycin analogues by S. pactum mutants. We also identified two new pactamycin analogues that have better selectivity indexes than pactamycin against malarial parasites.

Mechanism-Based Trapping of the Quinonoid Intermediate by Using the K276R Mutant of PLP-Dependent 3-Aminobenzoate Synthase PctV in the Biosynthesis of Pactamycin.

Mutational analysis of the pyridoxal 5'-phosphate (PLP)-dependent enzyme PctV was carried out to elucidate the multi-step reaction mechanism for the formation of 3-aminobenzoate (3-ABA) from 3-dehydroshikimate (3-DSA). Introduction of mutation K276R led to the accumulation of a quinonoid intermediate with an absorption maximum at 580 nm after the reaction of pyridoxamine 5'-phosphate (PMP) with 3-DSA. The chemical structure of this intermediate was supported by X-ray crystallographic analysis of the complex formed between the K276R mutant and the quinonoid intermediate. These results clearly show that a quinonoid intermediate is involved in the formation of 3-ABA. They also indicate that Lys276 (in the active site of PctV) plays multiple roles, including acid/base catalysis during the dehydration reaction of the quinonoid intermediate.

A single PLP-dependent enzyme PctV catalyzes the transformation of 3-dehydroshikimate into 3-aminobenzoate in the biosynthesis of pactamycin.

Natural amino donation: A PLP-dependent aminotransferase PctV, encoded in the pactamycin biosynthetic gene cluster, was found to catalyze the formation of 3-aminobenzoate from 3-dehydroshikimate with L-glutamate as the amino donor. The PctV reaction comprises a transamination and two dehydration reactions. This is the first report of a simple 3-ABA synthase in nature.

Biosynthetic studies and genetic engineering of pactamycin analogs with improved selectivity toward malarial parasites.

Pactamycin, one of the most densely functionalized aminocyclitol antibiotics, has pronounced antibacterial, antitumor, antiviral, and antiplasmodial activities, but its development as a clinical drug was hampered by its broad cytotoxicity. Efforts to modulate the biological activity by structural modifications using synthetic organic chemistry have been difficult because of the complexity of its chemical structure. However, through extensive biosynthetic studies and genetic engineering, we were able to produce analogs of pactamycin that show potent antimalarial activity, but lack significant antibacterial activity, and are about 10-30 times less toxic than pactamycin toward mammalian cells. The results suggest that distinct ribosomal binding selectivity or new mechanism(s) of action may be involved in their plasmodial growth inhibition, which may lead to the discovery of new antimalarial drugs and identification of new molecular targets within malarial parasites.

Deciphering pactamycin biosynthesis and engineered production of new pactamycin analogues.

Pactamycin is an aminocyclopentitol-derived natural product that has potent antibacterial and antitumor activities. Sequence analysis of an 86 kb continuous region of the chromosome from Streptomyces pactum ATCC 27456 revealed a gene cluster involved in the biosynthesis of pactamycin. Gene inactivation of the Fe-S radical SAM oxidoreductase (ptmC) and the glycosyltransferase (ptmJ), individually abrogated pactamycin biosynthesis; this confirmed the involvement of the ptm gene cluster in pactamycin biosynthesis. The polyketide synthase gene (ptmQ) was found to support 6-methylsalicylic acid (6-MSA) synthesis in a heterologous host, S. lividans T7. In vivo inactivation of ptmQ in S. pactum impaired pactamycin and pactamycate production but led to production of two new pactamycin analogues, de-6-MSA-pactamycin and de-6-MSA-pactamycate. The new compounds showed equivalent cytotoxic and antibacterial activities with the corresponding parent molecules and shed more light on the structure-activity relationship of pactamycin.

Cloning of the pactamycin biosynthetic gene cluster and characterization of a crucial glycosyltransferase prior to a unique cyclopentane ring formation.

The biosynthetic gene (pct) cluster for an antitumor antibiotic pactamycin was identified by use of a gene for putative radical S-adenosylmethionine methyltransferase as a probe. The pct gene cluster is localized to a 34 kb contiguous DNA from Streptomyces pactum NBRC 13433 and contains 24 open reading frames. Based on the bioinformatic analysis, a plausible biosynthetic pathway for pactamycin comprising of a unique cyclopentane ring, 3-aminoacetophenone, and 6-methylsalicylate was proposed. The pctL gene encoding a glycosyltransferase was speculated to be involved in an N-glycoside formation between 3-aminoacetophenone and UDP-N-acetyl-alpha-D-glucosamine prior to a unique cyclopentane ring formation. The pctL gene was then heterologously expressed in Escherichia coli and the enzymatic activity of the recombinant PctL protein was investigated. Consequently, the PctL protein was found to catalyze the expected reaction forming beta-N-glycoside. The enzymatic activity of the PctL protein clearly confirmed that the present identified gene cluster is for the biosynthesis of pactamycin. Also, a glycosylation prior to cyclopentane ring formation was proposed to be a general strategy in the biosynthesis of the structurally related cyclopentane containing compounds.

Directed biosynthesis of 5"-fluoropactamycin in Streptomyces pactum.

A new pactamycin analogue, 5"-fluoropactamycin, was prepared by directed biosynthesis. Supplementation of the fermentation medium of Streptomyces pactum, var. pactum with 3-amino-5-fluorobenzoic acid, an analogue of 3-aminobenzoic acid, an advanced precursor in pactamycin biosynthesis, resulted in co-production of pactamycin and the new pactamycin analogue. A similar feeding experiment with 3-amino-5-methylbenzoic acid did not result in formation of the corresponding methylated pactamycin analogue, but only in inhibition of pactamycin production. Comparison of antimicrobial and cytotoxic activities of pactamycin and 5"-fluoropactamycin showed no significant differences.

Use of a fractionated coupled transcription-translation system in the study of ribosomal resistance mechanisms in antibiotic-producing Streptomyces.

The coupled transcription-translation system, formerly involving extracts of Streptomyces lividans, has been developed such that it functions with ribosomes (or their subunits) from at least 20 different Streptomyces species. This fractionated system has been used to investigate the antibiotic responses of ribosomes from various Streptomyces which synthesize inhibitors of protein synthesis. Of the 11 organisms included in this study, two strains possessed ribosomes that were specifically resistant to the autogenous antibiotic. These were Streptomyces pactum and Streptomyces karnatakensis, both of which produce pactamycin. Ribosomal subunit exchange analysis further demonstrated that resistance to pactamycin in those strains is due to some property of the 30S ribosomal subunits.