Record Details
Biosynthesis and Rational Design of Novel Pactamycin Analogs
ScholarsArchive at Oregon State University
| Field | Value |
|---|---|
| Title | Biosynthesis and Rational Design of Novel Pactamycin Analogs |
| Names |
Abugrain, Mostafa
(creator) Mahmud, Taifo (advisor) |
| Date Issued | 2015-06-08 (iso8601) |
| Note | Graduation date: 2015 |
| Abstract | Pactamycin, a potent antitumor antibiotic produced by the soil bacterium Streptomyces pactum, is a structurally unique aminocyclopentitol-containing natural product. It consists of a highly functionalized cyclopentitol core unit, two aromatic rings [3- aminoacetophenone (3AAP) and 6-methylsalicylic acid (6MSA)], and a 1,1- dimethylurea moiety. Despite its potent biological activity, the development of this antibiotic was hampered by its high-toxicity profile. Earlier efforts to modulate its pharmacological properties by modifying the chemical structure using conventional synthetic chemistry were hampered by the complexity of the molecule, requiring alternative strategies for structure modifications, e.g., biosynthetic approaches. This dissertation describes an investigation of pactamycin biosynthesis in S. pactum and the development of new pactamycin analogs using biosynthetic approaches. Earlier studies have shown that the aminocyclopentitol unit of pactamycin is derived from glucose, possibly via N-acetylglucosamine (GlcNAc), whereas the 3AAP unit is derived from 3-aminobenzoic acid (3ABA). Although direct involvement of glucose and 3ABA in pactamycin has previously been established, the processes underlying their conversions to the aminocyclopentitol and 3AAP moieties were unknown. Using a combination of gene inactivation, chemical complementation, and biochemical studies, we demonstrated that 3ABA is processed by a set of discrete polyketide synthase (PKS) proteins, i.e., an adenosine monophosphate-forming acyl-coenzyme A (AMP-forming acyl-CoA) synthetase (PtmS), an acyl carrier protein (ACP) (PtmI), and a β-ketoacyl-ACP synthase (PtmK), to produce 3-[3-aminophenyl]3- oxopropionyl-ACP (3AP-3OP-ACP). We also found that the hydrolase PtmO is responsible for the cleavage of a β-ketoacyl product from ACP, which then undergoes a spontaneous decarboxylation. This study also revealed that neither free 3AAP nor its glycosylated form are directly involved in pactamycin biosynthesis. One of the most intriguing aspects of pactamycin biosynthesis is its high degree of tailoring modifications, e.g., N-carbamoylation, N-methylation, C-methylation, hydroxylation, an 6MSA attachment, which are all confined within the highly compacted core structure. Due to the promiscuity of some of the tailoring enzymes in the pactamycin pathway, the sequence or the timing of the tailoring processes were previously unclear. However, using a multiple gene inactivation strategy, we were able to establish the tailoring steps involved in pactamycin biosynthesis. Additionally, we produced two novel pactamycin analogs, TM-101 and TM-102. TM-101 was generated from a triple knockout mutant of ptmH (a radical S-adenosylmethionine (SAM) C-methyltransferase gene), ptmD (N-methyltransferase), and ptmQ (a PKS), whereas TM-102 was generated from a ΔptmD/ΔptmQ double knockout mutant. The chemical structures of TM-101 and TM-102 were elucidated by MS, ¹H NMR, ¹³C NMR, COSY, HMBC, and HSQC. Both compounds showed antimalarial activity but lacked significant antibacterial activity and were less toxic than pactamycin toward mammalian cells. Previous studies have also shown that the type I iterative PKS PtmQ is a 6MSA synthase that supplies 6MSA for pactamycin biosynthesis. However, the enzyme that is responsible for the attachment of 6MSA to the aminocyclitol unit was unknown. Through genetic and biochemical characterization, we discovered that PtmR, a β- ketoacyl-acyl carrier transferase (ACP) synthase (KAS) III-like protein, is responsible for the direct transfer of the 6-methylsalicylyl moiety from PtmQ to the aminocyclopentitol unit. The enzyme also recognizes a wide array of synthetically prepared acyl-N-acetylcysteamines (acyl-NACs) as substrates to generate a suit of new pactamycin derivatives with diverse functionalities. |
| Genre | Thesis/Dissertation |
| Topic | Novel Pactamycin Analogs |
| Identifier | http://hdl.handle.net/1957/56326 |
