Graduate Thesis Or Dissertation
 

Biosynthesis and Rational Design of Novel Pactamycin Analogs

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  • Pactamycin, a potent antitumor antibiotic produced by the soil bacterium Streptomycespactum, is a structurally unique aminocyclopentitol-containing natural product. Itconsists 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 thisantibiotic was hampered by its high-toxicity profile. Earlier efforts to modulate itspharmacological properties by modifying the chemical structure using conventionalsynthetic chemistry were hampered by the complexity of the molecule, requiringalternative strategies for structure modifications, e.g., biosynthetic approaches. Thisdissertation describes an investigation of pactamycin biosynthesis in S. pactum and thedevelopment of new pactamycin analogs using biosynthetic approaches.Earlier studies have shown that the aminocyclopentitol unit of pactamycin is derivedfrom glucose, possibly via N-acetylglucosamine (GlcNAc), whereas the 3AAP unit isderived from 3-aminobenzoic acid (3ABA). Although direct involvement of glucoseand 3ABA in pactamycin has previously been established, the processes underlyingtheir conversions to the aminocyclopentitol and 3AAP moieties were unknown. Usinga combination of gene inactivation, chemical complementation, and biochemicalstudies, we demonstrated that 3ABA is processed by a set of discrete polyketidesynthase (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 isresponsible for the cleavage of a β-ketoacyl product from ACP, which then undergoesa spontaneous decarboxylation. This study also revealed that neither free 3AAP nor itsglycosylated form are directly involved in pactamycin biosynthesis.One of the most intriguing aspects of pactamycin biosynthesis is its high degree oftailoring modifications, e.g., N-carbamoylation, N-methylation, C-methylation,hydroxylation, an 6MSA attachment, which are all confined within the highlycompacted core structure. Due to the promiscuity of some of the tailoring enzymes inthe pactamycin pathway, the sequence or the timing of the tailoring processes werepreviously unclear. However, using a multiple gene inactivation strategy, we wereable to establish the tailoring steps involved in pactamycin biosynthesis. Additionally,we produced two novel pactamycin analogs, TM-101 and TM-102. TM-101 wasgenerated 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 double knockout mutant. Thechemical structures of TM-101 and TM-102 were elucidated by MS, ¹H NMR, ¹³CNMR, COSY, HMBC, and HSQC. Both compounds showed antimalarial activity butlacked significant antibacterial activity and were less toxic than pactamycin towardmammalian cells.Previous studies have also shown that the type I iterative PKS PtmQ is a 6MSAsynthase that supplies 6MSA for pactamycin biosynthesis. However, the enzyme thatis 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 responsiblefor the direct transfer of the 6-methylsalicylyl moiety from PtmQ to theaminocyclopentitol unit. The enzyme also recognizes a wide array of syntheticallyprepared acyl-N-acetylcysteamines (acyl-NACs) as substrates to generate a suit of newpactamycin derivatives with diverse functionalities.
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