Thiazolyl peptide antibiotics, including thiostrepton, the thiocillins and the GE2270 family, disrupt bacterial protein synthesis selectively with nanomolar affinity either for a site on the 50S ribosomal subunit or on elongation factor EF-TU. We and others have recently shown that the pyridine/dihydropyridine ring-containing macrocyclic scaffold necessary for the antibiotic activity of this natural product class arises from posttranslational modification of nascent ribosomally generated preproteins. Thus, the C-terminal 14 residues of the prethiocillin 52mer undergo a cascade of 14 posttranslational modifications, including 6 Cys to thiazoles, two threonines to dehydrobutyrines and two serines to the central pyridine heterocycle. In the thiocillin producer B, subtilis ATCC 14579 we have shown that preprotein gene replacement will allow in vivo analysis of requirements for the posttranslational cascade. This proposal will investigate the timing, order, and mechanisms of the posttranslational cascade modifications with the ultimate aim of reprogramming the machinery to understand target specificity (50S ribosomal subunit vs EF-Tu) and to optimize antibiotic activities. This will include study of the 26-atom macrocyclic thiocillin and also cloning, expression, and evaluation of the gene clusters encoding the 29 atom macrocyclic GE 37468 (which targets EF-Tu not the 50S ribosomal subunit) and the 35 atom macrocyclic berninamycin A, in which the 7 C-terminal serines are processed posttranslationally, five of them to dehydroAla, one to an oxazole, and the other condensed to form the embedded pyridine: we wish to understand how the distinct posttranslational fates of these serines during antibiotic scaffold maturation are controlled.

Public Health Relevance

The thiazolyl peptide antibiotic family comprises more than 80 members with pyridine/dehydropyridine rings embedded in 26-atom macrocyclic frameworks. The size of the macrocyclic ring is a determinant of which steps in bacterial protein biosynthesis are selectively targeted. This project aims to delineate the chemical logic and enzymatic machinery of the cascade of at least 14 posttranslational modifications that morph the nascent ribosomal preproteins into the architecture of the mature antibiotic scaffolds.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM020011-42
Application #
8392263
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Gerratana, Barbara
Project Start
1987-09-30
Project End
2013-06-30
Budget Start
2012-12-01
Budget End
2013-06-30
Support Year
42
Fiscal Year
2013
Total Cost
$154,036
Indirect Cost
$63,297
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Tsodikov, Oleg V; Hou, Caixia; Walsh, Christopher T et al. (2015) Crystal structure of O-methyltransferase CalO6 from the calicheamicin biosynthetic pathway: a case of challenging structure determination at low resolution. BMC Struct Biol 15:13
Setser, Jeremy W; Heemstra Jr, John R; Walsh, Christopher T et al. (2014) Crystallographic evidence of drastic conformational changes in the active site of a flavin-dependent N-hydroxylase. Biochemistry 53:6063-77
Walsh, Christopher T; Wencewicz, Timothy A (2014) Prospects for new antibiotics: a molecule-centered perspective. J Antibiot (Tokyo) 67:7-22
Haynes, Stuart W; Gao, Xue; Tang, Yi et al. (2013) Complexity generation in fungal peptidyl alkaloid biosynthesis: a two-enzyme pathway to the hexacyclic MDR export pump inhibitor ardeemin. ACS Chem Biol 8:741-8
Walsh, Christopher T; Haynes, Stuart W; Ames, Brian D et al. (2013) Short pathways to complexity generation: fungal peptidyl alkaloid multicyclic scaffolds from anthranilate building blocks. ACS Chem Biol 8:1366-82
Gao, Xue; Jiang, Wei; Jiménez-Osés, Gonzalo et al. (2013) An iterative, bimodular nonribosomal peptide synthetase that converts anthranilate and tryptophan into tetracyclic asperlicins. Chem Biol 20:870-8
Walsh, Christopher T; O'Brien, Robert V; Khosla, Chaitan (2013) Nonproteinogenic amino acid building blocks for nonribosomal peptide and hybrid polyketide scaffolds. Angew Chem Int Ed Engl 52:7098-124
Parker, Jared B; Walsh, Christopher T (2013) Action and timing of BacC and BacD in the late stages of biosynthesis of the dipeptide antibiotic bacilysin. Biochemistry 52:889-901
Walsh, Christopher T; Wencewicz, Timothy A (2013) Flavoenzymes: versatile catalysts in biosynthetic pathways. Nat Prod Rep 30:175-200
Malcolmson, Steven J; Young, Travis S; Ruby, J Graham et al. (2013) The posttranslational modification cascade to the thiopeptide berninamycin generates linear forms and altered macrocyclic scaffolds. Proc Natl Acad Sci U S A 110:8483-8

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