Tel:	617-432-1715
	Fax:	617-432-0438
	E-Mail:	christopher_walsh@hms.harvard.edu
	Location:	Armenise Building, Room 616 200 Longwood Avenue Boston MA 02115
	Homepage:	http://walsh.med.harvard.edu/

The broad area of interest to this laboratory is the molecular basis of biological catalysis with focus on the structure and function of enzymes. Much of the current focus is on the biosynthesis and mechanism of action of antibiotics and bacterial siderophores. Several ongoing projects in the laboratory illustrate our approaches.

Vancomycin Biosynthesis

We are investigating the enzymatic pathway for biosynthesis of the vancomycin family of glycopeptide antibiotics. Vancomycin is a front line antibiotic for treatment of life-threatening gram-positive bacterial infections and many clinical isolates have become resistant to it, focussing interest on successors to vancomycin. The enzymatic pathway involves about two dozen enzymatic steps, including provision of nonproteinogenic amino acid monomers that get incorporated into the heptapeptide scaffold of the antibiotic, the assembly line for fashioning the heptapeptide as a series of elongating acyl-S-enzyme intermediates, cytochrome P450 oxidases that crosslink the scaffold, glycosyl transferases to add the sugar residues, and enzyme dedicated to the construction of the unusual aminodeoxy hexose vancosamine. We are studying all these different enzymatic steps with an eye to potential reprogramming of one or more steps for combinatorial biosynthesis.

Microcin B17

Certain strains of E. coli produce a peptide antibiotic Microcin B17 from an operon that encodes the 69 amino acid Microcin A orf that is the antibiotic precursor, orfs B-D that carry out posttranslational modification of A, orfs E,F for export of mature MccB17, and the immunity protein orf G. We are studying how orfs B,C,D carry out the posttranslational modification of 14 residues of orf A to produce four thiazoles and four oxazoles, heteroaromatic rings that are the key determinants of antibiotic activity. We are also studing how mature microcin interacts with its intracellular target DNA Gyrase to cause accumulation of double strand breaks in DNA.

Bacterial Siderophore Biogenesis

When bacteria are starved for iron, as occurs when they infect vertebrates, they turn on genes for biosynthesis of nonribosomal peptides, siderophores, that scavenge iron and then are taken back up by the bacteria. Siderophores are virulence factors by enabling pathogenic bacteria to multiply in hosts. We are examining the enzymatic assembly lines that make enterobactin, the E.coli siderophore, pyochelin from Pseudomonas aeruginosa, vibriobactin from Vibrio cholerae, and Yersiniabactin from Yersinia pestis. We are studying the enzymatic steps in the siderophore synthetase assembly lines and the modifications to the peptide backbones that create functionalgroups with high affinity for chelating iron, including hetrocycle formation and N- and C-methylations.

Macrocyclization of Nonribosomal Peptides.

A variety of peptide antibiotics are cyclic peptides (gramicidin, tyrocidine, bacitracin) or cyclic lipopeptides (surfactin, daptomycin). These are made on nonribosomal peptide synthetase assembly lines and cyclized by the last domain, a Thioesterase (TE) domain of the multimodular enzymatic assembly line. We are studying mechanism and structure of cyclizing TE domains and trying to use them in combinatorial biosynthesis.

Hybrid Polyketide/Nonribosmal Peptide Natural Products

Some antibiotics and siderophores are hybrids of polyketide and nonribosomal peptide constituents and are elongated on hybrid polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) assembly lines. We are studying three such assembly lines to decipher the rules for switching between PKS and NRPS modules in the biogenesis of epothilone (antitumor agent), rifamycin (antitubercular drug), and yersiniabactin.

References:

Mulichak AM, Losey HC, Walsh CT, Garavito RM. Structure of the UDP-glyucosyltransferase GftB that Modifies the Heptapeptide Aglycone in Biosynthesis of the Vancomycin Group of Antibiotics. Structure. 2001, 9, 547-57.

Keating TA, Marshall CG, Walsh CT. Reconstitution and characterization of the Vibrio cholerae vibriobactin synthetase from VibB, VibE, VibF, and VibH. Biochemistry. 2000 Dec 19;39(50):15522-30.

Chen H, Thomas MG, Hubbard BK, Losey HC, Walsh CT, Burkart MD. Deoxysugars in glycopeptide antibiotics: enzymatic synthesis of TDP-L-epivancosamine in chloroeremomycin biosynthesis. Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):11942-7.

Trauger JW, Kohli RM, Mootz HD, Marahiel MA, Walsh CT. Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase. Nature. 2000 Sep 14;407(6801):215-8.

Li YM, Milne JC, Madison LL, Kolter R, Walsh CT. From peptide precursors to oxazole and thiazole-containing peptide antibiotics: microcin B17 synthase. Science. 1996 Nov 15;274(5290):1188-93.

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