Beta-lactam compounds belong to the most important antibiotics in current use. The increasing occurrence of bacterial resistance against antibiotics, which threatens to move us back to pre-antibiotic era, calls for the development of new antibiotics. Beta-lactams are produced by fermentation in the microorganism, therefore knowledge about the biosynthetic enzymes involved is vital for the production of new antibiotics to escape resistance. This thesis presents structure-function studies on enzymes involved in the biosynthesis of the beta-lactam antibiotics cephalosporins and cephamycins DAOCS is a non-heme Fe(II) dioxygenase and catalyses the oxidative ring expansion of the penicillin nucleus into the nucleus of cephalosporins. Structures of DAOCS with substrates, product and cofactors obtained from two different crystal forms are presented here. The structural results suggest a mechanism for cephalosporin formation where 2-oxoglutarate and dioxygen need to react first to produce an oxidizing iron species, followed by reaction with the penicillin substrate. Structural differences in the two crystal forms indicate conformational flexibility in the C terminus and point to a role for the C terminus in catalysis. Biosynthesis of cephamycins (C7-methoxylated cephalosporins) is catalysed by two enzymes, cmcI and cmcJ, but the details of catalysis are largely unknown. The crystal structure of cmcI, presented here, is a hexamer consisting of a C-terminal Rossman domain and a smaller N-terminal domain. The N-terminal domain is involved in oligomerisation and the Rossmann domain binds SAM and SAH in a fashion common for methyltransferases, with a bound magnesium ion in the vicinity of SAM. The expected cephalosporin binding site is occupied by PEG, which ligates the magnesium ion. From docking studies of a cephalosoporin molecule to the cmcI-Mg2+-SAM structure, a model for substrate binding is proposed. Altogether, the results suggest cmcI is a methyltransferase that catalyses the second catalytic step in cephamycin biosynthesis
