Covering: up to May 2012 The β-lactam antibiotics and related β-lactamase inhibitors are amongst the most important small molecules in clinical use. Most, but not all, β-lactams including penicillins, cephalosporins, and clavulanic acid are produced via fermentation or via modification of fermented intermediates, with important exceptions being the carbapenems and aztreonam. The desire for more efficient routes to existing antibiotics and for access to new and synthetically challenging ones stimulates continued interest in β-lactam biosynthesis. We review knowledge of the pathways leading to β-lactam antibiotics focusing on the mechanisms, structures and biocatalytic applications of the enzymes involved.

The response of animals to hypoxia is mediated by the hypoxia‐inducible transcription factor. Human hypoxia‐inducible factor is regulated by four Fe(II)‐ and 2‐oxoglutarate‐dependent oxygenases: prolyl hydroxylase domain enzymes 1–3 catalyse hydroxylation of two prolyl‐residues in hypoxia‐inducible factor, triggering its degradation by the proteasome. Factor inhibiting hypoxia‐inducible factor catalyses the hydroxylation of an asparagine‐residue in hypoxia‐inducible factor, inhibiting its transcriptional activity. Collectively, the hypoxia‐inducible factor hydroxylases negatively regulate hypoxia‐inducible factor in response to increasing oxygen concentration. Prolyl hydroxylase domain 2 is the most important oxygen sensor in human cells; however, the underlying kinetic basis of the oxygen‐sensing function of prolyl hydroxylase domain 2 is unclear. We report analyses of the reaction of prolyl hydroxylase domain 2 with oxygen. Chemical quench/MS experiments demonstrate that reaction of a complex of prolyl hydroxylase domain 2, Fe(II), 2‐oxoglutarate and the C‐terminal oxygen‐dependent degradation domain of hypoxia‐inducible factor‐α with oxygen to form hydroxylated C‐terminal oxygen‐dependent degradation domain and succinate is much slower (approximately 100‐fold) than for other similarly studied 2‐oxoglutarate oxygenases. Stopped flow/UV‐visible spectroscopy experiments demonstrate that the reaction produces a relatively stable species absorbing at 320 nm; Mössbauer spectroscopic experiments indicate that this species is likely not a Fe(IV)=O intermediate, as observed for other 2‐oxoglutarate oxygenases. Overall, the results obtained suggest that, at least compared to other studied 2‐oxoglutarate oxygenases, prolyl hydroxylase domain 2 reacts relatively slowly with oxygen, a property that may be associated with its function as an oxygen sensor.Structured digital abstract MINT‐7987711: PHD2 (uniprotkb:Q9GZT9) enzymaticly reacts (MI:0414) CODD (uniprotkb:Q16665) by enzymatic study (MI:0415)

Factor‐inhibiting hypoxia‐inducible factor (FIH) is an Fe(II)/2‐oxoglutarate‐dependent dioxygenase that acts as a negative regulator of the hypoxia‐inducible factor (HIF) by catalysing β‐hydroxylation of an asparaginyl residue in its C‐terminal transcriptional activation domain (CAD). In addition to the hypoxia‐inducible factor C‐terminal transcriptional activation domain (HIF‐CAD), FIH also catalyses asparaginyl hydroxylation of many ankyrin repeat domain‐containing proteins, revealing a broad sequence selectivity. However, there are few reports on the selectivity of FIH for the hydroxylation of specific residues. Here, we report that histidinyl residues within the ankyrin repeat domain of tankyrase‐2 can be hydroxylated by FIH. NMR and crystallographic analyses show that the histidinyl hydroxylation occurs at the β‐position. The results further expand the scope of FIH‐catalysed hydroxylations.Database The coordinates for the structure have been deposited in the Protein Data Bank in Europe (PDBe; http://www.ebi.ac.uk/pdbe) under accession code 2y0iStructured digital abstract FIH and TNKS‐1 hydroxylate by enzymatic study (View Interaction 1, 2) FIH and Tankyrase‐2 bind by x‐ray crystallography (View interaction) FIH and Tankyrase‐2 hydroxylate by enzymatic study (View Interaction 1, 2, 3) FIH and TRPV4 hydroxylate by enzymatic study (View interaction) GABPB2 and FIH hydroxylate by enzymatic study (View interaction)

Marrying synthetic biology with synthetic chemistry provides a powerful approach toward natural product diversification, combining the best of both worlds: expediency and synthetic capability of biogenic pathways and chemical diversity enabled by organic synthesis. Biosynthetic pathway engineering can be employed to insert a chemically orthogonal tag into a complex natural scaffold affording the possibility of site-selective modification without employing protecting group strategies. Here we show that, by installing a sufficiently reactive handle (e.g., a C–Br bond) and developing compatible mild aqueous chemistries, synchronous biosynthesis of the tagged metabolite and its subsequent chemical modification in living culture can be achieved. This approach can potentially enable many new applications: for example, assay of directed evolution of enzymes catalyzing halo-metabolite biosynthesis in living cells or generating and following the fate of tagged metabolites and biomolecules in living systems. We report synthetic biological access to new-to-nature bromo-metabolites and the concomitant biorthogonal cross-coupling of halo-metabolites in living cultures.