A Substrate-triggered µ-Peroxodiiron(III) Intermediate in the 4-Choro-L-Lysine-Fragmenting Heme-Oxygenase-like Diiron Oxidase (HDO) BesC: Substrate Dissociation from, and C4 Targeting by, the Intermediate

ABSTRACTThe enzyme BesC from the β-ethynyl-L-serine biosynthetic pathway in Streptomyces cattleya fragments 4-chloro-L-lysine (produced from L-Lysine by BesD) to ammonia, formaldehyde, and 4-chloro-L-allylglycine and can analogously fragment L-Lys itself. BesC belongs to the emerging family of O2-activating non-heme-diiron enzymes with the "heme-oxygenase-like" protein fold (HDOs). Here we show that binding of L-Lys or an analog triggers capture of O2 by the protein’s diiron(II) cofactor to form a blue µ-peroxodiiron(III) intermediate analogous to those previously characterized in two other HDOs, the olefin-installing fatty acid decarboxylase, UndA, and the guanidino-N-oxygenase domain of SznF. The ∼ 5- and ∼ 30-fold faster decay of the intermediate in reactions with 4-thia-L-Lys and (4RS)-chloro-DL-lysine than in the reaction with L-Lys itself, and the primary deuterium kinetic isotope effects (D-KIEs) on decay of the intermediate and production of L-allylglycine in the reaction with 4,4,5,5-[2H]-L-Lys, imply that the peroxide intermediate or a successor complex with which it reversibly interconverts initiates the oxidative fragmentation by abstracting hydrogen from C4. Surprisingly, the sluggish substrate L-Lys can dissociate after triggering the intermediate to form, thereby allowing one of the better substrates to bind and react. Observed linkage between Fe(II) and substrate binding suggests that the triggering event involves a previously documented (in SznF) ordering of the dynamic HDO architecture that contributes one of the iron sites, a hypothesis consistent with the observation that the diiron(III) product cluster produced upon decay of the intermediate spontaneously degrades, as it has been shown to do in all other HDOs studied to date.

A metallo-β-lactamase enzyme for internal detoxification of the antibiotic thienamycin

Thienamycin, the first representative of carbapenem antibiotics was discovered in the mid-1970s from soil microorganism, Streptomyces cattleya, during the race to discover inhibitors of bacterial peptidoglycan synthesis. Chemically modified into imipenem (N-formimidoyl thienamycin), now one of the most clinically important antibiotics, thienamycin is encoded by a thienamycin gene cluster composed of 22 genes (thnA to thnV) from S. cattleya NRRL 8057 genome. Interestingly, the role of all thn-genes has been experimentally demonstrated in the thienamycin biosynthesis, except thnS, despite its annotation as putative β-lactamase. Here, we expressed thnS gene and investigated its activities against various substrates. Our analyses revealed that ThnS belonged to the superfamily of metallo-β-lactamase fold proteins. Compared to known β-lactamases such as OXA-48 and NDM-1, ThnS exhibited a lower affinity and less efficiency toward penicillin G and cefotaxime, while imipenem is more actively hydrolysed. Moreover, like most MBL fold enzymes, additional enzymatic activities of ThnS were detected such as hydrolysis of ascorbic acid, single strand DNA, and ribosomal RNA. ThnS appears as a MBL enzyme with multiple activities including a specialised β-lactamase activity toward imipenem. Thus, like toxin/antitoxin systems, the role of thnS gene within the thienamycin gene cluster appears as an antidote against the produced thienamycin.

Entropy drives selective fluorine recognition in the fluoroacetyl–CoA thioesterase from Streptomyces cattleya

Fluorinated small molecules play an important role in the design of bioactive compounds for a broad range of applications. As such, there is strong interest in developing a deeper understanding of how fluorine affects the interaction of these ligands with their targets. Given the small number of fluorinated metabolites identified to date, insights into fluorine recognition have been provided almost entirely by synthetic systems. The fluoroacetyl–CoA thioesterase (FlK) from Streptomyces cattleya thus provides a unique opportunity to study an enzyme–ligand pair that has been evolutionarily optimized for a surprisingly high 106 selectivity for a single fluorine substituent. In these studies, we synthesize a series of analogs of fluoroacetyl–CoA and acetyl–CoA to generate nonhydrolyzable ester, amide, and ketone congeners of the thioester substrate to isolate the role of fluorine molecular recognition in FlK selectivity. Using a combination of thermodynamic, kinetic, and protein NMR experiments, we show that fluorine recognition is entropically driven by the interaction of the fluorine substituent with a key residue, Phe-36, on the lid structure that covers the active site, resulting in an ∼5- to 20-fold difference in binding (KD). Although the magnitude of discrimination is similar to that found in designed synthetic ligand–protein complexes where dipolar interactions control fluorine recognition, these studies show that hydrophobic and solvation effects serve as the major determinant of naturally evolved fluorine selectivity.

Fluorothreonyl-tRNA deacylase prevents mistranslation in the organofluorine producerStreptomyces cattleya

Fluorine is an element with unusual properties that has found significant utility in the design of synthetic small molecules, ranging from therapeutics to materials. In contrast, only a few fluorinated compounds made by living organisms have been found to date, most of which derive from the fluoroacetate/fluorothreonine biosynthetic pathway first discovered inStreptomyces cattleya. While fluoroacetate has long been known to act as an inhibitor of the tricarboxylic acid cycle, the fate of the amino acid fluorothreonine is still not well understood. Here, we show that fluorothreonine can be misincorporated into protein in place of the proteinogenic amino acid threonine. We have identified two conserved proteins from the organofluorine biosynthetic locus, FthB and FthC, that are involved in managing fluorothreonine toxicity. Using a combination of biochemical, genetic, physiological, and proteomic studies, we show that FthB is atrans-acting transfer RNA (tRNA) editing protein, which hydrolyzes fluorothreonyl-tRNA 670-fold more efficiently than threonyl-RNA, and assign a role to FthC in fluorothreonine transport. Whiletrans-acting tRNA editing proteins have been found to counteract the misacylation of tRNA with commonly occurring near-cognate amino acids, their role has yet to be described in the context of secondary metabolism. In this regard, the recruitment of tRNA editing proteins to biosynthetic clusters may have enabled the evolution of pathways to produce specialized amino acids, thereby increasing the diversity of natural product structure while also attenuating the risk of mistranslation that would ensue.