Jonathan B. Spencer

Crystal structure and mechanism of a bacterial fluorinating enzyme

Fluorine is the thirteenth most abundant element in the earths crust, but fluoride concentrations in surface water are low and fluorinated metabolites are extremely rare. The fluoride ion is a potent nucleophile in its desolvated state, but is tightly hydrated in water and effectively inert. Low availability and a lack of chemical reactivity have largely excluded fluoride from biochemistry: in particular, fluorines high redox potential precludes the haloperoxidase-type mechanism used in the metabolic incorporation of chloride and bromide ions. But fluorinated chemicals are growing in industrial importance, with applications in pharmaceuticals, agrochemicals and materials products. Reactive fluorination reagents requiring specialist process technologies are needed in industry and, although biological catalysts for these processes are highly sought after, only one enzyme that can convert fluoride to organic fluorine has been described. Streptomyces cattleya can form carbon–fluorine bonds and must therefore have evolved an enzyme able to overcome the chemical challenges of using aqueous fluoride. Here we report the sequence and three-dimensional structure of the first native fluorination enzyme, 5′-fluoro-5′-deoxyadenosine synthase, from this organism. Both substrate and products have been observed bound to the enzyme, enabling us to propose a nucleophilic substitution mechanism for this biological fluorination reaction.

Spectroscopic and electronic structure studies of aromatic electrophilic attack and hydrogen-atom abstraction by non-heme iron enzymes

(4-Hydroxy)mandelate synthase (HmaS) and (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) are two α-keto acid dependent mononuclear non-heme iron enzymes that use the same substrate, (4-hydroxyphenyl)pyruvate, but exhibit two different general reactivities. HmaS performs hydrogen-atom abstraction to yield benzylic hydroxylated product (S)-(4-hydroxy)mandelate, whereas HPPD utilizes an electrophilic attack mechanism that results in aromatic hydroxylated product homogentisate. These enzymes provide a unique opportunity to directly evaluate the similarities and differences in the reaction pathways used for these two reactivities. An FeII methodology using CD, magnetic CD, and variable-temperature, variable-field magnetic CD spectroscopies was applied to HmaS and compared with that for HPPD to evaluate the factors that affect substrate interactions at the active site and to correlate these to the different reactivities exhibited by HmaS and HPPD to the same substrate. Combined with density functional theory calculations, we found that HmaS and HPPD have similar substrate-bound complexes and that the role of the protein pocket in determining the different reactivities exhibited by these enzymes (hydrogen-atom abstraction vs. aromatic electrophilic attack) is to properly orient the substrate, allowing for ligand field geometric changes along the reaction coordinate. Elongation of the FeIVO bond in the transition state leads to dominant FeIIIO•− character, which significantly contributes to the reactivity with either the aromatic π-system or the CH σ-bond.

Sequential removal of the benzyl-type protecting groups PMB and NAP by oxidative cleavage using CAN and DDQ

Abstract The selective cleavage of the PMB (4-methoxybenzyl) group in the presence of the NAP (2-naphthylmethyl) group was achieved using CAN with a range of mono-saccharides. The NAP group can then be removed selectively in the presence of a benzyl group using DDQ. This provides a strategy for sequential deprotection of hydroxyl groups.

Convenient synthesis of Cbz-protected β-amino ketones by a copper-catalysed conjugate addition reaction

A convenient synthesis of Cbz-protected β-amino ketones is reported. Benzyl carbamates and α,β-unsaturated ketones furnish the conjugate addition products in the presence of a Cu(II) catalyst under mild conditions. Other weakly basic nitrogen nucleophiles can also be used in this reaction.

Analysis of the Tetronomycin Gene Cluster: Insights into the Biosynthesis of a Polyether Tetronate Antibiotic

The biosynthetic gene cluster for tetronomycin (TMN), a polyether ionophoric antibiotic that contains four different types of ring, including the distinctive tetronic acid moiety, has been cloned from Streptomyces sp. NRRL11266. The sequenced tmn locus (113 234 bp) contains six modular polyketide synthase (PKS) genes and a further 27 open‐reading frames. Based on sequence comparison to related biosynthetic gene clusters, the majority of these can be assigned a plausible role in TMN biosynthesis. The identity of the cluster, and the requirement for a number of individual genes, especially those hypothesised to contribute a glycerate unit to the formation of the tetronate ring, were confirmed by specific gene disruption. However, two large genes that are predicted to encode together a multifunctional PKS of a highly unusual type seem not to be involved in this pathway since deletion of one of them did not alter tetronomycin production. Unlike previously characterised polyether PKS systems, oxidative cyclisation appears to take place on the modular PKS rather than after transfer to a separate carrier protein, while tetronate ring formation and concomitant chain release share common mechanistic features with spirotetronate biosynthesis.

Analysis of Specific Mutants in the Lasalocid Gene Cluster: Evidence for Enzymatic Catalysis of a Disfavoured Polyether Ring Closure

Lasalocid is a highly atypical polyether ionophoric antibiotic, firstly because it contains a type of aromatic ring normally associated with fungal polyketides, and secondly because the formation of its tetrahydropyran ring appears to contravene Baldwins rules, which predict the kinetically preferred routes for cyclisation reactions in organic chemistry. The lasalocid biosynthetic gene cluster has been cloned from Streptomyces lasaliensis, and the las locus (73 533 bp) was found to contain seven modular polyketide synthase (PKS) genes, including all the activities necessary for the synthesis of the aromatic moiety. Specific deletion from the gene cluster of the flanking lasC gene, which is predicted to encode a flavin‐linked epoxidase, abolished production both of lasalocid and of the minor cometabolite iso‐lasalocid without leading to accumulation of an identifiable intermediate; this suggests that oxidative cyclisation to form the polyether rings takes place on the PKS before release of the full‐length polyketide product. Meanwhile, a mutant in which the adjacent epoxide hydrolase lasB had been deleted produced iso‐lasalocid only. Iso‐lasalocid differs from lasalocid in the replacement of the tetrahydropyran ring by a tetrohydrofuran ring and represents the kinetically favoured product of cyclisation. The LasB epoxide hydrolase is therefore directly implicated in control of the stereochemical course of polyether ring formation during lasalocid biosynthesis.

Evidence that a Novel Thioesterase is Responsible for Polyketide Chain Release during Biosynthesis of the Polyether Ionophore Monensin

Polyether ionophores, such as monensin A, are known to be biosynthesised, like many other antibiotic polyketides, on giant modular polyketide synthases (PKSs), but the intermediates and enzymes involved in the subsequent steps of oxidative cyclisation remain undefined. In particular there has been no agreement on the mechanism and timing of the final polyketide chain release. We now report evidence that MonCII from the monensin biosynthetic gene cluster in Streptomyces cinnamonensis, which was previously thought to be an epoxide hydrolase, is a novel thioesterase that belongs to the α/β‐hydrolase structural family and might catalyse this step. Purified recombinant MonCII was found to hydrolyse several thioester substrates, including an N‐acetylcysteamine thioester derivative of monensin A. Further, incubation with a hallmark inhibitor of such enzymes, phenylmethanesulfonyl fluoride, led to inhibition of the thioesterase activity and to the accumulation of an acylated form of MonCII. These findings require a reassessment of the role of other enzymes implicated in the late stages of polyether ionophore biosynthesis.

A novel mycolactone from a clinical isolate of Mycobacterium ulcerans provides evidence for additional toxin heterogeneity as a result of specific changes in the modular polyketide synthase

Mycolactones are macrocyclic polyketide toxins produced by the pathogen Mycobacterium ulcerans, the etiologic agent of the emerging human disease known as Buruli ulcer. The disease is characterised by large necrotic skin lesions, and currently surgical intervention is the only realistic therapy. Mycolactone appears to play a key role in infection, since, in an animal model, subcutaneously injected purified mycolactone reproduces the pathology of the disease, while M. ulcerans strains deficient in mycolactone production do not provoke lesions. Mycolactones thus appear to provide the first example of a polyketide virulence factor in a human pathogen. Mycolactone also has immunosuppressive properties and appears to induce apoptosis. 5] The structures of mycolactones A and B have been determined 6] to be, respectively, the Zand Eisomers of a 12membered macrocyclic polyketide to which a second highly unsaturated polyketide chain is appended via an ester linkage (Scheme 1). The complete structures and their absolute configuration have been confirmed by chemical synthesis. 8] Further work has revealed the existence, in culture extracts of a typical strain of M. ulcerans, of small amounts of other mycolactones that differ from mycolactones A and B only in the side chain and whose structures very largely reflect the aberrant operation of a specific cytochrome P450 hydroxylase required for mycolactone biosynthesis. 10, 12] Scrutiny of 34 different clinical isolates also indicated very little heterogeneity, again restricted to the side chain, although the structures were not examined in detail. The genetic basis for mycolactone biosynthesis has recently been revealed. M. ulcerans contains a 174 kb megaplasmid that harbours, in addition to a number of auxiliary genes, several very large genes encoding type I modular polyketide synthases that closely resemble the actinomycete PKSs that

Biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics

The 2-deoxystreptamine-containing aminoglycosides are an important class of clinically valuable antibiotics. A deep understanding of the biosynthesis of these natural products is required to enable efforts to rationally manipulate and engineer the biological production of novel aminoglycosides. This review discusses the development of our biosynthetic knowledge over the past half-century, with emphasis on the relatively recent contributions of molecular biology to the elucidation of these biosynthetic pathways.

Glyceryl-S-acyl carrier protein as an intermediate in the biosynthesis of tetronate antibiotics.

The biosynthetic pathway to the unusual tetronate ring of certain polyketide natural products, including the antibiotics abyssomicin and tetronomycin (TMN) and the antitumour compound chlorothricin (CHL), is presently unknown. The gene clusters governing chlorothricin and tetronomycin biosynthesis both contain a gene encoding an atypical member of the FkbH family of enzymes, which has previously been shown to synthesise glyceryl‐S‐acyl carrier protein (ACP) as the first step in production of unusual extender units for modular polyketide biosynthesis. We show here that purified recombinant FkbH‐like protein, Tmn16, from the TMN gene cluster catalyses the efficient transfer of a glyceryl moiety from D‐1,3‐bisphosphoglycerate (1,3‐BPG) to either of the dedicated ACPs, Tmn7a and ChlD2, to form glyceryl‐S‐ACP, which directly implicates this compound as an intermediate in tetronate biosynthesis as well. Neither Tmn16 nor Tmn7a produced glyceryl‐S‐ACP when incubated, respectively, with analogous ACP and FkbH‐like proteins from a known extender‐unit pathway; this indicates a highly selective channelling of glycolytic metabolites into tetronate biosynthesis.