A B12-dependent radical SAM enzyme involved in oxetanocin A biosynthesis.

Oxetanocin A (OXT-A) is a potent antitumour, antiviral and antibacterial compound. Biosynthesis of OXT-A has been linked to a plasmid-borne Bacillus megaterium gene cluster that contains four genes: oxsA, oxsB, oxrA and oxrB. Here we show that both the oxsA and oxsB genes are required for the production of OXT-A. Biochemical analysis of the encoded proteins, a cobalamin (Cbl)-dependent S-adenosylmethionine (AdoMet) radical enzyme, OxsB, and an HD-domain phosphohydrolase, OxsA, reveals that OXT-A is derived from a 2'-deoxyadenosine phosphate in an OxsB-catalysed ring contraction reaction initiated by hydrogen atom abstraction from C2'. Hence, OxsB represents the first biochemically characterized non-methylating Cbl-dependent AdoMet radical enzyme. X-ray analysis of OxsB reveals the fold of a Cbl-dependent AdoMet radical enzyme, a family of enzymes with an estimated 7,000 members. Overall, this work provides a framework for understanding the interplay of AdoMet and Cbl cofactors and expands the catalytic repertoire of Cbl-dependent AdoMet radical enzymes.

Co-opting sulphur-carrier proteins from primary metabolic pathways for 2-thiosugar biosynthesis.

Sulphur is an essential element for life and is ubiquitous in living systems. Yet how the sulphur atom is incorporated into many sulphur-containing secondary metabolites is poorly understood. For bond formation between carbon and sulphur in primary metabolites, the major ionic sulphur sources are the persulphide and thiocarboxylate groups on sulphur-carrier (donor) proteins. Each group is post-translationally generated through the action of a specific activating enzyme. In all reported bacterial cases, the gene encoding the enzyme that catalyses the carbon-sulphur bond formation reaction and that encoding the cognate sulphur-carrier protein exist in the same gene cluster. To study the production of the 2-thiosugar moiety in BE-7585A, an antibiotic from Amycolatopsis orientalis, we identified a putative 2-thioglucose synthase, BexX, whose protein sequence and mode of action seem similar to those of ThiG, the enzyme that catalyses thiazole formation in thiamine biosynthesis. However, no gene encoding a sulphur-carrier protein could be located in the BE-7585A cluster. Subsequent genome sequencing uncovered a few genes encoding sulphur-carrier proteins that are probably involved in the biosynthesis of primary metabolites but only one activating enzyme gene in the A. orientalis genome. Further experiments showed that this activating enzyme can adenylate each of these sulphur-carrier proteins and probably also catalyses the subsequent thiolation, through its rhodanese domain. A proper combination of these sulphur-delivery systems is effective for BexX-catalysed 2-thioglucose production. The ability of BexX to selectively distinguish sulphur-carrier proteins is given a structural basis using X-ray crystallography. This study is, to our knowledge, the first complete characterization of thiosugar formation in nature and also demonstrates the receptor promiscuity of the A. orientalis sulphur-delivery system. Our results also show that co-opting the sulphur-delivery machinery of primary metabolism for the biosynthesis of sulphur-containing natural products is probably a general strategy found in nature.

Nucleophilic participation of reduced flavin coenzyme in mechanism of UDP-galactopyranose mutase.

UDP-galactopyranose mutase (UGM) requires reduced FAD (FAD(red)) to catalyze the reversible interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). Recent structural and mechanistic studies of UGM have provided evidence for the existence of an FAD-Galf/p adduct as an intermediate in the catalytic cycle. These findings are consistent with Lewis acid/base chemistry involving nucleophilic attack by N5 of FAD(red) at C1 of UDP-Galf/p. In this study, we employed a variety of FAD analogues to characterize the role of FAD(red) in the UGM catalytic cycle using positional isotope exchange (PIX) and linear free energy relationship studies. PIX studies indicated that UGM reconstituted with 5-deaza-FAD(red) is unable to catalyze PIX of the bridging C1-OP(ß) oxygen of UDP-Galp, suggesting a direct role for the FAD(red) N5 atom in this process. In addition, analysis of kinetic linear free energy relationships of k(cat) versus the nucleophilicity of N5 of FAD(red) gave a slope of ρ = -2.4 ± 0.4. Together, these findings are most consistent with a chemical mechanism for UGM involving an S(N)2-type displacement of UDP from UDP-Galf/p by N5 of FAD(red).

Enzyme-catalyzed transfer of a ketone group from an S-adenosylmethionine analogue: a tool for the functional analysis of methyltransferases.

S-adenosylmethionine (AdoMet or SAM)-dependent methyltransferases belong to a large and diverse family of group-transfer enzymes that perform vital biological functions on a host of substrates. Despite the progress in genomics, structural proteomics, and computational biology, functional annotation of methyltransferases remains a challenge. Herein, we report the synthesis and activity of a new AdoMet analogue functionalized with a ketone group. Using catechol O-methyltransferase (COMT, EC 2.1.1.6) and thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) as model enzymes, this robust and readily accessible analogue displays kinetic parameters that are comparable to AdoMet and exhibits multiple turnovers with enzyme. More importantly, this AdoMet surrogate displays the same substrate specificity as the natural methyl donor. Incorporation of the ketone group allows for subsequent modification via bio-orthogonal labeling strategies and sensitive detection of the tagged ketone products. Hence, this AdoMet analogue expands the toolbox available to interrogate the biochemical functions of methyltransferases.

Chemo-enzymatic detection of protein isoaspartate using protein isoaspartate methyltransferase and hydrazine trapping.

Isoaspartate formation is a ubiquitous post-translation modification arising from spontaneous asparagine deamidation or aspartate isomerization. The formation of isoaspartate inserts a methylene group into the protein backbone, generating a "kink", and may drastically alter protein structure and function, thereby playing critical roles in a myriad of biological processes, human diseases, and protein pharmaceutical development. Herein, we report a chemo-enzymatic detection method for the isoaspartate protein, which in particular allows the affinity enrichment of isoaspartate-containing proteins. In the initial step, protein isoaspartate methyltransferase selectively converts isoaspartates into the corresponding methyl esters. Subsequently, the labile methyl ester is trapped by strong nucleophiles in aqueous solutions, such as hydrazines to form hydrazides. The stable hydrazide products can be analyzed by standard proteomic techniques, such as matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry. Furthermore, the chemical trapping step allows us to introduce several tagging strategies for product identification and quantification, such as UV-vis and fluorescence detection through a dansyl derivative. Most significantly, the hydrazide product can be enriched by affinity chromatography using aldehyde resins, thus drastically reducing sample complexity. Our method hence represents the first technique for the affinity enrichment of isoaspartyl proteins and should be amendable to the systematic and comprehensive characterization of isoaspartate, particularly in complex systems.

Study of Uridine 5'-Diphosphate (UDP)-Galactopyranose Mutase Using UDP-5-Fluorogalactopyranose as a Probe: Incubation Results and Mechanistic Implications.

Uridine 5'-diphosphate-5-fluorogalactopyranose (UDP-5F-Galp, 7) was synthesized, and its effect on UDP-Galp mutase (UGM) was investigated. UGM facilitated the hydrolysis of 7 to yield UDP and 5-oxogalactose (24), but no 11 was detected. (19)F NMR and trapping experiments demonstrated that the reaction involves the initial formation of a substrate-cofactor adduct followed by decomposition of the resulting C5 gem-fluorohydrin to generate a 5-oxo intermediate (10). The results support the current mechanistic proposal for UGM and suggest new directions for designing mechanism-based inhibitors.