Hydrogenases use complex metal cofactors to catalyze the reversible formation of hydrogen. In [FeFe]-hydrogenases, the H-cluster cofactor includes a diiron subcluster containing azadithiolate, three CO, and two CN − ligands. During the assembly of the H cluster, the radical S-adenosyl methionine (SAM) enzyme HydG lyses the substrate tyrosine to yield the diatomic ligands. These diatomic products form an enzyme-bound Fe(CO) x (CN) y synthon that serves as a precursor for eventual H-cluster assembly. To further elucidate the mechanism of this complex reaction, we report the crystal structure and EPR analysis of HydG. At one end of the HydG (βα) 8 triosephosphate isomerase (TIM) barrel, a canonical [4Fe-4S] cluster binds SAM in close proximity to the proposed tyrosine binding site. At the opposite end of the active-site cavity, the structure reveals the auxiliary Fe-S cluster in two states: one monomer contains a [4Fe-5S] cluster, and the other monomer contains a [5Fe-5S] cluster consisting of a [4Fe-4S] cubane bridged by a μ 2 -sulfide ion to a mononuclear Fe 2+ center. This fifth iron is held in place by a single highly conserved protein-derived ligand: histidine 265. EPR analysis confirms the presence of the [5Fe-5S] cluster, which on incubation with cyanide, undergoes loss of the labile iron to yield a [4Fe-4S] cluster. We hypothesize that the labile iron of the [5Fe-5S] cluster is the site of Fe (CO) x (CN) y synthon formation and that the limited bonding between this iron and HydG may facilitate transfer of the intact synthon to its cognate acceptor for subsequent H-cluster assembly.radical SAM enzyme | tyrosine lyase | H-cluster biosynthesis T he assembly of the [FeFe]-hydrogenase diiron subcluster (1, 2) requires three maturase proteins, HydE, HydF, and HydG (3), and in vitro, they can assemble an active hydrogenase (4). The sequence and structure of the maturase HydE (5) indicates that it is a member of the radical S-adenosyl methionine (SAM) superfamily, although the biochemical function of HydE has not been experimentally determined. The GTPase HydF (6, 7) has been shown to transfer synthetic (8) or biologically derived (7, 9) diiron subclusters into apo-hydrogenase, suggesting that HydF functions as a template for diiron subcluster assembly. The tyrosine lyase HydG is also a member of the radical SAM superfamily and uses SAM and a reductant (such as dithionite) to cleave the Cα-Cβ bond of tyrosine, yielding p-cresol as the side chain-derived byproduct (10) and fragmenting the amino acid moiety into cyanide (CN − ) (11) and carbon monoxide (CO) (12), which are ultimately incorporated as ligands in the H cluster of the [FeFe]-hydrogenase HydA (4). Two site-differentiated [4Fe-4S] clusters in HydG have been identified using a combination of spectroscopy and site-directed mutagenesis (12-16). The cluster bound close to the N terminus ([4Fe-4S] RS ) by the CX 3 CX 2 C cysteine triad motif (SI Appendix, Fig. S1) is typical of the radical SAM superfamily (17, 18) and has been shown to catalyze the reductive cl...

Bacterial secondary metabolic pathways are responsible for the biosynthesis of thousands of bioactive natural products. Many enzymes residing in these pathways have evolved to catalyze unusual chemical transformations, which is facilitated by an evolutionary pressure promoting chemical diversity. Such divergent enzyme evolution has been observed in S-adenosyl-l-methionine (SAM)-dependent methyltransferases involved in the biosynthesis of anthracycline anticancer antibiotics; whereas DnrK from the daunorubicin pathway is a canonical 4-O-methyltransferase, the closely related RdmB (52% sequence identity) from the rhodomycin pathways is an atypical 10-hydroxylase that requires SAM, a thiol reducing agent, and molecular oxygen for activity. Here, we have used extensive chimeragenesis to gain insight into the functional differentiation of RdmB and show that insertion of a single serine residue to DnrK is sufficient for introduction of the monooxygenation activity. The crystal structure of DnrK-Ser in complex with aclacinomycin T and S-adenosyl-l-homocysteine refined to 1.9-Å resolution revealed that the inserted serine S297 resides in an α-helical segment adjacent to the substrate, but in a manner where the side chain points away from the active site. Further experimental work indicated that the shift in activity is mediated by rotation of a preceding phenylalanine F296 toward the active site, which blocks a channel to the surface of the protein that is present in native DnrK. The channel is also closed in RdmB and may be important for monooxygenation in a solvent-free environment. Finally, we postulate that the hydroxylation ability of RdmB originates from a previously undetected 10-decarboxylation activity of DnrK.

Summary Statement: The biosynthesis of lipoyl cofactors requires two lipoyl synthase mediated sulfur insertions. We report the crystal structures of a lipoyl synthase complexed with S-adenosylhomocysteine or 5'-methylthioadenosine. Models based on these structures identify likely substrate binding sites.Keywords: radical SAM, cofactors, crystal structure, enzyme catalysis, sulfur Abbreviations used: LipA, lipoyl synthase; BioB, biotin synthase; SAM, Sadenosylmethionine; LCD, lipoyl carrier domain; MTA, 5'-methylthioadenosine; RS, radical SAM; ACP, acyl carrier protein; SsLipA, Sulfolobus solfataricus LipA; TeLipA2 Thermosynechococcus elongatus LipA2; Ec, Escherichia coli; 5'-dA, 5'-deoxyadenosine. 2 ABSTRACTLipoyl cofactors are essential for living organisms and are produced by the insertion of two sulfur atoms into the relatively unreactive C-H bonds of an octanoyl substrate. This reaction requires lipoyl synthase, a member of the radical SAM enzyme superfamily. Herein we present crystal structures of lipoyl synthase with two [4Fe-4S] clusters bound at opposite ends of the TIM barrel, the usual fold of the radical SAM superfamily. The cluster required for reductive SAM cleavage conserves the features of the radical SAM superfamily, but the auxiliary cluster is bound by a CX 4 CX 5 C motif unique to lipoyl synthase. The fourth ligand to the auxiliary cluster is an extremely unusual serine residue. Site directed mutants show this conserved serine ligand is essential for the sulfur insertion steps. One crystallized LipA complex contains MTA, a breakdown product of SAM, bound in the likely SAM binding site. Modelling has identified an 18 Å deep channel, well-proportioned to accommodate an octanoyl substrate. These results suggest the auxiliary cluster is the likely sulfur donor, but access to a sulfide ion for the second sulfur insertion reaction requires the loss of an iron atom from the auxiliary cluster, which the serine ligand may enabled.3

Streptomyces soil bacteria produce hundreds of anthracycline anticancer agents with a relatively conserved set of genes. This diversity depends on the rapid evolution of biosynthetic enzymes to acquire novel functionalities. Previous work has identified S-adenosyl-l-methionine-dependent methyltransferase-like proteins that catalyze 4-O-methylation, 10-decarboxylation, or 10-hydroxylation, with additional differences in substrate specificities. Here we focused on four protein regions to generate chimeric enzymes using sequences from four distinct subfamilies to elucidate their influence in catalysis. Combined with structural studies we managed to depict factors that influence gain-of-hydroxylation, loss-of-methylation, and substrate selection. The engineering expanded the catalytic repertoire to include novel 9,10-elimination activity, and 4-O-methylation and 10-decarboxylation of unnatural substrates. The work provides an instructive account on how the rise of diversity of microbial natural products may occur through subtle changes in biosynthetic enzymes.

Hydrogenases are a potential source of environmentally benign bioenergy, using complex cofactors to catalyze the reversible reduction of protons to form hydrogen. AddressesChemistry and the Institute for Life Sciences, University of Southampton, Highfield Campus, Southampton SO17 1BJ, UK.Corresponding author: Peter L. Roach (plr2@soton.ac.uk). AbstractHydrogenases are a potential source of environmentally benign bioenergy, using complex cofactors to catalyze the reversible reduction of protons to form hydrogen. The most active subclass, the [FeFe]-hydrogenases, is dependent on a metallocofactor, the H cluster, that consists of a two iron subcluster ([2Fe] H ) bridging to a classical cubane cluster ([4Fe-4S] H ). The ligands coordinating to the diiron subcluster include an azadithiolate, three carbon monoxides, and two cyanides. To assemble this complex cofactor, three maturase enzymes, HydG, HydE and HydF are required. The biosynthesis of the diatomic ligands proceeds by an unusual fragmentation mechanism, and structural studies in combination with spectroscopic analysis have started to provide insights into the HydG mediated assembly of a [2Fe] H subcluster precursor.