Cobalamin-dependent radical S-adenosyl-l-methionine enzymes in natural product biosynthesis.

Covering: 2011 to 2018 This highlight summarizes the investigation of cobalamin (Cbl)- and radical S-adenosyl-l-methionine (SAM)-dependent enzymes found in natural product biosynthesis to date and suggests some possibilities for the future. Though some mechanistic aspects are apparently shared, the overall diversity of this family's functions and abilities is significant and may be tailored to the specific substrate and/or reaction being catalyzed. A little over a year ago, the first crystal structure of a Cbl- and radical SAM-dependent enzyme was solved, providing the first insight into what may be the shared scaffolding of these enzymes.

Resolution of the uncertainty in the kinetic mechanism for the trans-3-Chloroacrylic acid dehalogenase-catalyzed reaction.

trans- and cis-3-Chloroacrylic acid dehalogenase (CaaD and cis-CaaD, respectively) catalyze the hydrolytic dehalogenation of their respective isomers and represent key steps in the bacterial conversion of 1,3-dichloropropene to acetaldehyde. In prior work, a kinetic mechanism for the CaaD-catalyzed reaction could not be unequivocally determined because (1) the order of product release could not be determined and (2) the fluorescence factor for the enzyme species, E*PQ (where P = bromide and Q = malonate semialdehyde, the two products of the reaction) could not be assigned. The ambiguities in the model have now been resolved by stopped-flow experiments following the reaction using an active site fluorescent probe, αY60W-CaaD and 3-bromopropiolate, previously shown to be a mechanism-based inhibitor of CaaD, coupled with the rate of bromide release in the course of CaaD inactivation.

Investigation of enzymatic C-P bond formation using multiple quantum HCP nuclear magnetic resonance spectroscopy.

The biochemical mechanism for the formation of the C-P-C bond sequence found in l-phosphinothricin, a natural product with antibiotic and herbicidal activity, remains unclear. To obtain further insight into the catalytic mechanism of PhpK, the P-methyltransferase responsible for the formation of the second C-P bond in l-phosphinothricin, we utilized a combination of stable isotopes and two-dimensional nuclear magnetic resonance spectroscopy. Exploiting the newly emerged Bruker QCI probe (Bruker Corp.

Spectroscopic characterization and mechanistic investigation of P-methyl transfer by a radical SAM enzyme from the marine bacterium Shewanella denitrificans OS217.

Natural products containing carbon-phosphorus bonds elicit important bioactivity in many organisms. l-Phosphinothricin contains the only known naturally-occurring carbon-phosphorus-carbon bond linkage. In actinomycetes, the cobalamin-dependent radical S-adenosyl-l-methionine (SAM) methyltransferase PhpK catalyzes the formation of the second C-P bond to generate the complete C-P-C linkage in phosphinothricin.

Initial characterization of Fom3 from Streptomyces wedmorensis: The methyltransferase in fosfomycin biosynthesis.

Fosfomycin is a broad-spectrum antibiotic that is useful against multi-drug resistant bacteria. Although its biosynthesis was first studied over 40 years ago, characterization of the penultimate methyl transfer reaction has eluded investigators. The enzyme believed to catalyze this reaction, Fom3, has been identified as a radical S-adenosyl-L-methionine (SAM) superfamily member.

In vitro phosphinate methylation by PhpK from Kitasatospora phosalacinea.

Radical S-adenosyl-L-methionine, cobalamin-dependent methyltransferases have been proposed to catalyze the methylations of unreactive carbon or phosphorus atoms in antibiotic biosynthetic pathways. To date, none of these enzymes has been purified or shown to be active in vitro. Here we demonstrate the activity of the P-methyltransferase enzyme, PhpK, from the phosalacine producer Kitasatospora phosalacinea.

Binding energy in the one-electron reductive cleavage of S-adenosylmethionine in lysine 2,3-aminomutase, a radical SAM enzyme.

The common step in the actions of members of the radical SAM superfamily of enzymes is the one-electron reductive cleavage of S-adenosyl-l-methionine (SAM) into methionine and the 5'-deoxyadenosyl radical. The source of the electron is the [4Fe-4S]1+ cluster characterizing the radical SAM superfamily, to which SAM is directly ligated through its methionyl carboxylate and amino groups. The energetics of the reductive cleavage of SAM is an outstanding question in the actions of radical SAM enzymes.

Kinetic and stereochemical analysis of YwhB, a 4-oxalocrotonate tautomerase homologue in Bacillus subtilis: mechanistic implications for the YwhB- and 4-oxalocrotonate tautomerase-catalyzed reactions.

YwhB, a 4-oxalocrotonate tautomerase (4-OT) homologue in Bacillus subtilis, has no known biological role, and the gene has no apparent genomic context. The kinetic and stereochemical properties of YwhB have been examined using available enol and dienol compounds. The kinetic analysis shows that YwhB has a relatively nonspecific 1,3- and 1,5-keto-enol tautomerase activity, with the former activity prevailing.

S-adenosylmethionine as an oxidant: the radical SAM superfamily.

A recently discovered superfamily of enzymes function using chemically novel mechanisms, in which S-adenosylmethionine (SAM) serves as an oxidizing agent in DNA repair and the biosynthesis of vitamins, coenzymes and antibiotics. Members of this superfamily, the radical SAM enzymes, are related by the cysteine motif CxxxCxxC, which nucleates the [4Fe-4S] cluster found in each. A common thread in the novel chemistry of these proteins is the use of a strong reducing agent--a low-potential [4Fe-4S](1+) cluster--to generate a powerful oxidizing agent, the 5'-deoxyadenosyl radical, from SAM.

Inactivation of the phenylpyruvate tautomerase activity of macrophage migration inhibitory factor by 2-oxo-4-phenyl-3-butynoate.

Macrophage migration inhibitory factor (MIF) is an important immunoregulatory protein that has been implicated in several inflammatory diseases. MIF also has a phenylpyruvate tautomerase (PPT) activity, the role of which remains elusive in these biological activities. The acetylene compound, 2-oxo-4-phenyl-3-butynoate (2-OPB), has been synthesized and tested as a potential irreversible inhibitor of its enzymatic activity.

4-Oxalocrotonate tautomerase, its homologue YwhB, and active vinylpyruvate hydratase: synthesis and evaluation of 2-fluoro substrate analogues.

A series of 2-fluoro-4-alkene and 2-fluoro-4-alkyne substrate analogues were synthesized and examined as potential inhibitors of three enzymes: 4-oxalocrotonate tautomerase (4-OT) and vinylpyruvate hydratase (VPH) from the catechol meta-fission pathway and a closely related 4-OT homologue found in Bacillus subtilis designated YwhB. All of the compounds were potent competitive inhibitors of 4-OT with the monocarboxylated 2E-fluoro-2,4-pentadienoate and the dicarboxylated 2E-fluoro-2-en-4-ynoate being the most potent. Despite the close mechanistic and structural similarities between 4-OT and YwhB, these compounds were significantly less potent inhibitors of YwhB with K(i) values ranging from 5- to 633-fold lower than those determined for 4-OT.

Crystallization and X-ray diffraction analysis of ornithine cyclodeaminase from Pseudomonas putida.

Ornithine cyclodeaminase (OCD) is a member of the micro-crystallin protein family, the biological activity of which is the conversion of L-ornithine to L-proline and ammonia. In order to elucidate the functional groups of this enzyme that are involved in catalysis, the crystallization of OCD from Pseudomonas putida was undertaken. Using microbatch-under-oil screening at the high-throughput crystallization laboratory (HTC) at the Hauptman-Woodward Medical Research Institute Inc.

The roles of active-site residues in the catalytic mechanism of trans-3-chloroacrylic acid dehalogenase: a kinetic, NMR, and mutational analysis.

trans-3-Chloroacrylic acid dehalogenase (CaaD) converts trans-3-chloroacrylic acid to malonate semialdehyde by the addition of H(2)O to the C-2, C-3 double bond, followed by the loss of HCl from the C-3 position. Sequence similarity between CaaD, an (alphabeta)(3) heterohexamer (molecular weight 47,547), and 4-oxalocrotonate tautomerase (4-OT), an (alpha)(6) homohexamer, distinguishes CaaD from those hydrolytic dehalogenases that form alkyl-enzyme intermediates. The recently solved X-ray structure of CaaD demonstrates that betaPro-1 (i.

Reactions of 4-oxalocrotonate tautomerase and YwhB with 3-halopropiolates: analysis and implications.

4-Oxalocrotonate tautomerase (4-OT) and YwhB, a 4-OT homologue found in Bacillus subtilis, exhibit a low level hydratase activity that converts trans-3-haloacrylates to acetaldehyde, presumably through a malonate semialdehyde intermediate. The mechanism for the initial transformation of the 3-haloacrylate to malonate semialdehyde involves Pro-1 as well as an arginine, two residues that play critical roles in the 4-OT-catalyzed isomerization reaction and the YwhB-catalyzed tautomerization reaction. These residues are also critical for the trans-3-chloroacrylic acid dehalogenase (CaaD)-catalyzed conversion of trans-3-haloacrylates to malonate semialdehyde.

The 4-oxalocrotonate tautomerase- and YwhB-catalyzed hydration of 3E-haloacrylates: implications for the evolution of new enzymatic activities.

4-Oxalocrotonate tautomerase (4-OT) catalyzes the conversion of 2-oxo-4E-hexenedioate to 2-oxo-3E-hexenedioate through the intermediate, 2-hydroxy-2,4E-hexadienedioate. 4-OT and a homologue found in Bacillus subtilis (designated YwhB) share sequence identity and two key catalytic groups, Pro-1 and Arg-11, with the two subunits comprising trans-3-chloroacrylic acid dehalogenase (CaaD). 4-OT and YwhB have now been found to display a low-level hydratase activity, resulting in the dehalogenation of 3E-haloacrylates.

Reactions of trans-3-chloroacrylic acid dehalogenase with acetylene substrates: consequences of and evidence for a hydration reaction.

Various soil bacteria use 1,3-dichloropropene, a component of the commercially available fumigants Shell D-D and Telone II, as a sole source of carbon and energy. One enzyme involved in the catabolism of 1,3-dichloropropene is trans-3-chloroacrylic acid dehalogenase (CaaD), which converts the trans-isomers of 3-bromo- and 3-chloroacrylate to malonate semialdehyde. Sequence analysis suggested a relationship between the heterohexameric CaaD and the bacterial isomerase, 4-oxalocrotonate tautomerase (4-OT), thereby distinguishing CaaD from a number of dehalogenases whose mechanisms proceed through an alkyl- or aryl-enzyme intermediate.

Secretory leukocyte protease inhibitor: inhibition of human immunodeficiency virus-1 infection of monocytic THP-1 cells by a newly cloned protein.

The ability of the salivary protein, secretory leukocyte protease inhibitor (SLPI), to inhibit human immunodeficiency virus-1 (HIV-1) infection in vitro has been reported previously and has led to the suggestion that SLPI may be partially responsible for the low oral transmission rate of HIV-1. However, results contradictory to these findings have also been published. These discrepancies can be attributed to a number of factors ranging from the variability of macrophage susceptibility to HIV infection to the quality of commercially available preparations of SLPI.

The crystal structure of YdcE, a 4-oxalocrotonate tautomerase homologue from Escherichia coli, confirms the structural basis for oligomer diversity.

The tautomerase superfamily consists of three major families represented by 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), and macrophage migration inhibitory factor (MIF). The members of this superfamily are structurally homologous proteins constructed from a simple beta-alpha-beta fold that share a key mechanistic feature; they use an amino-terminal proline, which has an unusually low pK(a), as the general base in a keto-enol tautomerization. Several new members of the 4-OT family have now been identified using PSI-BLAST and categorized into five subfamilies on the basis of multiple-sequence alignments and the conservation of key catalytic and structural residues.