Enzymic Preparation of<u>L</u>-β-Lysine

Chirpich, Thomas P.; Herbst, M.; Edmunds, Henry N.; Baltimore, Barbara G.; Costilow, Ralph N.; Barker, H. A.

doi:10.1080/00327487308061488

Cited by 22 publications

(43 citation statements)

References 9 publications

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“…AdoMet was further purified by anion-exchange chromatography as reported previously (39). L-AzaAdoMet was synthesized and purified according to the method of Thompson et al (40).…”

Section: Methodsmentioning

confidence: 99%

Catalytic Roles for Carbon-Oxygen Hydrogen Bonding in SET Domain Lysine Methyltransferases

Couture

Hauk

Thompson

et al. 2006

Journal of Biological Chemistry

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SET domain enzymes represent a distinct family of protein lysine methyltransferases in eukaryotes. Recent studies have yielded significant insights into the structural basis of substrate recognition and the product specificities of these enzymes. However, the mechanism by which SET domain methyltransferases catalyze the transfer of the methyl group from S-adenosyl-L-methionine to the lysine ⑀-amine has remained unresolved. To elucidate this mechanism, we have determined the structures of the plant SET domain enzyme, pea ribulose-1,5 bisphosphate carboxylase/oxygenase large subunit methyltransferase, bound to S-adenosyl-L-methionine, and its non-reactive analogs Aza-adenosyl-L-methionine and Sinefungin, and characterized the binding of these ligands to a homolog of the enzyme. The structural and biochemical data collectively reveal that S-adenosyl-L-methionine is selectively recognized through carbon-oxygen hydrogen bonds between the cofactor's methyl group and an array of structurally conserved oxygens that comprise the methyl transfer pore in the active site. Furthermore, the structure of the enzyme co-crystallized with the product ⑀-N-trimethyllysine reveals a trigonal array of carbon-oxygen interactions between the ⑀-ammonium methyl groups and the oxygens in the pore. Taken together, these results establish a central role for carbon-oxygen hydrogen bonding in aligning the cofactor's methyl group for transfer to the lysine ⑀-amine and in coordinating the methyl groups after transfer to facilitate multiple rounds of lysine methylation.Protein lysine methylation has emerged as a prominent post-translational modification in gene regulatory and intracellular signaling pathways. In the nucleus, site-specific methylation of lysines within histones, transcription factors, and mitotic proteins governs a diverse array of processes within the nucleus including gene expression, DNA damage checkpoint control, cell cycle progression, and mitosis (1). In 2000, a breakthrough in our understanding of this modification occurred with the discovery of the first histone-specific protein lysine methyltransferases (PKMTs) 4 (2). These enzymes possess a conserved catalytic SET domain, a 120-residue motif that was named for three Drosophila gene regulatory factors, SU(VAR)3-9, E(Z), and TRX (3). This domain shares no apparent sequence or structural homology with other S-adenosyl-L-methionine (AdoMet)-dependent enzymes, establishing the SET domain family as a novel class of methyltransferases. This seminal discovery heralded the identification of a multitude of SET domain PKMTs, which methylate histone and non-histone substrates (1).Since their identification, crystal structures of several SET domain PKMTs in complex with various substrates and products have been reported, yielding insights into the catalytic mechanism and protein substrate specificity of this methyltransferase family. These structures include the histone methyltransferases SET7/9 (4 -8), SET8 (also known as PR-SET7) (9, 10), and Neurospora DIM-5 (11) as well as pea R...

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“…AdoMet was further purified by anion-exchange chromatography as reported previously (39). L-AzaAdoMet was synthesized and purified according to the method of Thompson et al (40).…”

Section: Methodsmentioning

confidence: 99%

Catalytic Roles for Carbon-Oxygen Hydrogen Bonding in SET Domain Lysine Methyltransferases

Couture

Hauk

Thompson

et al. 2006

Journal of Biological Chemistry

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“…Since the discovery of the first member, lysine 2,3-aminomutase, in 1970 (11), the radical AdoMet superfamily is now believed to comprise of thousands of proteins that participate in numerous biochemical processes in animals, plants, and microorganisms. These enzymes contain a motif of three cysteine residues (usually as CXXXCXXC) that nucleate a [4Fe-4S] cluster for binding of AdoMet via a unique Fe (Fe u ) site uncoordinated to the cysteine residue.…”

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confidence: 99%

Characterization of NocL Involved in Thiopeptide Nocathiacin I Biosynthesis

Zhang

Chen

Lin

et al. 2011

Journal of Biological Chemistry

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The radical S-adenosylmethionine (AdoMet) enzyme superfamily is remarkable at catalyzing chemically diverse and complex reactions. We have previously shown that NosL, which is involved in forming the indole side ring of the thiopeptide nosiheptide, is a radical AdoMet enzyme that processes L-Trp to afford 3-methyl-2-indolic acid (MIA) via an unusual fragmentation-recombination mechanism. We now report the expansion of the MIA synthase family by characterization of NocL, which is involved in nocathiacin I biosynthesis.

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“…One such pathway that operates in several bacterial species is the fermentation of lysine to yield acetate. Interestingly, the lysine fermentation pathway contains two analogous enzymes: lysine 5,6-aminomutase (5,6-LAM), which is AdoCbl-dependent (6,7), and lysine 2,3-aminomutase (2,3-LAM), which is an S-adenosylmethionine (AdoMet or SAM)-dependent ironsulfur enzyme (8)(9)(10). Both enzymes require pyridoxal 5Ј-phosphate (PLP) (8,11) in addition to AdoCbl or AdoMet, and both catalyze a 1,2 amino group shift with concomitant H atom migration (Fig.…”

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confidence: 99%

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

Berkovitch

Behshad

Tang

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

105

109

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Lysine 5,6-aminomutase is an adenosylcobalamin and pyridoxal-5 -phosphate-dependent enzyme that catalyzes a 1,2 rearrangement of the terminal amino group of DL-lysine and of L-␤-lysine. We have solved the x-ray structure of a substrate-free form of lysine-5,6-aminomutase from Clostridium sticklandii. In this structure, a Rossmann domain covalently binds pyridoxal-5 -phosphate by means of lysine 144 and positions it into the putative active site of a neighboring triosephosphate isomerase barrel domain, while simultaneously positioning the other cofactor, adenosylcobalamin, Ϸ25 Å from the active site. In this mode of pyridoxal-5 -phosphate binding, the cofactor acts as an anchor, tethering the separate polypeptide chain of the Rossmann domain to the triosephosphate isomerase barrel domain. Upon substrate binding and transaldimination of the lysine-144 linkage, the Rossmann domain would be free to rotate and bring adenosylcobalamin, pyridoxal-5 -phosphate, and substrate into proximity. Thus, the structure embodies a locking mechanism to keep the adenosylcobalamin out of the active site and prevent radical generation in the absence of substrate.A denosylcobalamin (AdoCbl; coenzyme B 12 ) is nature's biochemical radical reservoir, capable of catalyzing challenging chemical reactions by way of H atom abstraction and the generation of free-radical intermediates (1-3). AdoCbldependent isomerases catalyze 1,2 shifts between an H atom and a functional group such as -OH, -NH 3 ϩ , -(CO)S-coenzyme A, or other carbon-based groups. The catalytic power of AdoCbl lies in the homolytic cleavage of its weak (Ϸ30 kcal͞mol) organometallic C-Co bond, formed between an octahedral Co(III) center with five N coordinations and a 5Ј-deoxyadenosyl (Ado) axial ligand. C-Co bond homolysis results in the transient formation of cob(II)alamin and 5Ј-deoxyadenosyl radical (Ado • ). Ado • abstracts an H atom from the substrate, forming a substrate radical and 5Ј-deoxyadenosine (AdoH). To close the catalytic cycle, substrate reabstracts the H atom from AdoH, and recombination of cob(II)alamin and Ado • accompanies product formation. Amazingly, the enzymatic rate of C-Co bond homolysis is enhanced by a factor of Ϸ10 12 over nonenzymatic homolysis (4, 5). AdoCbl-dependent isomerases are often present in catabolic pathways and can serve to rearrange the substrate's carbon skeleton and͞or functional groups for further degradation. One such pathway that operates in several bacterial species is the fermentation of lysine to yield acetate. Interestingly, the lysine fermentation pathway contains two analogous enzymes: lysine 5,6-aminomutase (5,6-LAM), which is AdoCbl-dependent (6, 7), and lysine 2,3-aminomutase (2,3-LAM), which is an S-adenosylmethionine (AdoMet or SAM)-dependent ironsulfur enzyme (8-10). Both enzymes require pyridoxal 5Ј-phosphate (PLP) (8, 11) in addition to AdoCbl or AdoMet, and both catalyze a 1,2 amino group shift with concomitant H atom migration (Fig. 1A). In 5,6-LAM, AdoCbl is the source of the transient Ado • , whereas, in 2,3-L...

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Enzymic Preparation ofL-β-Lysine

Cited by 22 publications

References 9 publications

Catalytic Roles for Carbon-Oxygen Hydrogen Bonding in SET Domain Lysine Methyltransferases

Catalytic Roles for Carbon-Oxygen Hydrogen Bonding in SET Domain Lysine Methyltransferases

Characterization of NocL Involved in Thiopeptide Nocathiacin I Biosynthesis

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

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