Using the Structure‐Function Linkage Database to Characterize Functional Domains in Enzymes

Brown, Shoshana D.; Babbitt, Patricia C.

doi:10.1002/0471250953.bi0210s48

Cited by 5 publications

(6 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…32−37 The identity of pyruvate as the source of the carbons in the imidazoline ring was established using 13 C pyruvate isotopologues in vitro, which showed that C2 and C3 of pyruvate are incorporated into the tRNA base and C1 is lost as an unidentified side product, proposed to be either carbon dioxide or formate. 33 TYW1, like other enzymes in the 100,000-plus membered radical SAM superfamily, 38 uses a SAM-bound [4Fe−4S] cluster to reductively cleave SAM, forming methionine and a highly reactive 5′-deoxyadenosyl radical species (dAdo•) that initiates radical-based chemistry. 39 TYW1 also has an auxiliary iron−sulfur cluster, identified as a [4Fe−4S] cluster by an electron paramagnetic resonance (EPR) and Mossbauer study.…”

Section: ■ Introductionmentioning

confidence: 99%

“…TYW1, like other enzymes in the 100,000-plus membered radical SAM superfamily, uses a SAM-bound [4Fe–4S] cluster to reductively cleave SAM, forming methionine and a highly reactive 5′-deoxyadenosyl radical species (dAdo•) that initiates radical-based chemistry . TYW1 also has an auxiliary iron–sulfur cluster, identified as a [4Fe–4S] cluster by an electron paramagnetic resonance (EPR) and Mössbauer study .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Biochemical and Structural Characterization of a Schiff Base in the Radical-Mediated Biosynthesis of 4-Demethylwyosine by TYW1

et al. 2018

View full text Add to dashboard Cite

TYW1 is a radical S-adenosyl-l-methionine (SAM) enzyme that catalyzes the condensation of pyruvate and N-methylguanosine to form the posttranscriptional modification, 4-demethylwyosine, in situ on transfer RNA (tRNA). Two mechanisms have been proposed for this transformation, with one of the possible mechanisms invoking a Schiff base intermediate formed between a conserved lysine residue and pyruvate. Utilizing a combination of mass spectrometry and X-ray crystallography, we have obtained evidence to support the formation of a Schiff base lysine adduct in TYW1. When 13C labeled pyruvate is used, the mass shift of the adduct matches that of the labeled pyruvate, indicating that pyruvate is the source of the adduct. Furthermore, a crystal structure of TYW1 provides visualization of the Schiff base lysine–pyruvate adduct, which is positioned directly adjacent to the auxiliary [4Fe–4S] cluster. The adduct coordinates the unique iron of the auxiliary cluster through the lysine nitrogen and a carboxylate oxygen, reminiscent of how the radical SAM [4Fe–4S] cluster is coordinated by SAM. The structure provides insight into the binding site for tRNA and further suggests how radical SAM chemistry can be combined with Schiff base chemistry for RNA modification.

show abstract

Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Biochemical and Structural Characterization of a Schiff Base in the Radical-Mediated Biosynthesis of 4-Demethylwyosine by TYW1

et al. 2018

View full text Add to dashboard Cite

show abstract

“…The RS superfamily contains >100,000 members catalyzing a myriad of biologically important transformations. 13 The enzymes within this superfamily generally contain the CxxxCxxC motif, identified as the radical SAM signature sequence. 14 These conserved cysteines provide thiolate ligands to coordinate three iron atoms of a [4Fe-4S] cluster (referred to as the RS cluster) with the fourth iron of the cluster coordinated by the α-carboxylate and α-amino moieties of SAM (Fig.…”

mentioning

confidence: 99%

Biochemical and Spectroscopic Characterization of a Radical S-Adenosyl-l-methionine Enzyme Involved in the Formation of a Peptide Thioether Cross-Link

et al. 2016

View full text Add to dashboard Cite

Peptide-derived natural products are a class of metabolites that afford the producing organism a selective advantage over other organisms in their biological niche. While the polypeptide antibiotic produced by the non-ribosomal polypeptide synthetases (NRPS) are the most widely recognized, the ribosomally-synthesized and post-translationally-modified peptides (RiPPs) are an emerging group of natural products with diverse structures and biological functions. Both the NRPS derived peptides and the RiPPs undergo extensive post-translational modifications to produce structural diversity. Here we report the first characterization of the six cysteines in forty-five (SCIFF) [Haft, D.H. and Basu M.K. (2011) J Bacteriol 193, 2745–2755] peptide maturase Tte1186, which is a member of the radical SAM superfamily. Tte1186 catalyzes the formation of a thioether crosslink in the peptide Tte1186a encoded by an orf located upstream of the maturase, under reducing conditions in the presence of SAM. Tte1186 contains three [4Fe-4S] clusters that are indispensable for thioether crosslink formation; however, only one cluster catalyzes the reductive cleavage of SAM. Mechanistic imperatives for the reaction catalyzed by the thioether forming radical SAM maturases will be discussed.

show abstract

“…The S- adenosyl- l -methionine (AdoMet) radical enzyme superfamily has >300000 distinct members that carry out a plethora of diverse chemical transformations. 1 Most of the enzymes in this superfamily possess a characteristic CxxxCxxC motif housing a site-differentiated [4Fe-4S] cluster [reaction cluster (RC)] that in turn binds AdoMet at a unique iron. In the +1 oxidation state, the RC reductively cleaves AdoMet to form the 5′-deoxyadeonsyl radical intermediate (5′-dA • ).…”

mentioning

confidence: 99%

Deconvoluting the Reduction Potentials for the Three [4Fe-4S] Clusters in an AdoMet Radical SCIFF Maturase

et al. 2018

View full text Add to dashboard Cite

Enzymes in the S -adenosyl- l -methionine (AdoMet) radical enzyme superfamily are metalloenzymes that catalyze a wide variety of complex radical-mediated transformations with the aid of a [4Fe-4S] cluster, which is required for activation of AdoMet to generate the 5′-deoxyadenosyl radical to initiate the catalytic cycle. In addition to this cluster, some enzymes share an additional domain, the SPASM domain, that houses auxiliary FeS clusters whose functional significance is not clearly understood. The AdoMet radical enzyme Tte1186, which catalyzes a thioether cross-link in a cysteine rich peptide (SCIFF), has two auxiliary [4Fe-4S] clusters within a SPASM domain that are required for enzymatic activity but not for the generation of the 5′-deoxyadenosyl radical intermediate. Here we demonstrate the ability to measure independently the midpoint potentials of each of the three [4Fe-4S] clusters by employing Tte1186 variants for which only the first, second, or AdoMet binding cluster is bound. This allows, for the first time, assignment of reduction potentials for all clusters in an AdoMet radical enzyme with a SPASM domain. Our results show that the clusters have midpoint potentials that are within 100 mV of each other, suggesting that their electrochemical properties are not greatly influenced by the presence of the nearby clusters.

show abstract

Using the Structure‐Function Linkage Database to Characterize Functional Domains in Enzymes

Cited by 5 publications

References 22 publications

Biochemical and Structural Characterization of a Schiff Base in the Radical-Mediated Biosynthesis of 4-Demethylwyosine by TYW1

Biochemical and Structural Characterization of a Schiff Base in the Radical-Mediated Biosynthesis of 4-Demethylwyosine by TYW1

Biochemical and Spectroscopic Characterization of a Radical S-Adenosyl-l-methionine Enzyme Involved in the Formation of a Peptide Thioether Cross-Link

Deconvoluting the Reduction Potentials for the Three [4Fe-4S] Clusters in an AdoMet Radical SCIFF Maturase

Contact Info

Product

Resources

About