Structural basis for tRNA methylthiolation by the radical SAM enzyme MiaB.

Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio-N-isopentenyladenosine (msiA) modification occurs at position 37 (A37) in transfer RNAs that contain adenine in position 36 of the anticodon, and serves to promote efficient A:U codon-anticodon base-pairing and to prevent unintended base pairing by near cognates, thus enhancing translational fidelity. The msiA modification is installed onto isopentenyladenosine (iA) by MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase.

First Step in Catalysis of the Radical -Adenosylmethionine Methylthiotransferase MiaB Yields an Intermediate with a [3Fe-4S]-Like Auxiliary Cluster.

The enzyme MiaB catalyzes the attachment of a methylthio (-SCH) group at the C2 position of -(isopentenyl)adenosine (iA) in the final step of the biosynthesis of the hypermodified tRNA nucleotide 2-methythio--(isopentenyl)adenosine (msiA). MiaB belongs to the expanding subgroup of enzymes of the radical -adenosylmethionine (SAM) superfamily that harbor one or more auxiliary [4Fe-4S] clusters in addition to the [4Fe-4S] cluster that all family members require for the reductive cleavage of SAM to afford the common 5'-deoxyadenosyl 5'-radical (5'-dA) intermediate. While the role of the radical SAM cluster in generating the 5'-dA is well understood, the detailed role of the auxiliary cluster, which is essential for MiaB catalysis, remains unclear.

Parsing redox potentials of five ferredoxins found within Thermotoga maritima.

Most organisms contain multiple soluble protein-based redox carriers such as members of the ferredoxin (Fd) family, that contain one or more iron-sulfur clusters. The potential redundancy of Fd proteins is poorly understood, particularly in connection to the ability of Fd proteins to deliver reducing equivalents to members of the "radical SAM," or S-adenosylmethionine radical enzyme (ARE) superfamily, where the activity of all known AREs requires that an essential iron-sulfur cluster bound by the enzyme be reduced to the catalytically relevant [Fe S ] oxidation state. As it is still unclear whether a single Fd in a given organism is specific to individual redox partners, we have examined the five Fd proteins found within Thermotoga maritima via direct electrochemistry, to compare them in a side-by-side fashion for the first time.

Ferredoxins as interchangeable redox components in support of MiaB, a radical S-adenosylmethionine methylthiotransferase.

MiaB is a member of the methylthiotransferase subclass of the radical S-adenosylmethionine (SAM) superfamily of enzymes, catalyzing the methylthiolation of C2 of adenosines bearing an N -isopentenyl (i A) group found at position 37 in several tRNAs to afford 2-methylthio-N -(isopentenyl)adenosine (ms i A). MiaB uses a reduced [4Fe-4S] cluster to catalyze a reductive cleavage of SAM to generate a 5'-deoxyadenosyl 5'-radical (5'-dA•)-a required intermediate in its reaction-as well as an additional [4Fe-4S] auxiliary cluster. In Escherichia coli and many other organisms, re-reduction of the [4Fe-4S] cluster to the [4Fe-4S] state is accomplished by the flavodoxin reducing system.

Transformations of the FeS Clusters of the Methylthiotransferases MiaB and RimO, Detected by Direct Electrochemistry.

The methylthiotransferases (MTTases) represent a subfamily of the S-adenosylmethionine (AdoMet) radical superfamily of enzymes that catalyze the attachment of a methylthioether (-SCH) moiety on unactivated carbon centers. These enzymes contain two [4Fe-4S] clusters, one of which participates in the reductive fragmentation of AdoMet to generate a 5'-deoxyadenosyl 5'-radical and the other of which, termed the auxiliary cluster, is believed to play a central role in constructing the methylthio group and attaching it to the substrate. Because the redox properties of the bound cofactors within the AdoMet radical superfamily are so poorly understood, we have examined two MTTases in parallel, MiaB and RimO, using protein electrochemistry.

Identification of an intermediate methyl carrier in the radical S-adenosylmethionine methylthiotransferases RimO and MiaB.

RimO and MiaB are radical S-adenosylmethionine (SAM) enzymes that catalyze the attachment of methylthio (-SCH3) groups to macromolecular substrates. RimO attaches a methylthio group at C3 of aspartate 89 of protein S12, a component of the 30S subunit of the bacterial ribosome. MiaB attaches a methylthio group at C2 of N(6)-(isopentenyl)adenosine, found at nucleotide 37 in several prokaryotic tRNAs.