Ribonucleotide reductase (RNR) for dNTP production

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1978
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 395
Illegal AgeI site found at 582
Illegal AgeI site found at 924
Illegal AgeI site found at 1323
Illegal AgeI site found at 3143
1000
COMPATIBLE WITH RFC[1000]

This is a composite part, designed for the AdAPTED project of iGEM Athens 2021. This part was designed to be inserted into the pGGA plasmid, with a T7-LacO promoter that will lead to the overexpression of RNR. The RBS and Terminator used for this transcriptional unit were BBa_B0030 and BBa_B0015 respectively. After the transformation of E. coli BL21 a dNTP producing bacteria will occur.

Function

RNR catalyzes the conversion of all four ribonucleotides triphosphates (NTPs) into the corresponding dNTPs, therefore providing the building blocks for the synthesis and repair of DNA. This conversion is achieved by the reduction of the C2’-OH bond. This biochemical pathway is the only de novo dNTP production method. The reduction occurs for all nucleotides at a single active site. RNR is divided into three classes I, which is further divided into class Ia, Ib, and Ic, II, and III.

Protein Structure

Class I RNR is composed of two homoderic subunits α and β. When active, both in eucaryotes and procaryotes, the two proteins are associated in a dimeric or other oligomeric form, such as (alpha)n(beta)m. Based on the subclass of RNR, the metal centre required for the radical production, differs. Despite the differences apparent in the classes of RNR, all three contain a conserved cysteine residue at the active site. This cysteine residue is possibly converted into a thiyl radical, initiating the substrate turnover, by abstraction of a hydrogen atom from a ribose ring of the substrate. The substrate binding active site is located in the alpha 2 homodimer, encoded by nrdA. The binding site for the two irons is contained in the beta 2 homodimer, encoded by nrdB.

Ia RNR

RNR Ia is dependent from oxygen, contains a di-iron center (FeIII-O-FeIII), has two allosteric centers, can be inhibited by ATP, is distributed in Eukaryotes, eubacteria, archaea, bacteriophages, and virus, is either in the (alpha)2(beta)2 or (alpha)6(beta)6 form, and is encoded by the nrdA and nrdB genes.

nrdA

The alpha subunit contains the catalytic subunit, with the active site, for the nucleotide reduction. It also contains two allosteric sites for the allosteric regulation of RNR.

nrdB

The beta subunit contains the metallocofactor (di-iron), required for the reduction initiation.

SOURCE OF THIS PART

NCBI code: AAC75294.1 (nrdA) and AAC75295.1 (nrdB)

References

1. Torrents, E. (2014). Ribonucleotide reductases: essential enzymes for bacterial life. Frontiers in Cellular and Infection Microbiology, 4. doi:10.3389/fcimb.2014.00052
2. Matthias Kolberg, Kari R Strand, Pål Graff, K Kristoffer Andersson, Structure, function, and mechanism of ribonucleotide reductases, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Volume 1699, Issues 1–2, 2004, Pages 1-34, ISSN 1570-9639, https://doi.org/10.1016/j.bbapap.2004.02.007.