The Crystal Structure of Mycobacterium tuberculosis NrdH at 0.87 Å Suggests a Possible Mode of Its Activity

Abstract

Members of the NrdH family of redox proteins, which consists of small glutaredoxin-like proteins with thioredoxin-like activity, serve as the reducing partners of class Ib ribonucleotide reductases. Here, we report the crystal structure of NrdH from Mycobacterium tuberculosis, refined to a crystallographic R factor of 14.02% (Rfree = 15.53%) at 0.87 Å resolution. The tertiary structure of M. tuberculosis NrdH has a typical thioredoxin fold as expected. The extremely high resolution of the structure allows us to dissect the functionality of the protein in great depth. Structural superimposition of M. tuberculosis NrdH and thioredoxin reductase over the Escherichia coli thioredoxin reductase-thioredoxin complex suggests the ability of NrdH to accept electrons from M. tuberculosis thioredoxin reductase. This raises the important question of why glutaredoxins are unable to accept electrons from thioredoxin reductases and why thioredoxins are unable to reduce ribonucleotide reductases. Furthermore, forms of NrdH from other organisms have been shown to be a specific reductant of class Ib ribonucleotide reductases. We attempt to explain this substrate specificity by modeling the C-terminal peptide of a ribunucleotide subunit, NrdE, in the active site of NrdH using the already available Grx-NrdA-Cter-peptide structure. Statistical coupling analysis of NrdH, glutaredoxins, and thioredoxins reveals different sets of co-evolving contiguous clusters of amino acid residues, which might explain the differences in the biochemical properties of these structurally similar yet functionally distinct subclasses of proteins.

Streptococcus sanguinis Class Ib Ribonucleotide Reductase

“…1) (2, 55). A number of artificial and endogenous systems are capable of mediating this re-reduction step, including dithiothreitol (DTT),Trx/TrxR/NADPH, Grx/ GSH/GR/NADPH, and NrdH/TrxR/NADPH, where Trx is thioredoxin, Grx is glutaredoxin, and GR is glutaredoxin reductase (6,11,20,21,56). The observation that nrdH is colocalized in the same operon with nrdE and nrdF in S. sanguinis and many other organisms makes this protein the most reasonable candidate for the endogenous reductant.…”

The concerted action of a positive charge and hydrogen bonds dynamically regulates the pKa of the nucleophilic cysteine in the NrdH‐redoxin family

“…Recently, we determined a p K a of 6.2 and 6.3 for the N‐terminal cysteine of Corynebacterium glutamicum (Cg) and Mycobacterium tuberculosis NrdH‐redoxin, respectively . However, no structural information on the origin of this relatively low p K a is available since all the reported structures of NrdH‐redoxins are in the oxidized state (i.e., the active site cysteines are disulfide bonded) . In the other members of the thioredoxin family [e.g., thioredoxin (Trx), glutaredoxin (Grx), and mycoredoxin (Mrx)], the relatively low p K a of the nucleophilic cysteine is governed by the stabilizing effect of hydrogen bonds on the thiolate form of this cysteine .…”

“…5 However, no structural information on the origin of this relatively low pK a is available since all the reported structures of NrdH-redoxins are in the oxidized state (i.e., the active site cysteines are disulfide bonded). 2,[5][6][7] In the other members of the thioredoxin family [e.g., thioredoxin (Trx), glutaredoxin (Grx), and mycoredoxin (Mrx)], the relatively low pK a of the nucleophilic cysteine is governed by the stabilizing effect of hydrogen bonds on the thiolate form of this cysteine. 8,9 However, the contribution of hydrogen bonds to the lowering of the pK a of a cysteine is often not easily recognized from the crystal or solution structures.…”

Mycobacteriophage L5Gp56, a novel member of the NrdH family of redoxins

“…Homology modeling confirms that the thioredoxin fold is also present in L5 Gp50. The basic structure is similar to NrdH of E. coli (Stehr et al ., ) and M. tuberculosis (Phulera & Mande, ). However, the C‐terminal region of L5 NrdH encompassing the β4‐loop‐α3 domain appears to be structurally different.…”