Characterization of an alternate ribonucleotide reductase small subunit, NrdF1, from Mycobacterium tuberculosis

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides into deoxyribonucleotides (dNTPs) and are responsible for regulating a balanced supply of nucleotides for DNA replication and repair. These enzymes are grouped into three classes based on metallocofactor requirement, oxygen sensitivity and radical generation. Class I RNRs are multi-subunit complexes, which can exist as heterotetramers alpha2beta2, or as hexameric aggregates, as in mammalian cells alpha6beta6. The enzymes of this class are oxygen-dependent and require iron to generate the essential tyrosyl radical. The large subunit, alpha, contains the active site and allosteric sites, while the small subunit, beta, houses the iron center and generates the tyrosyl radical. Recent evidence show organisms contain more than one class of RNRs and/or multiple variants of the small subunit protein, which regulate dNTP pools under changing environmental conditions.; Mycobacterium tuberculosis contains both a class I and a class II RNR. The class I RNR system composed of the nrdE, alpha subunit and the nrdF2, beta subunit. M. tuberculosis encodes two additional small (beta) subunit variants ( nrdF1, nrdB). Under normal growth conditions, the class I NrdEF2 system is active. However, microarray studies have revealed that nrdF1, is upregulated in response to DNA damaging agents. All class I RNR small subunits contain 16 amino acids, important for RNR complex formation and catalysis, which are conserved in NrdF1.; The crystal structure of the NrdF2 small subunit was determined to 2.2 A resolution. Comparative modeling suggests that radical transfer would be the same in the NrdF1 alternate small subunit. Studies done to determine hydroxyurea sensitivity illustrate the tyrosyl radical of the NrdF1 dimer is more resistant to the radical scavenger, hydroxyurea and appears to be better protected within the subunit. This suggests a more buried tyrosyl environment, and hence an overall difference in active site structure. In order to determine whether NrdF1 is truly a functional small subunit, we studied the ability of NrdF1 to form an active complex with NrdE (NrdEF1). We show that NrdF1 can bind to NrdE to form a catalytically active NrdEF1 complex, in the absence of NrdF2. Activity measurements illustrate that although the four substrates are the same, NrdEF1 and NrdEF2 have distinct kinetic parameters and appear to be important under different conditions.; Activity assays performed in the presence of multiple nucleotide effectors display an increase in the activity of the NrdEF RNR enzyme. This new finding suggests that this class of RNRs contain an additional allosteric binding site. These studies provide a basis for understanding how organisms alter RNR expression and/or activity to regulate nucleotide pools in response to different environmental conditions.