The Ribonucleotide Reductase System of Lactococcus lactis

Abstract

Escherichia coli contains the genetic information for three separate ribonucleotide reductases. Two of them (class I enzymes), coded by the nrdAB and nrdEF genes, respectively, contain a tyrosyl radical, whose generation requires oxygen. The NrdAB enzyme is physiologically active. The function of the nrdEF gene is not known. The third enzyme (class III), coded by nrdDG, operates during anaerobiosis. The DNA of Lactococcus lactis contains sequences homologous to the nrdDG genes. Surprisingly, an nrdD ؊ mutant of L. lactis grew well under standard anaerobic growth conditions. The ribonucleotide reductase system of this mutant was shown to consist of an enzyme of the NrdEF-type and a small electron transport protein. The coding operon contains the nrdEF genes and two open reading frames, one of which (nrdH) codes for the small protein. The same gene organization is present in E. coli. We propose that the aerobic class I ribonucleotide reductases contain two subclasses, one coded by nrdAB, active in E. coli and eukaryotes (class Ia), the other coded by nrdEF, present in various microorganisms (class Ib). The NrdEF enzymes use NrdH proteins as electron transporter in place of thioredoxin or glutaredoxin used by NrdAB enzymes. The two classes also differ in their allosteric regulation by dATP.Ribonucleotide reductases are essential enzymes that catalyze the reduction of ribonucleoside di-or triphosphates and thereby provide the building blocks required for DNA replication and repair. Three different classes of enzymes are known (1), each with a distinct protein structure but all requiring a protein radical for catalysis and all regulated by similar allosteric effects.Class I reductases are aerobic enzymes present in all higher organisms and certain microorganisms, among them Escherichia coli (2). This bacterium actually has the potential to produce two separate class I enzymes. One of them, coded for by the nrdA and -B genes (3) is the functional enzyme during the growth of E. coli and has been the prototype for all class I enzymes. The second enzyme is coded for by the nrdE and -F genes (4), first discovered in Salmonella typhimurium (5), and is normally not fully functional. Expression of the chromosomal nrdEF genes thus is not sufficient to complement mutations in nrdAB (5). The two enzymes show a limited sequence similarity but contain certain strategical amino acids in identical positions. They differ to some extent in their allosteric regulation and with respect to their hydrogen donors (6). A functionally active reductase of the NrdEF-type was recently found in Mycobacterium tuberculosis (7). Mycoplasma genitalium contains the nrdEF genes but not the nrdAB genes (8).All class I enzymes consist of two proteins that are named R1 and R2 for NrdAB enzymes (2) and R1E and R2F (6) for the NrdEF enzymes. Each protein has specific functions: R1 and R1E contain the binding sites for both substrates and allosteric effectors and carry out the actual reduction of the ribonucleotide. R2 and R2F contain diferric iron centers...

The Manganese-containing Ribonucleotide Reductase ofCorynebacterium ammoniagenes Is a Class Ib Enzyme

“…6a). This type of allosteric regulation is typical of class Ib enzymes and differs from that of class Ia enzymes (10,12) .…”

“…The NrdH product has been found to be a specific electron donor for the class Ib enzyme of S. typhimurium and L. lactis (12,37). The deduced NrdI product comprises 144 amino acid residues and has a predicted molecular mass of 15.8 kDa.…”

“…These enzymes share with class I enzymes the subunit composition and distinct sequence similarity, including all highly conserved residues, such as the iron ligands, the tyrosyl radical, and active site cysteines. Thus, the discovery of these enzymes led to the division of the class I RNR in two subclasses, classes Ia and Ib (12). The class Ia reductase is encoded by the nrdA and nrdB genes, coding for the homodimeric proteins R1 and R2, respectively, and the class Ib reductase is encoded by the nrdE and nrdF genes, coding for the homodimeric proteins R1E and R2F, respectively.…”

“…Moreover, the physiological role of these "silent" enzymes is still unknown. However, the Lactococcus lactis RNR was found to be a func-tionally active reductase of the class Ib type (12), and the purified enzyme from Mycobacterium tuberculosis also turned out to belong to this class (13,48). Class Ib genes have also been described in Bacillus subtilis, Mycoplasma genitalium, and M. pneumoniae (14 -16).…”

Streptococcus sanguinis Class Ib Ribonucleotide Reductase

“…In many, but not all, of the class Ib RNRs, NrdH can re-reduce the disulfide in ␣ produced concomitant with dNDP formation (Fig. 1) and thus is required for multiple turnovers (6,11,(17)(18)(19). NrdH itself can be re-reduced in vitro by TrxR and NADPH in E. coli, B. anthracis, Staphylococcus aureus, and Corynebacterium glutamicum (6,11,20,21).…”