Adenosylcobalamin-dependent ribonucleoside triphosphate reductase from Lactobacillus leichmannii. Rapid, improved purification involving dGTP-based affinity chromatography plus biophysical characterization studies demonstrating enhanced, "Crystallographic level" purity

Ribonucleoside triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillu s leichmannii is a 5'-deoxyadenosylcobalamin-dependent (AdoCbl; Coenzyme B- 12) enzyme. RTPR is also a prototypical adenosylcobalamin-dependent ribonuc leotide reductase, one that, as its name indicates, converts ribonucleoside triphosphates (NTP) to deoxyribonucleoside triphosphates (cNTP). Upon subs trate binding to RTPR, AdoCbl's cobalt-carbon bond is cleaved to generate c ob(II)alamin, 5'-deoxyadenosine, and the cysteine (C408) derived thiyl radi cal. Five key cysteines (Cys 119, 408, 419, 731, and 736), from among the t en total cysteines, are involved in RTPR's catalytic mechanism. A critical examination of the RTPR isolation and purification literature su ggested that the purification protocol currently used results in RTPR which contains 20-40% microheterogeneity, along with minor contamination by othe r proteins. In addition, no report of crystalline RTPR has ever appeared. T he literature indicates that irreversible cysteine oxidation (e.g., to -SO2 H or -SO3H) is one highly plausible reason for the microheterogeneity of RT PR. The literature also indicates that improvement in the level of enzyme p urity is the most effective next step in coaxing enzymes to crystallize tha t have previously failed to do so. A shortened, improved purification of RTPR has been developed, one involvin g a shorter purification time, a lower pH, a higher concentration of the mo re effective reductant DTT (all designed to help protect the cysteines from oxidation), and a final step utilizing our recently reported, improved dGT P-based affinity chromatography resin. The resultant RTPR is similar to 20- 30% higher in both specific activity and in its ability to undergo single t urnovers, and is homogeneous by mass spectrometry and dynamic light scatter ing. Additionally, the revised purification procedure eliminates > 30 prote ins present in 2-3% amounts along with damaged RTPR that does not bind prop erly (i.e. tightly) to the dGTP-affinity resin. Finally, dGTP-based affinit y chromatography purified RTPR has yielded the first reported, albeit small , single crystals of RTPR.