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 Lactobacillus leichmannii is a 5'-deoxyadenosylcobalamin-dependent (AdoCbl; Coenzyme B12) enzyme. RTPR is also a prototypical adenosylcobalamin-dependent ribonucleotide reductase, one that, as its name indicates, converts ribonucleoside triphosphates (NTP) to deoxyribonucleoside triphosphates (dNTP). Upon substrate binding to RTPR, AdoCbl's cobalt-carbon bond is cleaved to generate cob(II)alamin, 5'-deoxyadenosine, and the cysteine (C408) derived thiyl radical. Five key cysteines (Cys 119, 408, 419, 731, and 736), from among the ten total cysteines, are involved in RTPR's catalytic mechanism. A critical examination of the RTPR isolation and purification literature suggested that the purification protocol currently used results in RTPR which contains 2040% microheterogeneity, along with minor contamination by other proteins. In addition, no report of crystalline RTPR has ever appeared. The literature indicates that irreversible cysteine oxidation (e.g., to -SO2H or -SO3H) is one highly plausible reason for the microheterogeneity of RTPR. The literature also indicates that improvement in the level of enzyme purity is the most effective next step in coaxing enzymes to crystallize that have previously failed to do so. A shortened, improved purification of RTPR has been developed, one involving a shorter purification time, a lower pH, a higher concentration of the more effective reductant DTT (all designed to help protect the cysteines from oxidation), and a final step utilizing our recently reported, improved dGTP-based affinity chromatography resin. The resultant RTPR is approximately 20-30% higher in both specific activity and in its ability to undergo single turnovers, and is homogeneous by mass spectrometry and dynamic light scattering. Additionally, the revised purification procedure eliminates > 30 proteins present in 2-3% amounts along with damaged RTPR that does not bind properly (i.e. tightly) to the dGTP-affinity resin. Finally, dGTP-based affinity chromatography purified RTPR has yielded the first reported, albeit small, single crystals of RTPR.
Suto
R K
RK
Department of Biochemistry, Colorado State University, Fort Collins 80523, USA.
Whalen
M A
MA
Finke
R G
RG
D000494:Allosteric Regulation / Q000187:drug effects; D000495:Allosteric Site / Q000187:drug effects; D002846:Chromatography, Affinity / Q000379:methods*; D002847:Chromatography, Agarose; D003038:Cobamides / Q000737:chemistry / Q000378:metabolism*; D003460:Crystallization; D003461:Crystallography; D003848:Deoxyguanine Nucleotides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007778:Lactobacillus / Q000201:enzymology*; D013058:Mass Spectrometry; D008956:Models, Chemical; D012117:Resins, Synthetic / Q000737:chemistry; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000302:isolation & purification*; D012542:Scattering, Radiation; D019346:Sodium Acetate / Q000494:pharmacology; D013379:Substrate Specificity
D003038:Cobamides; D003848:Deoxyguanine Nucleotides; D012117:Resins, Synthetic; D019346:Sodium Acetate; C029603:deoxyguanosine triphosphate; D012264:Ribonucleotide Reductases; C057295:ribonucleoside-triphosphate reductase; C000913:cobamamide