Isolation of the gene for the B12-dependent ribonucleotide reductase from Anabaena sp. strain PCC 7120 and expression in Escherichia coli - PubMed (original) (raw)

Isolation of the gene for the B12-dependent ribonucleotide reductase from Anabaena sp. strain PCC 7120 and expression in Escherichia coli

Florence K Gleason et al. J Bacteriol. 2002 Dec.

Abstract

The gene for ribonucleotide reductase from Anabaena sp. strain PCC 7120 was identified and expressed in Escherichia coli. This gene codes for a 1,172-amino-acid protein that contains a 407-amino-acid intein. The intein splices itself from the protein when it is expressed in E. coli, yielding an active ribonucleotide reductase of 765 residues. The mature enzyme was purified to homogeneity from E. coli extracts. Anabaena ribonucleotide reductase is a monomer with a molecular weight of approximately 88,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Superose 12 column chromatography. The enzyme reduces ribonucleotides at the triphosphate level and requires a divalent cation and a deoxyribonucleoside triphosphate effector. The enzyme is absolutely dependent on the addition of the cofactor, 5'-adenosylcobalamin. These properties are characteristic of the class II-type reductases. The cyanobacterial enzyme has limited sequence homology to other class II reductases; the greatest similarity (38%) is to the reductase from Lactobacillus leichmannii. In contrast, the Anabaena reductase shows over 90% sequence similarity to putative reductases found in genome sequences of other cyanobacteria, such as Nostoc punctiforme, Synechococcus sp. strain WH8102, and Prochlorococcus marinus MED4, suggesting that the cyanobacterial reductases form a closely related subset of the class II enzymes.

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Figures

FIG. 1.

(A) Alignment of the Anabaena strain PCC 7120 RNR with the reductase from L. leichmannii. The five cysteines corresponding to those in the active site of the Lactobacillus enzyme are indicated with a ▾. The site of the intein insertion in the Anabaena enzyme is indicated by a filled arrow. The alignment was generated with the GAP program (Genetics Computer Group, University of Wisconsin). (B) Intein sequence expressed by the Anabaena nrdJ gene. The putative endonuclease active site is underlined.

FIG. 2.

SDS-PAGE of an Anabaena RNR preparation. Lane 1, molecular weight standards for myosin (220,000), phosphorylase b (97,000), bovine serum album (66,000), ovalbumin (45,000), and carbonic anhydrase (30,000); lane 2, DEAE pool; lane 3, chelating pool; lane 4, octyl pool; lane 5, Reactive Red 120 agarose pool, 3 μg of protein; lane 6, Reactive Red 120 agarose pool, 6 μg of protein.

FIG. 3.

Estimation of the _M_r of native Anabaena RNR by FPLC on a Superose 12 column. The column was calibrated with four standards (λ): sweet potato β-amylase (Mr, 200,000), yeast alcohol dehydrogenase (Mr, 150,000), bovine serum albumin (Mr, 66,000), and egg albumin (Mr, 45,000). The column was equilibrated and run as described in the text under “Methods.” Anabaena RNR elutes at the position indicated by *, with a calculated molecular weight of 88,000.

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Isolation of the gene for the B12-dependent ribonucleotide reductase from Anabaena sp. strain PCC 7120 and expression in Escherichia coli - PubMed (original) (raw)