Characterization of a periplasmic S1-like nuclease coded by the Mesorhizobium loti symbiosis island.
DNA sequences encoding hypothetical proteins homologous to S1 nuclease from Aspergillus oryzae are found in many organisms including fungi, plants, pathogenic bacteria, and eukaryotic parasites. One of these is the M1 nuclease of Mesorhizobium loti which we demonstrate herein to be an enzymatically active, soluble, and stable S1 homolog that lacks the extensive mannosyl-glycosylation found in eukaryotic S1 nuclease homologs. We have expressed the cloned M1 protein in M. loti and purified recombinant native M1 to near homogeneity and have also isolated a homogeneous M1 carboxy-terminal hexahistidine tag fusion protein. Mass spectrometry and N-terminal Edman degradation sequencing confirmed the protein identity. The enzymatic properties of the purified M1 nuclease are similar to those of S1. At acidic pH M1 is 25 times more active on single-stranded DNA than on double-stranded DNA and 3 times more active on single-stranded DNA than on single-stranded RNA. At neutral pH the RNase activity of M1 exceeds the DNase activity. M1 nicks supercoiled RF-I plasmid DNA and rapidly cuts the phosphodiester bond across from the nick in the resultant relaxed RF-II plasmid DNA. Therefore, M1 represents an active bacterial S1 homolog in spite of great sequence divergence. The biochemical characterization of M1 nuclease supports our sequence alignment that reveals the minimal 21 amino acid residues that are necessarily conserved for the structure and functions of this enzyme family. The ability of M1 to degrade RNA at neutral pH implies previously unappreciated roles of these nucleases in biological systems.
Pimkin
Maxim
M
Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
Miller
C Glenn
CG
Blakesley
Lauryn
L
Oleykowski
Catherine A
CA
Kodali
Nagendra S
NS
Yeung
Anthony T
AT
D020561:Alphaproteobacteria / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D017124:Conserved Sequence; D003461:Crystallography; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004720:Endonucleases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D044404:Genomic Islands; D006863:Hydrogen-Ion Concentration; D015636:Magnesium Chloride / Q000737:chemistry; D008969:Molecular Sequence Data; D019897:Periplasm / Q000201:enzymology; D010957:Plasmids / Q000737:chemistry; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D015636:Magnesium Chloride; D012965:Sodium Chloride; D012313:RNA; D004720:Endonucleases