Nuclease P1 from Penicillium Citrinum

Background

Nuclease P1 (EC3.1.30.1; Nuclease 5'-Nucleotidehydrolase, 3'-Phosphohydrolase) is an extracellular enzyme first identified by Kuninaka, et al. in 1957 in an aqueous extract of the mold Penicillium citrinum. It has been shown by Fujimoto, et al (1974) to be a single strand-specific nuclease which exhibits high selectivity for single-stranded nucleic acids and single-stranded regions in double-stranded nucleic acids. Nuclease P1 is one of the most widely used single-strand specific nucleases in molecular biology; its selective activity has found useful applications in studies on nucleic acid structure.

Mode of Action

Nuclease P1 exhibits both phosphodiesterase and monoesterase activities. It hydrolyzes both 3'-5'-phosphodiester bonds in RNA and heat denatured DNA and 3'-phosphomonoester bonds in mono- and oligonucleotides terminated by 3'-phosphate without base specificity. Although the enzyme is essentially base-unspecific, it has been shown that the cleavage rates do depend on the identity of the base 5' to the cleaved bond (Box, et al, 1993; Romier et al, 1998).

Nuclease P1 attacks RNA and single-stranded DNA by concerted endo- and exonucleolytic action capable of hydrolyzing to the level of 5'-mononucleotides (Figure 1). Rapid fragmentation of polynucleotides occurs, forming both mononucleotides and oligonucleotides. The oligonucleotides produced in the initial stages of hydrolysis have 3'-OH and 5'-PO4 termini.

Highly purified nuclease P1 exhibits phosphomonoesterase activity to yield nucleoside and inorganic phosphate (Figure 2). Ribonucleoside 3'-monophosphates are hydrolyzed 20-50 times faster than the corresponding 2-deoxyribonucleotides. The base preference of the enzyme for 3'-ribonucleotides is G>A>C>U, whereas that for 3'-deoxynucleotides is C≥T>A≥G.

Both phosphodiesterase and phosphomonoesterase activities of nuclease P1 are zinc-dependent. The enzyme contains, per monomer, three zinc ions which are bound in a cleft forming the active site which is not accessible for regular double-stranded substrates (Romier et al, 1998). Two of the zinc ions form a closely-spaced dinuclear pair buried at the bottom of the pocket which activate the attacking water. The third zinc ion is located further apart and is more exposed to solvent. The enzyme attacks preferentially the linkage between the 3'-hydroxyl group of a nucleotide and the 5'-phosphoryl group of the adjacent nucleotide (Figure 3). The third, more exposed zinc ion stabilizes the leaving 3'-oxyanion.

The hydroxide ion (shaded sphere) bridging Zn1 and Zn3, and properly oriented by Asp45, is attacking the phosphate in-line with the P-O38-bond. In the resulting penta-coordinated transition state the attacking hydroxide and the leaving O38 occupy apical positions. Zn2+ is stabilizing the leaving O-38-oxyanion, while R48 neutralizes the additional negative charge of the transition state. Steric hindrance between the Rp-sulfur of a phophorothioate or of a phosphorodithioate with Zn2 will prevent cleavage. (Adapted from Romier et al. 1998. Recognition of Single-Stranded DNA by Nuclease P1: High Resolution Crystal Structures of Complexes with Substrate Analogs. Proteins: Structure, Function, and Genetics. 32:414-424.)

P1 is unable to cleave phosphodiester bonds with an abasic 5'nucleotide, whereas removal of the base of the 3'-nucleotide does not affect hydrolysis (Weinfield, et al, 1993). The aromaticity of the 5'-base affects the activity, indicating that it may be involved in a stacking interaction with an aromatic protein side chain. The importance of the 5'-base was reinforced by the finding that although P1 is essentially unspecific, the enzyme shows some dependence of its cleavage rates on the identity of the base 5' to the cleaved bond.

Figure 1: Phosphodiesterase Activity of Nuclease P1

Figure 2: Phosphomonoesterase Activity of Nuclease P1

Figure 3: Proposed catalytic mechanism of P1 nuclease.

Structure

Nuclease P1 is a single polypeptide chain with an estimated molecular weight of 36 kDa. It is a glycoprotein containing mannose, galactose and glucosamine present in a ratio of approximately 6:2:1 (Fujimoto, 1975); it has a high content of hydrophobic amino acids like tyrosine and tryptophan. Direct sequencing of nuclease P1 by Maekawa et al (1991) has shown that it contains 270 amino acids with a calculated molecular weight of 29,221. The considerably higher molecular weight of 36,000-37,000 observed on SDS gels is likely accounted for by four N-glycosylations, at asparagines residues 92, 138, 184, and 197. These post-translational modifications produce a 19% increase in mass as carbohydrate. There is no free sulfhydryl in nuclease P1, but two disulfide bridges exist between Cys-80 and Cys-85 and Cys-72 and Cys-217

The three-dimensional structure of nuclease P1 has been determined at 2.8A resolution from tetragonal crystals (Volbeda et al. 1991) and at 2.2 A resolution from orthorhombic crystals (Suck et al. 1993). The 3-D structure is represented below (Figure 4).

Figure 4: 3-D structure of Nuclease P1

Physico-chemical Properties

The isoelectric point of nuclease P1 is around 4.5. The optimal temperature of phosphodiesterase and monoesterase activities is 70oC; increased thermal activity is attributed to the presence of high amounts of hydrophobic amino acids in the enzyme structure. The enzyme is stable between pH 5 and 8 and at temperatures below 60oC. Hydrolysis of natural single-stranded DNA and RNA is maximal around pH 5; poly(A) and poly(C) are rapidly hydrolyzed at pH 6, but are highly resistant at pH 4.5. Poly(U) and poly(I) are easily cleaved between pH 4 and 5, but very slowly at pH 6. Phosphomonoesterase activity toward 3'-ribonucleotides is optimal around pH 4.5. The activity on 3'-rCMP and 3'-rUMP is highest at pH 6, and at pH 7.2 and 8.5, the enzyme attacks 3'-rAMP and 3'-rGMP, respectively, very rapidly.

Denaturing reagents like guanidine hdyrochloride strongly inhibit activity; denaturation however seems to be reversible. The enzyme is also inactivated by dialysis and pH 5 and reactivated almost completely by addition of 1 mM Zn2+. Dialysis removes one of the three zinc ions, with total inactivation being observed on addition of 0.02 M EDTA at pH 5; partial restoration of activity is obtained by adding Zn2+.

Applications

Nuclease P1 has historically been used for the industrial production of 5'-mononucleotides from yeast RNA. The 5'mononucleotides, in particular 5'-GMP, exhibit flavor-enhancing properties in food. 5'GMP acts synergistically with monosodium glutamate and thus can largely replace monosodium glutamate in various food products. In view of the high demand for 5'-nucleotides in the food and beverage industries, attempts have been made to immobilize P1 nuclease in the commercial production of 5'-nucleotides.

In recent years, nuclease P1 has found extensive use as an analytical tool in nucleic acid research. It has been used for; (i) structural studies and base composition analysis of nucleic acids, including sequence analysis of end-group-labeled RNA and analysis of t-RNA structure (Chango, 2009; Hua, 2007, Shimelis and Giese, 2006, Katayama-Fujimura, 1984; Aultman, KS, 1982; Furuichi, 1975; Silberklang, 1977) , (ii) removal of nucleic acids during protein purification (Zabriske, 1988), and (iii) analysis of DNA damage (Lai, 2008; Zhou et al, 2005; Jiang, 2003; Godschalk, 2002; Budzinski, 1992). Recently, nuclease P1 has played a central role in the development of methods for studies involving t-RNA dependent amino acid biosynthesis and t-RNA dependent trans-amidation (Sheppard et al., 2008), isolation of bulky aromatic/heterocyclic carcinogen-DNA adducts such as dG-C8-MeIQx and dG-C8-PhIP (Neale, 2008), quantitative determination of DNA interstrand cross-links and monoadducts induced in human cells by agents like UVA irradiation and psoralens (Lai, 2008; Cao, 2008), and determination 2'-deoxyinosine in tissue DNA for studies on DNA deamination (Kim, 2006a, 2006b).

References

Aultman, K.S. and Chang, S. H. 1982. Partial P1 nuclease digestion as a probe of tRNA structure. Eur. J. Biochem. 124:471-476.

Boxm, HC, et al. 1993. The differential lysis of phosphoester bonds by nuclease P1. Biochim. Biophys. Acta. 1161:291-294

Budzinski, E.E., et al. 1992. Analysis of DNA Damage at the Dinucleoside Monophosphate Level: Application to the Formamido Lesion. Radiation Research. 132(3):288-295.

Cao, H, et al. 2008. LC-MS/MS for the detection of DNA interstrand cross-links formed by 8-methoxypsolaren and UVA irradiation in human cells.

Chango, A. 2009. Simultaneous determination of genomic DNA methylation and uracil misincorporation. Med. Princ. Pract. 18(2): 81-84.

Desai, N.A. and V. Shankar. 2003. Single-strand specific nucleases. FEMS Microbiology Reviews. 26:457-491.

Fujimoto, M, et al. 1975. Some physical and chemical properties of nucelae P1. Agric. Biol. Chem. 39:1991-1997.

Godschalk, et. al. 2002. Comparison of multiple DNA adduct types in tumor adjacent human lung tissue: effect of cigarette smoking. Carcinogenesis. 23(12):2081-2086.

Hua NP and T. Naganuma. 2007. Application of CE for determination of DNA base composition. Electrophoresis. 3:366-372.

Jiang, Q, et al. 2003. 5-Chlorouracil, a marker of DNA damage from hypochlorous acid during inflammation. A gas chromatography-mass spectrometry assay. J. Biol. Chem. 278(35):32834-32840.

Katayama-Fujimura Y, et. al. 1984. Estimation of DNA base composition by high performance liquid chromatography of its nuclease P1 hydrolysate. Agric. Biol. Chem. 48:3169-3172.

Khan, F et al. 2005. Enhanced recognition of hydroxyl radical modifed plasmid DNA by circulating cancer antibodies. J. Exp. Clin. Cancer Res. 24(2):289-296.

Kuninaka, A, et al. 1961. Studies on 5'-phosphodiesterase in microorganisms. Part II. Properties and applications of Penicillium citrinum 5'-phosphodiesterase. Agric. Biol. Chem. 25:693-701.

Lim, K.S. et. al. 2006. Quantitative gas chromatography mass spectrometric analysis of 2'-deoxyinosine in tissue DNA. Nat. Protoc. 1(4):1995-2002.

Lim, K.S. et. al. 2006. Potential artifacts in the measurement of DNA deamination. Free Radic. Biol. Med. 40(11):1939-1948.

Maekawa, K, et al. 1991. Primary structure of nuclease P1 from Penicillium citrinum. Eur. J. Biochem. 200:651-661.

Neale, JR, et al. 2008. Methods for aromatic and heterocyclic amine carcinogen-DNA adduct analysis by liquid chromatography-tandem mass spectrometry. Polycycl. Aromat. Compd. 28(4-5): 402-417.

Romier C, et al. 1998. Recognition of single-stranded DNA by nuclease P1: high resolution cyrstal structures of complexes with substrate analogs. 32(4):414-424.

Sheppard, K, et al. 2008. Assays for transfer RNA-dependent amino acid biosynthesis. Methods. 44(2):139-145.

Shimelis, O and R.W. Giese. 2006. Nuclease P1 digestion/high-performance liquid chromatography, a practical method for DNA quantitation. J. Chromatogr A. 1117 (2): 132-136.

Silberklang, M., et al. 1077. The use of nuclease P1 in sequence analysis of end group labeled RNA. Nucleic Acids Res. 4:4091-4108.

Suck D, et al. 1993. The three-dimensional structures of Penicillium P1 and Aspergillus S1 nucleases. J. Cell. Biochem. Suppl. 17C:154.

Volbeda, A, et al. 1991. Crystal structure of Penicillium citrinum P1 nuclease at 2.8A resolution. EMBO J. 10:1607-1618.

Weinfield, M, et al. 1993. Influence of nucleic acid base aromaticity on substrate reactivity with enzymes acting on single-stranded DNA. Nucleic Acids Res. 21:621-626.

Zabriske, D. W. and DiPaolo, M. 1988. Removal of nucleic acid contaminants using nuclease enzymes during protein isolation. Biotechnol. Bioeng. 32:100-104.

Zhou, G. D., et. al. 2005. Tissue-specific attenuation of endogenous DNA I-compounds in rats by carcinogen azoxymethane: possible role of dietary fish oil in colon cancer prevention. Cancer Epidemiol Biomarkers Prev. 14(5):1230-1235.