.I. Mol. Biol. (1991) 222, 27-30

Crystallization and Preliminary Crystallographic Analysis of a Novel Nuclease from Serratia marcescens Mitchell D. Millerl, Michael J. Bemedikl, Merry C. Sullivan2 Nancy S. Shipley’ and Kurt L. Krause172 ‘Department of Biochemical and Biophysical Sciences Wniversity of Houston, Houston, TX 77204-5934, U.S.A 2Division of Atherosclerosis and Lipoprotein Research Department of Medicine, Baylor College of Medicine Houston, TX 77030, U.S.A. (Received 10 April

1991; accepted 19 July 1991)

Crystals have been obtained of the extracellular endonuclease from the bacterial pathogen Serratia marcescerw. This magnesium-dependent enzyme is equally active against single and double-stranded DNA, as well as RNA, without any apparent base preference. The Serratia nuclease is not homologous with staphylococcal nuclease, the only other broad specificity endonuclease for which a structure exists, nor is it homologous with other nucleases that have been solved by X-ray diffraction. The structure of this enzyme should, therefore, provide new information about this class of enzyme. At present we have succeeded in obtaining large, high quality crystals using ammonium sulfate. They crystallize in the orthorhombic space group P2,2,2,, with cell dimensions a = 106.7 A, b = 74.5 A, c = 6S9 8, and diffract to beyond 2 A. Low-resolution native data sets have been recorded and a search is under way for heavy-atom derivatives. Keywords: crystallography;

structure;

temperature (Biedermann et al., 1989). When incubated with substrate, the enzyme catalyzes the production of 5’ monophosphate-terminated oligonucleotides, i.e. the 3’ O-P bond is broken during the reaction (Eaves & Jeffries, 1963; Nestle & Roberts, 1969a,b; Fig. 1). It has been reported to possess equal catalytic activity toward DNA and RNA and it acts equally well against both single and double-stranded nucleic acid material. Also the Serratia nuclease acts without any apparent base preference. Some mononucleotides such as ATP and thymidine 3’,5’-bisphosphate have been shown to inhibit the enzyme’s activity and may serve as competitive inhibitors of the enzyme (Yonemura et al., 1983). The nucleic acid sequence of the Serratia nuclease was determined by Benedik and co-workers (Ball et al., 1987). Previously, several groups studying this enzyme had reported somewhat conflicting protein sequence information, but the DNA sequences from five different strains currently under study show at most one amino acid variation from the sequence reported (M. Benedik, personal communication). The Serratia nuclease is similar to the Staphylococcus nuclease in that it readily digests both DNA and RNA, but the Staphylococcus nuclease is much smaller at 13,000 M, and it

We report the crystallization and preliminary characterization of a broad specificity endonuclease, the Serratia marceacens extracellular nuclease. In the area of broad specificity nucleases, that is nucleases that act readily on single and doublestranded DNA and RNA, only one structure, the from secreted nuclease extracellularly Staphylococcus, has been solved (Cotton et al., 1979; Loll k Lattman, 1989). The Sewatia nuclease, while possessing some properties similar to those of the Staphylococcus nuclease, displays a quite different chemical behavior, and its solution should reveal new information about this class of enzymes. Serratia marcescens, a Gram-negative enteric bacteria of increasing pathogenic importance (Bergamo & Thirumoorthi, 1989; Saito et al., 1989), secretes several proteins extracellularly: two lipases (Heller, 1979; Givskov et al., 1988), two chitinases (Monreal & Reese, 1969; Jones et al., 1986; Harpster & Dunsmuir, 1989), two proteases (Lee et al., 1984; Molla et al., 1987), and as noted above, a nuclease. The Serratia nuclease is composed of a single polypeptide chain 245 residues long with a molecular weight of 26,700 (Ball et al., 1987). Its properties include a requirement for divalent magnesium and a pH optimum of 8. The enzyme contains two disulfide bonds and shows long-term stability at room 0022%2836/91/21002744

$03.00/O

endonuclease; Serratia marcescens

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0 1991 Academic Press Limited

28

M. D. Miller I -0

-P=

0

R i bonuclease

A

Staphylococcus nuclease cleavage

6

H

Figure 1. Location of the cleavage site in the Serratia nuclease reaction along with cleavage sites in commonly studied nuclease reactions for comparison.

requires calcium for its activity and not magnesium. In addition, a comparison of the sequences of the two enzymes reveals only a 17 y. sequence identity. The most interesting difference between the two enzymes involves the fact that they catalyze the cleavage of different bonds in nucleic acids. While the Staphylococcus nuclease catalyzes the formation of 3’ monophosphate-terminated nucleotides, and therefore must catalyze the cleavage of the 5’ O-P bond, the Serratia nuclease, as noted above, catalyzes the cleavage of the 3’ O-P bond (Fig. 1). Determination of the tertiary structure of the Serratia enzyme would likely contribute to the understanding of these differences. Since the Serratia nuclease lacks sequence homology with Staphylococcus nuclease, and since it behaves in a chemically distinct manner from that nuclease, it is unclear whether any similar structural motifs will be employed by the enzymes. As a result, nothing as yet can be stated about whether the reaction proceeds via nucleophilic attack of water on phosphorus facilitated by a general base or if a covalent intermediate is likely. No work has been done on the stereochemistry of the reaction of the Serratia nuclease. Elegant triple isotope experiments performed by Gerlt and co-workers on the Staphylococcus nuclease documented inversion of the phosphate in that system (Mehdi & Gerlt, 1982). Crystal structures of many other nucleases have been produced. A listing of these would include several ribonucleases, including ribonuclease A (and variants) (Wyckoff et al., 1970; Wlodawer L%SjSlin, 1983; Williams et al., 1987), the ribonuclease T, and barnase families (Hill et al., 1983), and ribonuclease H (Yang et al., 1990). Other important nuclease structures solved by crystallography include DNase I (Oefner $ Suck, 1986), EcoRI (McClarin et al., 1986; Kim et al., 1990), the X-5’ exonuclease found in the Klenow fragment of DNA polymerase I (Ollis et al., 1985) and the P, nuclease (Volbeda et al.,

et al.

1991). A comparison of the sequences of these nucleases with the Serratia nuclease reveals no sequence identities over 20% (Devereux et al., 1984; J. E. Chappelear, R. C. Cottingham & G. E. Fox, personal communication). Although there is apparently little direct sequence homology between the nucleases solved to date and the Serratia nuclease, some of these enzymes do share similar properties. The subgroup of endonucleases of about 250 residues in length that cleave the 5’ phosphate-3’ hydroxyl bond in DNA, using Mg’+ as a cofactor, and with a pH optimum near 8, would include DNase I and EcoRI. DNase I is an endonuclease of 30,400 M, that normally uses calcium as a cofactor, but is most active in the presence of Ca2+ and Mg2+. It cleaves single and double-stranded DNA, although doublestranded DNA is the preferred substrate. Its structure consists of two outer predominantly helical layers inside of which are two six-stranded /?-sheets. DNase I is only 157% homologous with the Serratia nuclease, but this does not exclude the possibility of structural homology. EcoRI is a DNA endonuclease restriction involved in doublestranded site-specific cleavage of DNA, and as such it displays functional properties quite different from the Serratia nuclease. The EcoRI structure is composed of two domains, each consisting of an a//? subdomain along with an extension arm that wraps around the DNA. The overall complex displays 2-fold symmetry. A comparison of the sequences of EcoRI and Serratia nuclease reveals 156% direct homology.

(a)

Puri$cation and crystallization Serratia nuclease

of

the

Purification of the Serratia nuclease was accomplished by using a genetically modified strain of S. marcescens and standard ion exchange chromatography. Briefly, a protease-deficient S. marcescens strain SM6prt :: TnS-A was transformed with a plasmid encoding the nuclease gene and carrying an operator constitutive mutation (pUC19Nuc40c), thus producing broth highly enriched for nuclease. After incubation at 30°C for 14 hours, the cells were spun out and discarded. Next, the broth was brought to 40% ammonium sulfate saturation and the precipitate collected and discarded. Then the nuclease pellet was precipitated out of solution at 87% ammonium sulfate. The nuclease pellet was collected and applied to a Whatman P-11 phosphocellulose column and eluted with a gradient. The resulting nuclease was nearly homogeneous on SDS/ polyacrylamide gel electrophoresis chromatography and displayed wild-type activity in standard nuclease assays. Crystallization was accomplished at 4°C using a two-step batch dialysis. First, an 8 mg/ml solution of the nuclease was prepared in 100 m&%-sodium phosphate (pH 60). This solution was predialyzed overnight versus 50 mM-sodium phosphate, 1.0 M-

Communications

29

Figure 2. Crystals of the nuclease from S. marcescens grown from ammonium sulfate. Typical dimensions of the large crystals are W5 mm x 64 mm x 0.4 mm. (0 ammonium sulfate (pH 6.0). Next, the dialysate was to 50 mM-sodium phosphate, 1.7 Mchanged ammonium sulfate (pH 60). After 10 to 14 days, large clear crystals were formed of typical dimensions 0.5 mm x 94 mm x 64 mm (Fig. 2). Using an Enraf Nonius FR590 sealed tube instrument at 40 kV, 50 mA, precession photographs of all principal zones were obtained (Fig. 3). Results to date reveal that the crystals occupy an orthorhombic lattice with unit cell dimensions a=106*7A, b=74.5A, c=6%9A (lA=@lnm). The reciprocal lattice displays mmm symmetry and systematic absences are present along all three reciprocal axes, suggesting that the crystals occupy the space group P2,2,2, (Fig. 3). The crystals exhibit excellent stability in the X-ray beam and diffract to beyond 2.0 A. Packing analysis suggests that two nuclease molecules are present per asymmetric unit. With a molecular weight of 26,700, the V, value is 2.6 A3/dalton, or 52% solvent in the unit cell (Matthews, 1968). Inspection of the principal zones, especially the h&l and Okl, reveals at low resolution, h + k or a pseudocentering pattern where reflection k+ 1 odd are usually absent. The best explanation we have at) present for this is that one of the nuclease molecules within the asymmetric unit is located at a, posit,ion which, in projection along the a or c axis, is close to being centered. In addition to the precession work, we collected a 4 A data set on the native crystals using a Rigaku AFC5R diffractometer running at 50 kV, 180 mA. Analysis of this data set confirmed our choice of lattice and Laue group and also confirmed that the centering conditions that appear at low resolution along two of the principal zones are not met within the .general set of reflections. Currently highresolution data collection using the wild-type nuclease crystals is underway. Since no homologous st’ructures exist. we are screening heavy-atom compounds as possible derivatives in anticipation of a muhiple isomorphous replacement solution to the phases.

Figure 3. Precession photograph of the 8. marcescens nuclease displaying 2 principal zones. (a) Note the systematic absences along a* and c* as well as the mm symmetry in this h01 photograph. At low resolution pseudocentering is apparent, especially along c*. This 8 h exposure was made on an Enraf Nonius FR590 operating at 40 kV and 50 mA. The crystal to film distance was 75 mm; c* is vertical and a* is horizontal. (b) Pseudocentering is more prominent in the h&l zone. The crystal to film distance was 100 mm; b* is vertical and u* is horizontal. This work was made possible in part by a grant from the Keck Foundation and matching funds from the University of Houston along with a Clinical Investigat,or Award DK01928 to K.L.K., and by grant GM36891 from the National Institutes of Health and a Texas Advanced Research Program Award 36521178 to M..J.B.

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by R. Huber