Purification and characterization of urease from

132

Purification and characterization of urease from Schizosaccharomyces pombe

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by HARVARD UNIVERSITY on 08/23/13 For personal use only.

Mark W. Lubbers, Susan B. Rodriguez, Neville K. Honey, and Roy J. Thornton

Abstract: The urease from the ascomycetous fission yeast Schizosaccharomyces pombe was purified about 4000-fold (34% yield) to homogeneity by acetone precipitation, ammonium sulfate precipitation, DEAE-Sepharose ion-exchange column chromatography, and if required, Mono-Q ion-exchange fast protein liquid chromatography. The enzyme was intracellular and only one species of urease was detected by nondenaturing polyacrylamide gel electrophoresis (PAGE). The native enzyme had a Mr of 212 kDa (Sepharose CL6B-200 gel filtration) and a single subunit was detected with a Mr of 102 kDa (PAGE with sodium dodecyl sulfate). The subunit stoichiometry was not specifically determined, but the molecular mass estimations indicate that the undissociated enzyme may be a dimer of identical subunits. The specific activity was 700-800 kmol urea. min-I . mg protein-', the optimum pH for activity was 8.0, and the K,,, for urea was 1.03 mM. The sequence of the amino terminus was M e t - G l n - P r o - k g - G l u - L e u - H i s - L y s - L e u - T ~ u - l aand the sequence of two tryptic peptides of the enzyme were Phe-Ile-Glu-Thr-Asn-Glu-Lys and Leu-Tyr-Ala-Pro-Glu-Asn-Ser-ProGly-Phe-Val-Glu-Val-Leu-Glu-Gly-Glu-Ile-Glu-Leu-Leu-Pro-Asn-Leu-Pro. The N-terminal sequence and physical and kinetic properties indicated that S. pombe urease was more like the plant enzymes than the bacterial ureases. Key words: urease, Schizosaccharomycespombe, fission yeast, ascomycetous yeast.

Resume : L'urCase de la levure ascomyckte Schizosaccharomyces pombe qui se divise par fission a CtC purifiCe environ 4000 fois B 1'homogCnCitt (rendement 34%) par prkcipitation B l'acktone, prkcipitation au sulfate d'ammonium, chromatographie sur colonne Cchangeuse d'ions sur DEAE-Sepharose et, si nkcessaire, par chromatographie liquide rapide des protCines (Cchangeuse d'ions) sur Mono Q. L'unCase Ctait intracellulaire et un seul type a CtC d6tectC par Clectrophorbse sur gel de polyacrylamide (PAGE) non-dCnaturant. L'enzyme d'origine avait un MI de 212 kDa (filtration sur Sepharose CL6B-200) et une seule sous-unit6 a CtC d6tectCe avec un MI de 102 kDa (PAGE avec dodecylsulfate de sodium). La stoechiomCtrie de la sous-unit6 n'a pas CtC dCterminCe specifiquement mais les estimations des masses molCculaires ont indiqd que l'enzyme non-dissociCe pounait &treun dimkre de deux sous-unitCs identiques. L'activitC spkcifique Ctait de 700-800 pmol urCe . min-l. mg protCine-l, le pH optimal d'activitk se situait i 8,O et le K,,, pour l'uree Ctait de 1,03 mM. La sCquence de la partie terminale aminCe Ctait M e t - G l n - P r o - A r g - G l u - L e u - H i s - L y s - L e u - T p a et la sCquence de deux peptides tryptiques de l'enzyme Ctait Phe-Ile-Glu-Thr-Asn-Glu-Lyset Leu-Tyr-Ala-Pro-Glu-Asn-Ser-Pro-Gly-PheVal-Glu-Val-Leu-Glu-Gly-Glu-Ile-Glu-Leu-Leu-o-Asn-Leu-Pro. La sCquence de la portion N-terminale et les propriCtCs physiques et cinktiques indiquent que I'unrCase de S. pombe est plus apparentee aux enzymes des vCgCtaux qu'aux unrkases des bactkries. Mots elks : urCase, Schizosaccharomyces pombe, levure qui se divise par fission, levure ascomycbte. [Traduit par la rCdaction]

Introduction the (urea amidohydrolase' EC 3'5.1'5) hydrolysis of urea to yield ammonia and carbamate, which

Received July 4, 1995. Revision received October 19, 1995. Accepted October 20, 1995.

M.W. Lubbers,lS.B. Rodriguez, N.K. Honey, and R. J. T h ~ r n t o nDepartment .~ of Microbiology and Genetics, School of Biological Sciences, Massey University, Palmerston North, New Zealand. Author to whom all correspondence should be sent. Present address: New Zealand Dairy Research Institute, Pnvate Bag 11 029, Palmerston North, New Zealand. Present address: Department of Biological and Physical Sciences, Indiana University, P.O. Box 9003, Kokomo, Indiana 46904, U.S.A. Can. J. Microbial. 42: 132-140 (1996). Printed in Canada / ImprimC au Canada

spontaneously hydrolyzes to carbonic acid and a second molecule of ammonia (Andrews et al. 1984). Urease is found in plants, algae, invertebrates, yeasts, filamentous fungi, and bacteria, including archaebacteria (Mobley and Hausinger 1989). However, as only certain genera or species within each division have urease activity, it is often a useful criterion for taxonomic assignment (Christensen 1946; Seeliger 1956; Sen and Komagata 1979; Booth and Vishniac 1987). The urease from jack bean (Canavalia ensiformis) was the first enzyme to be crystallized (Sumner 1926) and it remains the best characterized urease. The 590-kDa jack bean urease is a hexamer of 9 1-kDa subunits, with two nickel ions per subunit (Andrews et al. 1984).Urease is implicated in the pathogenesis of several clinical conditions and the enzymes from a number of the bacteria involved have been characterized; the bacteria are mostly gram negative and include Proteus, Helicobacter, Klebsiella spp., Providencia stuartii, Morganella morganii,

Lubbers et al.


Ureaplasma urealyticum, and Yersinia enterocolitica (reviewed by Mobley and Hausinger 1989; Hu and Mobley 1990; Skumik et al. 1993). In addition, the ureases from some other bacteria have been characterized, including the gram-positive bacteria Staphylococcus spp. (Schafer and Kaltwasser 1994; Christians et al. 1991), Bacillus sp. strain TB-90 (Maeda et al. 1994), and Lactobacillus spp. (Kakimoto et al. 1989, 1990). The bacterial enzymes are generally 200-250 kDa in size (Mobley and Hausinger 1989) and composed of one large (a,61-73 kDa) and two small (p, 8-17 kDa; y, 6-11 kDa) subunits (Mobley and Hausinger 1989; Jones and Mobley 1989; Kakimoto et al. 1989,1990; Hu et al. 1990; Christians et al. 1991; Thirkell et al. 1989; Maeda et al. 1994). A notable exception is the enzyme from Helicobacter species, which has only two subunits (61-66 kDa and 26-31 kDa) and native size estimates of 535-650 kDa (Dunn et al. 1991; Hawtin et al. 1990; Labigne et al. 1991; Costas et al. 1991; Evans et al. 1991; Austin et al. 1992; Ferrero and Labigne 1993). There have been a few reports of bacterial ureases with a single subunit; however, these may be the result of small subunits having been overlooked on sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) gels (Mobley and Hausinger 1989; Thirkell et al. 1989; Christians et al. 1991). Despite the differences between the bacterial ureases and jack bean urease, regions of the subunits have significant amino acid sequence similarities (Jones and Mobley 1989; Blanchard 1990; Hu et al. 1990; Muirooney and Hausinger 1990; Labigne et al. 1991; Morsdorf and Kaltwasser 1990; Maeda et al. 1994; Skumik et al. 1993). Expression of the genes encoding the urease subunits has been shown in several bacteria (Jones and Mobley 1988; Mulrooney et al. 1988; Mulrooney and Hausinger 1990; Lee et al. 1990, 1992; Gatermann and Marre 1989; Hu et al. 1992), soy bean (Holland and Polacco 1992), and Aspergillus nidulans (Mackay and Pateman 1980) to be insufficient for production of catalytically active urease and up to three or more accessory genes are required for co- and (or) post-translational modifications, such as nickel incorporation into the apoenzyme. The kinetic properties of bacterial ureases can vary considerably, with estimates of K,,, ranging from 0.1 to >I00 mM urea, pH optima from 2 to 8.7, and specific activities from as low as 30 to as high as 180 000 pmol urea.min-I .mg protein-' (Kakimoto et al. 1989; Mobley and Hausinger 1989). Comparatively little is known of the structure and kinetics of eucaryotic microbial ureases. The enzymes from two species of the filamentous ascomyceteAspergillus (Creaser and Porter 1985; Smith et al. 1993) have been purified, and the enzyme from the basidiomycetous yeast Rhodosporidium paludigenum (Phillips et al. 1990) has been partially purified. These ureases, like the plant enzyme, have a single subunit with sizes ranging from 40 to 83 kDa. The subunit stoichiometries of these eukaryotic ureases vary and may be trimer, hexamer, and possibly octamer, with native enzyme sizes between 240 and 569 kDa. No sequence data are available for any eucaryotic microbial urease. Urease activity is common among basidiomycetous yeasts and related species but rare amongst ascomycetous yeasts (Sen and Komagata 1979; Booth and Vishniac 1987). An exception is the ascomycetous fission yeast Schizosaccharomycespombe, which has one of the highest urease activities of all yeasts (Booth and Vishniac 1987; Phillips et al. 1990). It would be

interesting to determine if S. pombe urease has a similar structure and kinetic properties as urease from another ascomycete, the filamentous fungus Aspergillus, or if S. pombe urease is more similar to the bacterial or plant enzymes. We report here the purification to homogeneity, characterization, and N-terminal sequence of S. pombe urease, the first such report on any yeast urease.

Materials and methods Schizosaccharomyces pombe strain and culture conditions Schizosaccharomyces pombe 972 (Gutz et al. 1974) was a gift from P. Thuriaux. Liquid cultures were prepared in yeast extract (YE) medium (Moreno et al. 1991) and incubated at 30°C with reciprocal shaking at 180 rpm.

Urease assay Urease activity was measured by following the release of ammonia from urea as described by Wong and Shobe (1974). All assays were done in duplicate or triplicate. The amount of ammonia released by urease activity was determined by subtraction of a reaction in which the substrate (urea) was omitted. Reagent blanks were prepared as described by Wong and Shobe (1974).

Preparation of crude cell extracts Schizosaccharomyces pombe cells from a stationary-phase culture (approximately 108 CFU . mL-l) in YE were harvested by centrifugation (4000 x g for 10 min at 4OC), resuspended in about half the original volume of ice-cold water and recentrifuged. Cells were kept at 0-4°C for all subsequent procedures. The cell pellet was resuspended in twice the pellet volume of 0.02 M potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 1 mM 2-mercaptoethanol (PEB) and disrupted by two passages through a French press at a pressure of 9000 psi (1 psi = 6.895 kPa). The disrupted cells were centrifuged at 110 000 x g for 1 h to remove all cell debris, including membranes, if the crude extract was to be used directly; or at 3 1 000 x g for 40 min, if further purification was to be carried out.

Determination of urease location in S. pombe cultures An aliquot of an early stationary-phase S. pombe culture (approximately lo8 CFU - mL-l) was filtered through a 0.45-pm pore size membrane filter. The filtrate was kept for analysis (culture medium) and the rest of the culture was centrifuged (6000 x g, five min, 4°C). The cell pellet was resuspended in PEB and washed by shaking vigorously for 5 min. An aliquot of the cell suspension was filtered as above and the filtrate was kept for analysis (wash buffer). The rest of the cell suspension was centrifuged as above. The cell pellet was resuspended in PEB and an aliquot was kept for analysis (washed whole cells). The rest of the cells were disrupted by two passes through a French pressure cell and centrifuged as above. An aliquot of the supernatant (crude lysate), which would have contained organelles, membranes, and cellular debris, was kept for analysis. The remainder of the supernatant was centrifuged (1 10 000 x g, 60 min, 4°C) and the pellet (cell debris) and supernatant (soluble fraction) were examined for urease activity.

Purification of S. pombe urease All steps were carried out at 0-4°C unless stated otherwise. The pH of crude cell extracts was adjusted to pH 7.0 with 3 N KOH and proteins were precipitated with acetone using the following cuts: 0-40, 40-50, and 50-60% v/v acetone. Precipitated proteins were recovered by centrifugation at 20 000 x g for 10 min at 0°C. Urease precipitated in the 50-60% v/v acetone cut was dissolved in PEB (8 mL . L starting culture-l) to give a protein concentration of about 5 mg . mL-l. The redissolved urease was precipitated by ammonium


Can. J. Microbiol. Vol. 42, 1996 sulfate at 35-45% saturation. Precipitated proteins were recovered as above, resuspended in 0.66 mL PEB . L starting culture-', and dialyzed with PEB. A DEAE-SepharoseTM (Pharmacia) column (8 x 1.6 cm) was equilibrated in PEB. Then the urease sample was loaded and washed (flow rate, approximately 10 mL . h-l) with several column volumes of 0.2 M PEBS (PEB containing 0.2 M NaCl). The urease was eluted with a linear 0.2-0.35 M NaCl gradient (in PEB, 80 mL total volume, 10 rnL . h-l) and 2-mL fractions were collected. The most active fractions were pooled and then concentrated and desalted by ultrafiltration through a XM 50 membrane (molecular mass cutoff, 50 kDa; Amicon). Further purification was done, where indicated, by fast protein liquid chromatography (FPLC) as follows: the concentrated and desalted urease, in PEB, was applied to a MonoQ HR 515 ion-exchange column (equilibrated with 0.2 M PEBS at room temperature, Pharmacia) and eluted with a 30-mL linear 0.2-0.5 M NaCl gradient (in PEB), at a flow rate of 0.5 mL . min-'.

Native molecular mass determination The native molecular mass was determined by gel filtration using a calibrated Sepharose CL6B-200 (Sigma) column (1.6 x 92 cm packed gel matrix). The resin was washed, resuspended, degassed, equilibrated, and run in PEBS buffer (PEB containing 0.1 M NaCl) at 4OC using the procedures suggested in the Pharmacia handbook Gelfiltration: theory and practice. The inclusion of 0.1 M NaCl in the gel filtration buffer should have minimized aggregation of urease with contaminants (Jones and Mobley 1988). The column was eluted in an upward flow configuration, to reduce packing of the gel matrix, at a constant operating pressure of 100 cm of water and a flow rate of 12 mL . h-', which is within the flow rate recommended for determination of molecular mass using CL6B-200 (maximum, 20 mL . h-I; Sigma Technical Bulletin No. GF-3). The flow of the column was not stopped from the time the column was poured until the end of all molecular mass determinations. The column was calibrated using blue dextran (2000 kDa), thyroglobulin (669 kDa), apoferritin (443 kDa), p-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), and carbonic anhydrase (29 kDa) standards from a MW-GF 1000 kit (Sigma) according to the manufacturers instructions. The elution volume (V,) of blue dextran, determined by monitoring the absorbance of fractions at 620 nm, was taken as the void volume (V,) of the column. Each standard was separately run through the column and the absorbance at 280 nm was used to follow the elution of the proteins. All samples were dissolved in PEBS, centrifuged at 13 000 x g for 5 min at 4OC to remove particulate matter, adjusted to 2.0 mLvolume, and loaded onto the column. Fractions (2.04 mL) were collected once the applied sample had reached the column surface. The amount of protein standard applied to the column was 10-16 mg for thyroglobulin, bovine serum albumin and apofemtin; 4 mg for carbonic anhydrase; 6-8 mg for alcohol dehydrogenase and p-amylase; and 5 mg for blue dextran. The total protein content of S.pombe urease samples (partially purified by acetone precipitation and dissolved in PEBS; specific activity about 1 U . mg-l) applied to the column was about 8 mg. Schizosaccharomycespornbe urease samples were loaded and fractions collected, as for the standards.

pH optimum of S. pombe urease The urease activity of a S. pombe crude extract was measured over a final pH range of 4.2-10.2. Acetate (pH 4.2-6.7) and phosphate (pH 6.1-10.2) substrate buffers were used. Inhibition of urease by fully protonated phosphoric acid (Mobley and Hausinger 1989) was avoided by using acetate buffers for the low pH range. Alternative buffers for the high pH range, such as Tris-HC1 or CHES (2-(Ncyclohexylamino)ethane sulfonic acid), inhibited the color development of the ammonia assay, and therefore, were not used. All buffers were 0.02 M and contained 1 mM EDTAand 50 mM urea. Any change in pH caused by the addition of enzyme was measured. Apart from the substrate buffer, all other assay conditions were standard.

K,,, determination A S. pombe crude extract was assayed in substrate buffers (0.02 M phosphate, pH 7.5) containing 0.1,O. 15,0.25,0.5,0.75, 1.O, 1.5,2.0, 5.0, 10.0, or 25.0 mM urea. All buffers contained 1 mM EDTA. Activity was determined, in duplicate, for 0, 15,30,45,90, and 120 s incubation at 25OC. 2-Mercaptoethanol was not included because it may competitively inhibit urease, although it did not affect activity at a high (50 mM) urea concentration. The K,,, was estimated from an Eadie-Hofstee plot.

Preparation and purification of urease peptides About 300 pg of purified S. pombe urease in 150 yL of 0.1 M ammonium bicarbonate was heated to 80°C for 5 min. The denatured urease was incubated with 3 pg trypsin (L-l-tosylamide-2phenylethyl chloromethyl ketone treated, Sigma) for 4.5 h at 37OC. The resulting peptides were separated and recovered by reversedphase high performance liquid chromatography (HPLC) using a HY-TACH C18 column. Peptides were eluted with a 2-90% v/v acetonitrile gradient in 0.1% formic acid over 1 h at a flow rate of 1 mL . min-I at 50°C. Peptides were detected by absorbance at 214 nm and peaks corresponding to eluted peptides were collected.

Protein sequencing Purified proteins and peptides were sequenced by the sequential automated Edman degradation method with an Applied BiosystemsTM model 470A apparatus.

Protein assay The concentration of protein in solution was determined using the Coomassie blue dye binding method as described by Bradford (1976) (protein concentrations, 20-200 pg . mL-l) or the modification described by Spector (1978) (protein concentrations, 10-50 pg . mL-I). Bovine serum albumin (BSA fraction V; Sigma) was used to prepare the standard curve.

Polyacrylamide gel electrophoresis The buffer system of Laemmli (1970) was used for both native (nondenaturing) and SDS (denaturing and reducing) PAGE, except that SDS was not included in the loading buffer, gel, or running buffer for native PAGE. Molecular masses of proteins were determined by comparison to a standard curve (log M, versus relative mobility). Proteins were visualized by silver staining according to the method of Morrissey (198 1). Urease activity in native PAGE gels was visualized as described by de Llano et al. (1989).

Results and discussion Location of urease in S. pombe An intracellular location for S. pombe urease was demonstrated by fractionating a culture of S. pombe using filtration and centrifugation. The urease activity was associated with the soluble, nonparticulate fraction of the cytoplasm. No activity was detected in the culture medium, nor was any activity removed from the cell surface by vigorous shaking in PEB (wash buffer). The total activity of the washed whole cells was 55 pmol .min-1; however, when the cells were disrupted in the French press, the activity rose to 934 pmol.min-I (crude lysate). Negligible activity was associated with the cell debris, including membrane fragments, which was removed from the crude lysate by ultracentrifugation. All of the activity was accounted for in the nonparticulate cell lysate (soluble fraction), indicating that the enzyme is intracellular and not membrane associated. This is in agreement with the intracellular location determined for ureases from bacteria and jack bean


Lubbers et al.

(Mobley and Hausinger 1989). An extracellular location of urease, which could be extracted from the cell surface by washing, has only been demonstrated for Helicohacter species (Bode et al. 1989; Dunn et al. 1990, 1991; Austin et al. 1991; Hawtin et al. 1990). The whole-cell urease activity of S. pombe was less than 6% of the cell-free extract activity, indicating that the ratelimiting step in hydrolysis of extracellular urea is probably the entry of urea into the cell. It is less likely that ammonia diffusion out of the cell (necessary for detection by the assay) was rate limiting, because the permeability of the membrane to urea is probably lower than the permeability to ammonia, as demonstrated for bacteria (Jahns et al. 1988). Aspergillus nidulans mutants that lack active transport of urea can no longer grow on urea below a concentration of 3 mM, and growth at slightly higher urea concentrations is probably facilitated by a second passive transport system (Pateman et al. 1982). Wild-type S. pombe grows on 2 mM urea (Kinghom and Fluri 1984), so a urea transport system probably exists for this yeast.

Fig. 1. Native PAGE of S. pombe urease. The gels were 6% w/v polyacrylamide. (A) Crude S. pombe cell extract (200 pg protein) stained for urease activity. (B) Analysis of the various purification steps in the isolation of S. pombe urease. The protein loaded per lane was as follows: lanes 1 and 5 , 3 pg crude extract; lane 2 , 3 pg acetone precipitate; lanes 3 and 6, 3.6 pg ammonium sulfate precipitate; lanes 4 and 7,0.6 pg pooled DEAE-Sepharose chromatography fractions. Proteins in lanes 1-4 were silver stained. Lanes 5-7 were stained for urease activity. Urease activity was not readily visible in lanes 5 and 6 because the enzyme specific activity was low at these stages of purification.

Urease isozymes One band of urease activity was observed on native PAGE of a crude S. pombe extract (Fig. 1A). This suggests that only a single urease was present, although the possibility of multiple forms with the same electrophoretic mobility cannot be excluded. A mutation in any one of the four S. pombe urease genes completely abolishes the enzyme activity (Kinghorn and Fluri 1984), consistent with a single urease species. Effect of urea, nickel, and manganese on urease production Nickel has been found in all ureases examined and a mechanism involving nickel has been proposed for urease catalysis (Hausinger 1987; Mobley and Hausinger 1989; Christians et al. 1991; Zemer 1991; Kakimoto et al. 1989; Thirkell et al. 1989; Hawtin et al. 1991; Park et al. 1994). Growth in the presence of urea, nickel sulfate, and manganese sulfate can increase the urease activity in bacteria (e.g., Bast 1988; Kakimoto et al. 1990; Rando et al. 1990; Schneider and Kaltwasser 1984; Mackerras and Smith 1986), algae (Rees and Bekheet 1982), and yeasts (Booth and Vishniac 1987). Complex media may contain insufficient available nickel for the synthesis of fully active urease and nickel-chelating compounds may be present in media (Rando et al. 1990; Hu and Mobley 1993). Kakimoto et al. (1990) demonstrated that 0.005% manganese sulfate tetrahydrate + 0.005% nickel sulfate in the growth medium can act synergisticallyto increase the urease activity of Lactohacillus fermentum approximately twofold. No significant difference in the total urease activity of S.pombe was observed when crude extracts were prepared as described in Materials and methods from cells grown in YE, YE + 0.1% urea, or YE + 0.1% urea + 0.005% manganese sulfate tetrahydrate + 0.005% nickel sulfate. This suggests that the YE medium used for growth of S. pombe cells in the present study contained sufficient available nickel for urease synthesis and also that S. pombe urease activity is not induced by urea, consistent with previous findings that urea is not required for synthesis of urease in S. pombe (Fluri and Kinghom 1985). Schizosaccharomyces pombe urease is also not controlled by nitrogen repression (Fluri and Kinghom 1985). Neurospora crassa

urease, as in S. pombe, is neither induced nor repressed (Haysman and Howe 1971). Urease activity in A. nidulans is not inducible, but it is controlled by nitrogen repression (Mackay and Pateman 1982).

Enzyme purification Preliminary experiments established that the protease inhibitor phenylmethylsulfonyl fluoride (0.02 mM included in the PEB buffer) did not affect the yield of S. pombe urease activity in the crude cell extract or the stability of the enzyme. The crude enzyme extract was reasonably stable, retaining over 90% of the original activity after storage for 7 days at 4OC or overnight at 18-22°C. There was no advantage in using S. pombe cells in log phase rather than stationary phase. Acetone and ammonium sulfate precipitations followed by DEAE-Sepharose ion-exchange chromatography were sufficient to obtain >90% pure S. pombe urease for purification 1, as estimated from stained protein bands on native and SDSpolyacrylamide gels (Fig. lB, Fig. 2, and Table 1). The purification 2 preparation contained a significant amount of contaminating protein after the DEAE-Sepharose purification step, which was removed by FPLC ion-exchange using a Mono-Q column (Table 1). The degree of purification required to give pure S. pomhe urease in the present study was about 4000-fold. A similar high purification factor was required to obtain pure urease from Aspergillus niger (Smith et al. 1993) and A. nidulans (Creaser and Porter 1985). Urease from most other organisms, including

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Can. J . Microbial. Vol. 42, 1996

Table 1. Purification of urease from Schizosaccharomyces pombe.


Total activity (p,mol. min-I)

Total protein (mg)

Step

1

2

1

2

Crude extract Acetone Ammonium sulfate DEAE-Sepharose Mono-Q FPLC

252 152 143 85.5

1199 613 248 197 62.3

1428 114 5.40 0.12

5850 579 16.1 0.437 0.076

-

-

Specific activity (pmol . min-I . mg protein-')

1

2

Purification (n-fold)

1

2

Yield (%) I

2

Note: Values are for two purifications, 1 and 2. Purification 1 was on 6 L of culture, the preparation was estimated >90% pure after the DEAE-Sepharose step and a WLC purification step was not done. Purification 2 was on 21 L of culture and a final FPLC Mono-Q ion-exchange step was used to purify the enzyme to homogeneity.

Fig. 2. SDS-PAGE of S. pombe urease purified by acetone precipitation, ammonium sulfate precipitation, and DEAE-Sepharose ion-exchange chromatography. A 7.5% w/v polyacrylarnide denaturing (SDS) gel was used. Proteins were silver stained. Molecular mass standards are marked.

the yeast R. paludigenum (Phillips et al. 1990), appears to be present at much higher levels within the cell (Mobley and Hausinger 1989).

Subunit size and native molecular mass of S. pombe urease A single major subunit of 102 kDa was detected for S. pombe urease by SDS-PAGE (Fig. 2). To determine if the barely visible minor bands (Fig. 2) were contaminant proteins or small subunits of S. pombe urease, the enzyme was further purified using Mono-Q FPLC ion-exchange and examined on a 15% W/Vacrylamide gel. Only the 102-kDa protein band was seen, indicating that the enzyme has only one subunit. The native molecular mass of S. pombe urease was determined by gel filtration. The void volume (V,, 73.0 mL) of the calibrated gel filtration column and the elution volume of P-amylase (V,, 124.0 mL) were periodically checked and remained unchanged throughout the course of the experiment. Three independently prepared urease extracts were used for a total of four determinations. Urease assays for each of the four

determinations were done in triplicate and the average urease activity for each fraction was used to graphically estimate the elution volume Ve (122.8 mL) from the point of maximum activity (Fig. 3A). The calculated elution parameter V,V,-l, was 1.682 for all four determinations. The native molecular mass, estimated from the standard curve VeVo-I versus log M,. (regression coefficient r = 0.999), was about 212 kDa (Fig. 3B). The subunit stoichiometry of ureases has generally been determined by relating the native molecular mass of the urease to the subunit sizes and the relative abundance of the different subunits estimated from SDS-polyacrylamide gels (Mobley and Hausinger 1989). The jack bean urease has a native molecular mass of 590 kDa and a single subunit of 91 kDa, indicating that the native enzyme is a hexamer (Andrews et al. 1984; Takashima et al. 1988). The urease of Helicobacter species is composed of two subunits of about 61-66 kDa and 26-31 kDa with an estimated 1:1 stoichiometry (Dunn et al. 1990,1991; Labigne et al. 1991; Hu and Mobley 1990; Evans et al. 1991; Hawtin et al. 1990; Costas et al. 1991). Most size estimates of Helicobacter urease are about 535-650 kDa and, therefore, it has been assumed that the native enzyme has six copies of each of the subunits, like jack bean urease (Dunn et al. 1991; Hu and Mobley 1990; Evans et al. 1991; Austin et al. 1992). However, many other bacteria, including Proteus, Klebsiella, Lactobacillus, and Staphylococcus spp., Providencia stuartii, Morganella morganii, Ureaplasma urealyticum, Yersinia enterocolitica, and Bacillus sp. strain TB-90 appear to have urease composed of one large (or, 61-73 kDa) and two small (p, 8-17 kDa and y, 6-11 kDa) subunits (Mobley and Hausinger 1989; Jones and Mobley 1989; Skurnik et al. 1993; Kakimoto et al. 1989, 1990; Hu et al. 1990; Christians et al. 1991; Thirkell et al. 1989; Maeda et al. 1994; Schafer and Kaltwasser 1994; Jose et al. 1994). For most bacteria, it has been suggested that two copies of the large (a)subunit and two or four copies of each of the small subunits (p, 7 ) are present in the native enzyme (Mobley and Hausinger 1989; Thirkell et al. 1989; Maeda et al. 1994). The enzymes from Staphylococcus xylosus and Staphylococcus saprophyticus may have a ( 4 3 ~ subunit )~ structure (Schafer and Kaltwasser 1994; Jose et al. 1994).The nickel-containing catalytic subunit of bacterial ureases is the large subunit (Sriwathana and Mobley 1993; Thirkell et al. 1989; Hawtin et al. 1991; Labigne et al. 1991; Park and Hausinger 1993). Therefore, while the Helicobacter

Lubbers et al.


Fig. 3. Sepharose CL6B-200 gel filtration column chromatography of S. pombe urease. (A) Elution profile of S. pombe urease. Each curve represents a separate passage of partially purified urease; three independent urease isolations were used for the four determinations (two determinations were on the same urease preparation). Urease activity for each point is the average of three determinations. The interpolated peak of the curves represents the V, of urease, marked with an arrow. (B) VeV,-l elution parameter as a function of molecular mass for the size standards used to calibrate the column: thyroglobulin (669 m a ) , apoferritin (443 kDa), P-amylase (200 m a ) , alcohol dehydrogenase (150 kDa), albumin (66 kDa) and carbonic anhydrase (29 m a ) . Arrows indicate VeV,-l and the corresponding molecular mass for S.pombe urease.

EMuent volume (mL)

and jack bean ureases have six copies of the catalytic subunit, other bacteria may have two or four copies of the catalytic subunit. However, recently it has been proposed that bacterial

Fig. 4. Activity of S. pombe urease at various pH. Crude S. pombe urease was assayed in phosphate (0) and acetate (A) buffers. One hundred percent activity was 0.41 pmol . min-1. mL-l. Values are the means of two determinations and had a range of variation of less than 5%.

ureases could be a trimer of catalytic units (P.A. Kaplus, cited as a personal communication in Park et al. 1994). If this is correct, then all known ureases may only be active as trimers or hexamers of the catalytic subunit. The native size of S.pombe urease (212 kDa) and the subunit size (102 kDa) indicate that the enzyme could be a dimer of the single subunit. However, this has not been rigorously tested and the inaccuracies inherent in molecular mass determinations may have resulted in incorrect subunit stoichiometry estimations of S. pombe urease, as well as many bacterial ureases. A single type of urease subunit appears to be a conserved feature among all eukaryotes, although the subunit size estimates are smaller than for S. pombe and vary from 40 kDa (A. nidulans, Creaser and Porter 1985), 72 kDa (R. paludigenum, Phillips et al. 1990), 80 kDa (Ustilago violacea, Baird and Garber 1981), 83 kDa (A. niger, Smith et al. 1993), to 91 kDa (jack bean, Takishima et al. 1988). The subunit stoichiometries of the eukaryotic ureases also vary and may be trimer (A. niger, Smith et al. 1993), hexamer (jack bean, Andrews et al. 1984), and possibly octamer (R. paludigenum, Phillips et al. 1990). The native size of S. pombe urease is similar to the size of urease from other ascomycetes (240 kDa, A. nidulans, Creaser and Porter 1985; 250 kDa, A. niger, Smith et al. 1993), while jack bean urease is 590 kDa (Andrews et al. 1984) and urease from the basidiomycetous yeast R. paludigenum is 569 kDa (Phillips et al. 1990). Since we have shown that S. pombe urease has one subunit, only a single gene would be required to encode the urease. Four independent genes have been identified that are essential for urease activity of permeabilized S. pombe cells (Kinghorn and

Can. J. Microbiol. Vol. 42, 1996


Fig. 5. Comparison of the N-terminal sequence of S. pombe urease with other ureases. Gaps introduced for the alignment are indicated by dashes; residue positions matching S. pombe are indicated by colons.

Schizosaccharomyces pombe Jack bean Helicobacter pylori Helicobacterfelis Escherichia coli Proteus vulgaris Proteus mirabilis Klebsiella aerogenes Klebsiella pneumoniae Yersinia enterocolitica Bacillus sp. strain TB-90 Lactobacillus femzentum Ureaplasma urealyticum Staphylococcus xylosus

Fluri 1984) or cell-free extracts (Lubbers 1993). Therefore, three of the S.pornbe urease genes appear to be accessory genes required for urease activity but not required to encode the enzyme subunit. Four urease genes have also been reported for A. nidulans (Mackay and Pateman 1982) and Neurospora crassa (Haysman and Howe 1971; Benson and Howe 1978). For A. nidzdans, one of the genes appears to encode an urea active transport protein, one gene encodes the urease subunit, and at least one gene may be involved in the synthesis or incorporation of an essential nickel cofactor (Mackay and Pateman 1980,1982; Pateman et al. 1982).Schizosaccharomyces pornbe urease seems to be constitutive, so there is not an obvious regulatory role for any of the urease genes. A urea transport role can also be discounted because mutants of any of the four urease genes lack intracellular urease activity (Kinghom and Fluri 1984; Lubbers 1993). The four urease genes of S . pornhe are not necessarily equivalent to the A. nidulans genes, because the methods for isolating the respective urease mutants were different. However, as in Aspergillus, one or more of the S. pornbe genes may be involved in the synthesis or incorporation of a nickel cofactor.

Kinetic characteristics Schizossacharomycespombeurease was active over a broad pH range (pH 4-10) with maximum activity at about pH 8.2 (Fig. 4). The pH optima for most other fungal ureases are similar to that for S . pornbe, being within the range of pH 8.0-8.5, e.g., Aspergillus species (Zawada and Sutcliffe 1981; Creaser and Porter 1985; Smith et al. 1993) and R. paludigenurn (Phillips et al. 1990). An exception is Ustilago violacea urease, which has a pH optimum of 7.0. Ureases from most bacteria, with a few exceptions, and jack bean also have slightly lower pH optima, at about pH 7.0-7.75 (Mobley and Hausinger 1989; Christians et al. 1991; Thirkell et al. 1989). The K, of S . pornbe urease, estimated from an EadieHofstee plot (regression coefficient r = 0.998), was 1.0 mM urea. An impure urease preparation was used for this determination; however, it has been shown that for microbial ureases the K, values determined for crude cell extracts agree with the values for purified extracts and, therefore, impure urease can be used for valid K, determinations (Mobley and Hausinger 1989).The K,reported for A. nidulans urease (1.3 mM, Creaser

and Porter 1985) is close to the value we observed for S. pornbe. Jack bean (Andrews et al. 1984), Ustilago violacea (Baird and Garber 1981), R. paludigenum (Phillips et al. 1990), and A. niger ureases have a K, for urea of 2.8-3.8 mM. It appears that all the eukaryotes so far examined have quite similar K, values. In contrast, the K, of bacterial ureases range from 0.1 mM to >I00 mM (Mobley and Hausinger 1989). The specific activity of S . pombe urease was about 700-800 U .mg protein-'. The specific activities of the Aspergillus enzymes are similar: 670 U.mg-I for A. nidulans (Creaser and Porter 1985) and 1341 U.mg-l for A. niger (Smith et al. 1993). However, R. paludigenurn has a much less active urease (62.5 U . mg-1) and jack bean urease is much more active (3500 U . mg-I, Andrews et al. 1984).

Sequence of urease N-terminus and peptides The N-terminal sequence of purified S. pombe urease, determined for two samples (47 and 300 pg), was MQPRELHKLTLHQLGS. The sequence data confirmed that only a single subunit type was present in the native urease. Purified urease was digested with bovine trypsin, the resulting peptides were purified by HPLC, and the sequence of three peptides, designated T21, T40, and T43, was determined: T21, FIETNEK; T40, LYAPENSPGFVEVLEGEIELLPNLP; T43, ELHKLTLHNLGSLA. The sequence of T43 overlaps the directly sequenced N-terminus, giving 18 residues of Nterminus sequence. The urease sequences were compared with other urease sequences in the Genbank, EMBL, SwissProt, and PIR data bases using the FASTA program (Deveraux et al. 1984). No significant identity was found between the peptides T40 and T21 and other urease sequences. However, a high degree of identity was noted between the N-terminal sequence of S . pornbe urease and the N-terminal sequences bf urease subunits from other sources. The N-terminal regions of the urease sequences were aligned using the PILEUP program (Deveraux et al. 1984). For the bacterial ureases, which have two or three urease subunits, the subunit that corresponds to the jack bean N-terminus was used for the sequence comparison. The best alignment for S. pombe urease was obtained if a two amino acid gap is placed at the third and fourth residue positions (Fig. 5). These two residues, absent in S . pomhe urease,

Lubbers et al.


are highly conserved amongst the other ureases. Overall, the 20 N-terminal residues were highly conserved, with most substitutions being conservative (Fig. 5). The urease sequences with the greatest similarity to the S. pombe enzyme are from the only other eukaryote sequence reported Cjack bean) and Helicobacter spp. All of the bacteria used for the sequence comparison have a urease consisting of three subunits except the Helicobacter, which has a two subunit enzyme. One might speculate that the Helicobacter-type urease has a closer evolutionary relationship to the eukaryotic ureases Cjack bean and S. pombe) than the other bacteria examined.

Conclusions Schizosaccharomyces pombe urease was purified and characterized. Overall, the kinetic (K,, pH optimum, specific activity) and structural properties (single subunit, native molecular mass, amino acid sequence) were more similar to the small number of other eukaryotic ureases studied than to the bacterial ureases and were especially close to those of the ascomycete Aspergillus urease. Previous studies have demonstrated a role for urease in the purine degradation pathway of A. nidulans (Scazzochio and Darlington 1968) and S.pomhe (Kinghorn and Fluri 1984). We speculate that the similarity of the urease from these organisms may indicate a similar physiological role for the enzyme. It appears that even though ascomycetous yeasts do not generally have urease (Sen and Komagata 1979; Booth and Vishniac 1987), the ascomycetous yeast S. pombe has a urease that most closely resembles the enzyme of the filamentous fungal ascomycetes. We have shown that only one of the four urease genes of S. pombe is required to encode the single urease subunit. The other three urease genes may encode accessory functions required for urease activity in S. pomhe. We are presently trying to isolate the four urease genes from S. pomhe.

References

Andrews, R.K., Blakeley, R.L., and Zemer, B. 1984. Urea and urease. In Advances in inorganic biochemistry Vol. 6. Edited by G.L. Eichhorn and L. G. Marzilli. Elsevier, New York. pp. 245-283. Austin, J.W., Doig, P., Stewart, M., and Trust, T.J. 1991. Macromolecular structure and aggregation states of Helicobacter pylori urease. J. Bacteriol. 173: 5663-5667. Austin, J.W., Doig, P., Stewart, M., and Trust, T.J. 1992. Structural comparison of urease and a GroEL analog from Helicobacterpylori. J. Bacteriol. 174: 7470-7473. Baird, M.L., and Garber, E.D. 1981. The genetics and biochemistry of urease in Ustilago violacea. Biochem. Genet. 19: 1101-1 114. Bast, E. 1988. Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatiaceae). Arch. Microbiol. 150: 6-10. Benson, E.W., and Howe, H.B. 1978. Reversion and interallelic complementation at four urease loci in Neurospora crassa. Mol. Gen. Genet. 165: 277-282. Blanchard, A. 1990. Ureaplasma urealyticum urease genes: use of a UGA tryptophan codon. Mol. Microbiol. 4: 669-676. Bode, G., Malfertheiner, P., Nilius, M., Lehnhardt, G., and Ditschuneit, H. 1989. Ultrastructural localisation of urease in outer membrane and periplasm of Campylobacter pylori. J. Clin. Pathol. 42: 778-779.

Booth, J.L., and Vishniac, H.S. 1987. Urease testing and yeast taxonomy. Can. J. Microbiol. 33: 396-404. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. Christensen, W.B. 1946. Urea decomposition as a means of differentiating Proteus and Paracolon cultures from each other and from Salmonella and Shigella types. J. Bacteriol. 52: 461-466. Christians, S., Jose, J., Schafer, U., and Kaltwasser, H. 1991. Purification and subunit determinationof the nickel-dependentStaphylococcus xylosus urease. FEMS Microbiol. Lett. 80: 271-276. Costas, M., Owen, R. J., Morgan, D.D., and Goodwin, C.S. 1991.Loss of urease activity in Helicobacter mustelae. Lett. Appl. Microbiol. 13: 261-264. Creaser, E.H., and Porter, R.L. 1985. The purification of urease from Aspergillus nidulans. Int. J. Biochem. 17: 1339-1341. de Llano, J.J.M., Garcia-Segura, J.M., and Gavilanes, J.G. 1989. Selective silver staining of urease activity in polyacrylamide gels. Anal. Biochem. 177: 37-40. Deveraux, J., Haeberli, P., and Smithies, 0 . 1984. A comprehensive set of sequence analysis programs for the vax. Nucleic Acids Res. 12: 387-395. Dunn, B.E., Campbell, G.P., Perez-Perez, G.I., and Blaser, M. J. 1990. Purification and characterization of urease from Helicobacter pylori. J. Biol. Chem. 265: 9464-9469. Dunn, B.E., Sung, C.-C., Taylor, N.S., and Fox, J.G. 1991. Purification and characterization of Helicobacter mustelae urease. Infect. Immun. 59: 3343-3345. Evans, D.J., Evans, D.G., Kirkpatrick, S.S., and Graham, D.Y. 1991. Characterization of the Helicobacter pylori urease and purification of its subunits. Microb. Pathog. 10: 15-26. Ferrero, R.L., and Labigne, A. 1993. Cloning, expression and sequencing of Helicobacter felis urease genes. Mol. Microbiol. 9: 323-333. Fluri, R., and Kinghom, J.R. 1985. Induction control of purine catabolism in Schizosaccharomycespombe. Curr. Genet. 9: 573-578. Gatermann,S., andMarre, R. 1989. Cloning andexpression of Staphylococcus saprophyticus urease gene sequences in Staphylococcus carnosus and contribution of the enzyme to virulence. Infect. Immun. 57: 2998-3002. Gutz, H., Heslot, H., Leupold, U., and Loprieno, N. 1974. Schizosaccharomyces pombe. In Handbook of genetics. Vol. 1. Edited by R.C. King. Plenum, New York. pp. 395-446. Hausinger, R.P. 1987. Nickel utilization by microorganisms. Microbiol. Rev. 51: 22-42. Hawtin, P.R., Stacey, A.R., and Newell, D.G. 1990. Investigation of the structure and localization of the urease of Helicobacter pylori using monoclonal antibodies. J. Gen. Microbiol. 136: 1995-2000. Hawtin, P.R., Delves, H.T., and Newell, D.G. 1991. The demonstration of nickel in the urease of Helicobacterpylori by atomic absorption spectroscopy. FEMS Microbiol. Lett. 77: 51-54. Haysman, P., and Howe, H.B. 1971. Some genetic and physiological characteristics of urease-defective strains of Neurospora crassa. Can. J. Genet. Cytol. 13: 256-269. Holland, M.A., and Polacco, J.C. 1992. Urease-null and hydrogenasenull phenotypes of a phylloplane bacterium reveal altered nickel metabolism in two soybean mutants. Plant Physiol. 98: 942-948. Hu, L.-T., and Mobley, H.L.T. 1990. Purification and N-terminal analysis of urease from Helicobacter pylori. Infect. Immun. 58: 992-998. Hu, L.-T., and Mobley, H.L.T. 1993.Expression of catalytically active recombinant Helicobacter pylori urease at wild-type levels in Escherichia coli. Infect. Immun. 61: 2563-2569. Hu, L.-T., Nicholson, E.B., Jones, B.D., Lynch, M.J., and Mobley, H.L.T. 1990. Morganella morganii urease: purification, characterization,and isolation of gene sequences.J. Bacteriol. 172: 3073-3080.


Can. J. Microbiol. Vol. 42. 1996 Hu, L.-T., Foxall, P.A., Russell, R., and Mobley, H.L.T. 1992. Purification of recombinant Helicobacter pylori urease apoenzyme encoded by ureA and ureB. Infect. Immun. 60: 2657-2666. Jahns, T., Zobel, A., Kleiner, D., and Kaltwasser, H. 1988. Evidence for carrier-mediated, energy-dependent uptake of urea in some bacteria. Arch. Microbiol. 149: 377-383. Jones, B.D., and Mobley, H.L.T. 1988. Proteus rnirabilis urease: genetic organization,regulation, and expression of structural genes. J. Bacteriol. 170: 3342-3349. Jones, B.D., and Mobley, H.L.T. 1989. Proteus rnirabilis urease: nucleotide sequence determination and comparison with jack bean urease. J. Bactenol. 171: 6414-6422. Jose, J., Schafer, U.K., and Kaltwasser,H. 1994. Threonine is present instead of cysteine at the active site of urease from Staphylococcus xylosus. Arch. Microbiol. 161: 384-392. Kakimoto, S., Sumino, Y., Akiyama, S., and Nakao, Y. 1989. Purification and characterization of acid urease from Lactobacillus reuteri. Agric. Biol. Chem. 53: 1119-1 125. Kakimoto, S., Sumino, Y., Kawahara, K., Yamazaki, E., and Nakatsui, I. 1990. Purification and characterization of acid urease from Lactobacillus fermenturn. Appl. Microbiol. Biotechnol. 32: 538-543. Kinghom, J.R., and Fluri, R. 1984. Genetic studies of urine breakCurr. Genet. down in the fission yeast ~chizosaccharomyces~ornbe: 8: 99-105. Labigne, A., Cussac, V., and Courcoux, P. 1991. Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity. J. Bacteriol. 173: 1920-1931. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-685. Lee, M.H., Mulrooney, S.B., and Hausinger, R.P. 1990. Purification, characterization,and in vivo reconstitution of Klebsiella aerogenes urease apoenzyme. J. Bacteriol. 172: 4427-4431. Lee, M.H., Mulrooney, S.B., Renner, M.J., Markowicz, Y., and Hausinger, R.P. 1992. Klebsiella aerogenes urease gene cluster: sequence of ureD and demonstration that four accessory genes (ureD, ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis. J. Bacteriol. 174: 4324-4330. Lubbers, M.W. 1993. Genetic and biochemical studies on the urease enzyme system of Schizosaccharornyces pombe. Ph.D. thesis, Massey University, Palmerston North, New Zealand. Mackay, E.M., and Pateman, J.A. 1980. Nickel requirement of a urease-deficientmutant in Aspergillus nidulans. J. Gen. Microbiol, 116: 249-251. Mackay, E.M., and Pateman, J.A. 1982. The regulation of urease activity in Aspergillus nidulans. Biochem. Genet. 20: 763-776. Mackerras, A.H., and Smith, G.D. 1986. Urease activity of the cyanobacterium Anabaena cylindrica. J. Gen. Microbiol. 132: 2749-2752. Maeda, M., Hidaka, M., Nakamura, A., Masaki, H., and Uozumi, T. 1994. Cloning, sequencing,and expression of thermophilic Bacillus sp. strain TB-90 urease gene complex in Escherichia coli. J. Bacten01. 176: 432-442. Mobley, H.L.T., and Hausinger, R.P. 1989. Microbial ureases: significance,regulation, and molecular characterization.Microbiol. Rev. 53: 85-108. Moreno, S., Klar, A,, and Nurse, P. 1991. Molecular genetic analysis of fission yeast Schizosaccharomyces pornbe. Methods Enzymol. 194: 795-823. Morrissey,J.H. 1981. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117: 307-310. Morsdorf, G., and Kaltwasser,H. 1990.Cloning of the genes encoding urease from Proteus vulgaris and sequencing of the structuralgenes. FEMS Microbiol. Lett. 66: 67-74.

Mulrooney, S.B., and Hausinger, R.P. 1990. Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation. J. Bacteriol. 172: 583775843, Mulrooney, S.B., Lynch, M. J., Mobley, H.L.T., and Hausinger, H.L.T. 1988. Purification, characterization, and genetic organization of recombinant Providencia stuartii urease expressed by Escherichia coli. J. Bactenol. 170: 2202-2207. Park, I.-S., and Hausinger, R.P. 1993. Site-directed mutagenesis of Klebsiella aerogenes urease: identificationof histidineresidues that appear to function in nickel ligation, substrate binding, and catalysis. Protein Sci. 2: 1034-1041. Park, I.-S., Carr, M.B., and Hausinger, R.P. 1994. In vitro activation of urease apoprotein and role of UreD as a chaperone required for nickel metallocenter assembly. Proc. Natl. Acad. Sci. U.S.A. 91: 3233-3237. Pateman, J.A., Dunn, E., and Mackay, E.M. 1982. Urea and thiourea transport in Aspergillus nidulans. Biochem. Genet. 20: 777-790. Phillips, A,, Pretorius, G.H.J., and Du Toit, P.J. 1990. A survey of yeast ureases and characterization of partially purified Rhodosporidiurnpaludigenum urease. FEMS Microbiol. Lett. 79: 21-26. Rando, D., Steglitz,U., Morsdorf, G., and Kaltwasser,H. 1990. Nickel availability and urease expression in Proteus rnirabilis. Arch. Microbiol. 154: 428-432. Rees, T.A.V., and Bekheet, I.A. 1982. The role of nickel in urea assimilation by algae. Planta, 156: 385-387. Scazzocchio,C., and Darlington,A. J. 1968. The induction andrepression of the enzymes of purine breakdown in Aspergillus nidulans. Biochim. Biophys. Acta, 166: 557-568. Schafer, U.K., and Kaltwasser,H. 1994. Urease from Staphylococcus saprophyticus: purification, characterization and comparison to Staphylococcus xylosus urease. Arch. Microbiol. 161: 393-399. Schneider, J., and Kaltwasser, H. 1984. Urease from Arthrobacter oxydans, a nickel-containing enzyme. Arch. Microbiol. 139: 355-360. Seeliger, H.P.R. 1956. Use of a urease test for the screening and identification of Cryptococci. J. Bacteriol. 72: 127-131. Sen, K., and Komagata, K. 1979. Distribution of urease and extracellular DNase in yeast species. J. Gen. Appl. Microbiol. 25: 127-136. Skumik, M., Batsford, S., Mertz, A., Schiltz, E., and Toivanen, P. 1993. The putative cationic antigen of Yersinia enterocolitica is a urease P-subunit. Infect. Immun. 61: 2498-2504. Smith, P.T., King, A.D., and Goodman, N. 1993. Isolation and characterization of urease from Aspergillus niger. J. Gen. Microbiol. 139: 957-962. Spector, T. 1978. Refinement of the coomassie blue method of protein quantitation. Anal. Biochem. 86: 142-146. Sriwanthana,B., and Mobley, H.L.T. 1993. Proteus rnirabilis urease: histidine 320 of UreC is essential for urea hydrolysis and nickel ion binding within the native enzyme. Infect. Immun. 61: 2570-2577. Sumner, J. B. 1926. The isolation and crystallization of the enzyme urease. J. Biol. Chem. 69: 435-441. Takishima, K., Suga, T., and Mamiya, G. 1988. The structure of jack bean urease. The complete amino acid sequence,limited proteolysis and reactive cysteine residues. Eur. J. Biochem. 175: 151-165. Thirkell, D., Myles, A.D., Precious, B.L., Frost, J.S., Woodall, J.C., Burdon, M.G., and Russell, W.C. 1989. The urease of Ureaplasma urealyticum. J. Gen. Microbiol. 135: 315-323. Wong, B.L., and Shobe, C.R. 1974. Single-steppurification of urease by affinity chromatography. Can. J. Microbiol. 20: 623-630. Zawada, J.W., and Sutcliffe, J.F. 1981. A possible role for urease as a storage protein in Aspergillus tamarii. Ann. Bot. 48: 797-810. Zemer, B. 1991. Recent advances in the chemistry of an old enzyme, urease. Bioorganic Chem. 19: 116-131.