Characterization of urease from Sporosarcina ureae

Arch Microbiol (1992) 157:411-416

Archives of

Microbiolegy 9 Springer-Verlag 1992

Characterization of urease from Sporosarcina ureae Deborah D. McCoy, Aysegul Cetin, and Robert P. Hausinger Departments of Microbiology and Biochemistry, Michigan State University, East Lansing, MI 48824, USA Received September 23, 1991/Accepted December 26, 1991

Abstraet. Alkaline stable (pH 7.75-12.5) urease from Sporosarcina ureae was purified over 400-fold by ion exchange and hydrophobic interaction chromatography. The cytoplasmic enzyme was remarkably active with a specific activity of greater than 9300 gmol urea degraded rain -1 mg protein -1 at pH 7.5, where it has optimal activity. Although S. ureae is closely related to Bacillus pasteurii, known to possess a homopolymeric urease containing 1 nickel per subunit [M r = 65000], the S. ureae enzyme is comprised o f three subunits [apparent Mr = 63100 (e), 14500 (fl), and 8500 (7)] in an estimated e/~72 stoichiometry and contains 2.1 _+ 0.6 nickel ions per eft72 unit as measured by atomic absorption spectrometry. Stationary phase cultures sometimes possessed low levels of urease activity, but the specific activity of cell extracts of partially purified urease preparations from such cultures could be elevated by heat treatment, dilution, or dialysis to values comparable to those observed in samples from exponentially grown cells.

Key words: Urease - Sporosarcina ureae Alkaline stable - Enzyme activation

Nickel -

Bacterial ureases are important enzymes in environmental transformations of certain nitrogenous compounds, in ruminant metabolism, and in the development of certain human and animal diseases (reviewed by Mobley and Hausinger 1989). The enzyme has been purified from several microorganisms, but the best characterized bacterial urease is that from Klebsiella aerogenes. This heteropolymeric enzyme consists of three subunits of apparent Mrs = 72000 (e), 11000 (/~), and 9000 (7) in an ~2fl474 stoichiometry, and contains 4 nickel ions per native molecule (Todd and Hausinger 1987). The mechanism of inhibition has been characterized for several inhibitiors (Todd and Hausinger 1989, 1991), and it was shown that there are two active sites per enzyme; i.e., 2

Correspondence to." R. P. Hausinger

nickel per catalytic center. Sequencing of the K. aerogenes urease gene cluster revealed the presence of other genes in addition to the three urease structural genes; these accessory genes were shown to be essential for incorporating Ni into apoenzyme (Mulrooney and Hausinger 1990). The enzymic and genetic properties o f K. aerogenes urease serve as a paradigm for most other bacterial ureases, however, reports in the literature indicated the existence of a distinct class of ureases. Purified ureases from Bacillus pasteurii (Christians and Kaltwasser 1986) and Brevibacterium ammoniagenes (Nakano et al. 1984) were reported to be homopolymeric with subunit Mrs = 65000 and 67000, respectively, and each enzyme was found to contain a single nickel ion per subunit. We were interested in characterizing the properties of a mononickel urease and sought to purify an example of this class of enzyme. We chose to study urease from Sporosarcina ureae because this microbe was known to be very closely related to B. pasteurii (Pechman et al. 1976), it had been shown to possess high levels of a nickelcontaining urease (Schneider and Kaltwasser 1984), and its urease had not yet been characterized. We found that, in contrast to the B. pasteurii and B. ammoniagenes enzymes, S. ureae urease is heteropolymeric and contains 2 nickel per large subunit.

Materials and methods Bacterial growth conditions Sporosarcina ureae 634, originally described by MacDonald and

MacDonald (1962), was obtained from Professor Tom Corner (Michigan State University). Cultures were grown at 25-30 ~ with vigorous aeration in a medium described by Goldman and Wilson (1977), but with the following modifications: K-glutamate (30 g/l) served as the main carbon and energy source, 25 mM NH4C1 served as the main nitrogen source, NiSO4 96 H20 (4 mg/1) was provided, and the medium was supplemented with 10% brain heart infusion. In a typical large-scale growth, a 10-liter culture was grown to 0 D 6 o 0 = 2, yielding 70-100 g of cells (wet weight). Cells were washed once in CED buffer [50 mM 2-N-eyclohexylaminoethanesulfonic acid (CHES), 1 mM EDTA, 1 mM dithiothreitol, pH 9.5], resuspended in an equal volume of CED buffer, and stored frozen at -20 ~

412

Assay

Nickel content

Urease activity was measured by quantitating the rate of ammonia released from urea by formation of indophenol which was monitored at 625 nm (Weatherburn 1967). The standard assay buffer consisted of 200mM urea, 50 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) 0.5 mM EDTA (pH 7.5). Although the enzyme does not exhibit long-term stability at this pH, insignificant losses were observed over the assay period. Reactions were initiated by addition of enzyme, the concentration of released ammonia was measured in timed aliquots, and rates were determined by linear regression analysis. One unit (U) of urease activity is defined as the amount of enzyme required to degrade 1 gmol urea per min at 30 ~ in the standard assay buffer. Protein content was determined by using a commercially available protein assay (Bio-Rad Company, Richmond, Calif., USA) and using bovine serum albumin as the standard.

The nickel content of purified urease was determined by using a Varian Spectra AA-400Z atomic absorption spectrometer equipped with a graphite furnace, autosampler, and Zeeman background correction. Samples were hydrolyzed in 1 N HNO3, dried, dissolved in 50 mM HNO3 and analyzed by measuring peak height during atomization.

Urease purification Thawed cells were treated on ice for 30-60 min with lysozyme (1 mg/ml), adjusted to 1 mM toluenesulfonyl fluoride, and disrupted by three passages through a French pressure cell (American Instrument Co., Silver Spring, Md., USA) at 1.2 x 108 Pa. The broken cell mixture was centrifuged at 100000 x g for 90 min at 4 ~ and the supernatant solution was applied to a column (2.5 x 12 cm) of DEAE-Sepharose equilibrated in CED buffer at 4 ~ The urease was eluted with a 200-ml linear gradient of 0 to 1.0 M KC1 in CED buffer, resulting in a peak of activity at approximately 0.5 M KC1. Peak fractions were adjusted to 2 M KC1 and loaded onto a preequilibrated phenyl-Sepharose column (2.5 x 15 cm) at 4 ~ After a wash with 50 ml of 2.0 M KC1 in CED buffer, urease was removed with a single-step elution by using CED buffer. Peak phenyl-Sepharose fractions were combined, diluted 4-fold with CED buffer, and applied to a Mono-Q HR 10/10 column equilibrated in this buffer. The activity was eluted at 0.5 M KC1 by using a multi-step KC1 gradient in CED buffer. The active fractions were pooled and either dialyzed against CED buffer or diluted at least 2-fold with HED buffer [50 mM HEPES, 1 mM EDTA, 1 mM dithiothreitol (pH 8.0)]. The sample was applied to a Mono-Q HR 5/5 column equilibrated in HED buffer, and urease was eluted at 450 mM KC1 by using a/multi-step KC1 gradient in this buffer. For some preparations, additional purification was achieved by inserting a phenyl-Superose chromatographic step between the two Mono-Q columns. This step involved adjusting the sample to 2 M KC1 in CED buffer, loading onto a preequilibrated phenyl-Superose HR 5/5 column, and eluting with a linear decreasing KC1 gradient in CED buffer. Urease activity eluted at approximately 1 M in KC1. All resins were obtained from Pharmacia (Uppsala, Sweden).

Polyacrylamide gel electrophoresis Electrophoresis was carried out by using the buffers of Laemmli (1970), a 10-18% polyacrylamide gradient resolving gel, and a 4.5% stacking gel. Samples were run after denaturing at 100 ~ for 5 min. The gels were stained with Coomassie brillant blue and scanned by using a Gilford Response spectrophotometer (Gilford Instrument Laboratories, Oberlin, Ohio, USA) at 595 nm.

Amino terminal sequence analysis The enzyme subunits were resolved in a 0.75 mm denaturing gel, as described above, and transferred to a sheet of Pro-Blot membrane (Applied Biosystems, Foster City, Calif., USA) by standard procedures (Matsudaira 1990). The bands were visualized by Coomassie blue staining, cut from the paper, and analyzed by using an Applied Biosystems 477A automated sequencer in the Michigan State University Macromolecular Structure Facility.

p H Studies The enzyme stability was assessed at various pH values by incubating urease in 1 mM EDTA, 10 mM buffer at 0 ~ for 60 min or 20 h, then diluting enzyme 20-fold into the standard assay buffer at 30 ~ Buffers to test stability included succinate (pH 4.8-5.8), 2-(N-morpholino)ethanesulfonic acid (MES, pH 5.3-6.6), HEPES (pH 6.5-7.8), 3-tris(hydroxymethyl)methylamino-l-propanesulfonie acid (pH 7.7-8.6), CHES (pH 8.9-10.0), cyclohexylaminopropanesulfonic acid (CAPS, pH 10.4-11.4), and phosphate (pH 11.5-13.2). The effects of pH on Km and V~,axwere established by assaying urease in 100 mM buffer containing 20 to 200 mM urea at 30 ~ Buffers included succinate (5.6 6.3), MES (pH 5.9-6.8), HEPES (pH 6.6-8.0), (N-hydroxyethyl)piperazine-N'-2-hydroxypropane sulfonic acid (pH 7.2-8.6), CHES (pH 8.9-10.2), and CAPS (pH 9.9-11.1). The kinetic parameters were calculated by using the method of Wilkinson (1961).

Activation studies For thermal activation, urease samples were incubated m CED buffer at 30, 37, 50, or 60 ~ and aliquots were withdrawn at selected timepoints and monitored for urease activity by using the standard assay at 30 ~ For dilution activation, urease samples were diluted 1000-fold into 100 mM HEPES, 1 mM EDTA, pH 7.5 buffer at 30 ~ At selected timepoints, 1 ml aliquots were withdrawn and mixed with 1 ml 400 mM urea (yielding the standard assay conditions) and urease activity was assessed.

Results

Urease purification A f t e r h a r v e s t i n g the cells, the s p e n t m e d i u m was a s s a y e d for u r e a s e a n d f o u n d to possess negligible levels o f activity. P u r i f i c a t i o n o f the e n z y m e f r o m d i s r u p t e d cells b y a f o u r - c o l u m n p r o c e d u r e is s u m m a r i z e d in T a b l e 1. T h e e n z y m e was n o t p u r i f i e d to h o m o g e n e i t y in this process, h o w e v e r , c o m p a r i s o n o f gel scans f r o m several p r e p a r a t i o n s clearly d e m o n s t r a t e d t h a t three p e p t i d e s were a l w a y s a s s o c i a t e d with urease activity a n d a l w a y s o c c u r r e d in the s a m e i n t e n s i t y r a t i o , whereas, the intensity of other minor peptides varied among preparations. I n t e g r a t i o n o f the gel scans d e m o n s t r a t e d t h a t urease a c c o u n t e d for 4 5 - 8 2 % o f the t o t a l p r o t e i n in these p r e p a r a t i o n s ; for the p r e p a r a t i o n s u m m a r i z e d in T a b l e 1 the e s t i m a t e d p u r i t y was 7 0 % . I n c l u s i o n o f p h e n y l S u p e r o s e c h r o m a t o g r a p h y in the p u r i f i c a t i o n p r o t o c o l led to a p r e p a r a t i o n t h a t was e s t i m a t e d b y gel s c a n n i n g to be > 9 5 % h o m o g e n e o u s ; h o w e v e r , the e n z y m e suffered p a r t i a l i n a c t i v a t i o n d u r i n g this p r o c e s s l e a d i n g to recov e r y o f o n l y 4 % o f the initial activity a n d the specific activity d r o p p e d to 2,075 U / r a g .

413 Table 1. Purification of urease from Sporosarcina ureae Purification step

Cell extracts DEAESepharose PhenylSepharose Mono-Q (10/10) Mono-Q (5/5)

Specific activity (U/mg)

Purification (-fold)

22.6 93.3 290 5170 9340

1

Total activity (U)

Enzyme recovery (%)

1 4.1

28100 27900

100 99

12.8

23600

84

16500 10500

59 37

229 413

2

Fig. 2. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of S. ureae urease. S. ureae urease (5 p.g) is shown in lane 1. Molecular weight markers include phosphorylase b, Mr = 92500; bovine serum albumin, M, = 66200; ovalbumin, Mr = 45000; carbonic anhydrase, Mr = 31000; soybean trypsin inhibitor, Mr = 21500; and l y s o z y m e , M r = 14400 (lane 2) as well as myoglobin (Mr = 16950) and its cyanogen bromide fragments, M,s = 14400, 8160, 6210, and 2510 (lane 3)

General urease properties Sporosarcina ureae urease was stable over a pH range of 7.75-12.5 for up 20 h at 0 ~ but rapidly lost activity at higher or lower pH values (Fig. 1). Maximal enzyme activity was observed at pH 7.5; decreased activity at higher or lower pH was found to be primarily due to a reduction in Vma x rather than an increase in K m. At pH 7.5, the the purified enzyme possessed a Vm,x > 12000U/mg and a K m of 6 0 _ 14mM. As shown in Fig. 2, the enzyme possessed three subunits of apparent Mrs = 63100 + 2000 (~), 14500 + 1000 (fl), and 8500 _+ 1000 (7). The integrated intensities of the three Coomassie blue-stained bands divided by their relative mass yielded an ~ : fl : ? ratio of 1.20 : 0.80: 2.00. Sequences of the three S. ureae urease subunits are provided in Fig. 3 and compared to other ureases which have been sequenced. Five preparations ofurease ranging in purity from 70-95% were analyzed by atomic absorption analysis; using appropriate corrections for sample purity the nickel content of urease was 2.1 _+ 0.6 nickel per ~fl72 unit (M r 94600).

1200

100-

f >

o 9 0

80-

9

o

.~

60-

8 40-

"i 2O

a

A

I'I

13

pH Fig. 1. pH stability of Sporosarcina ureae urease. Partially purified urease (1140 U/mg) was incubated at 0 ~ in buffers at the indicated pH values for 1 h (e) or 20 h (o), then urease activity was assessed in standard assay conditions at pH 7.5

3

Activation o f urease

In contrast to the well-behaved properties of samples from the typical cultures described above, occasional preparations of urease from stationary phase cultures exhibited anomalous behaviour in which the specific activity increased when the samples were heated, diluted, or dialyzed. This elevation of specific activity was due to an increase in total units due to enzyme activation rather than to removal of contaminating protein. Thermal or dilution activation was observed both in cell extracts and, if the temperature was maintained at < 4 ~ throughout the isolation, in partially purified samples from these preparations. An example of thermal activation in a partially purified urease sample is shown in Fig. 4. The specific activity was observed to double after 30 min incubation at 60 ~ whereas, the increase was more gradual at 50 ~ and only a slight increase was seen at 37 ~ or 30 ~ Activation did slowly occur in the latter two samples; when they were assayed after 18 h, the specific activities were 1380 and 1075 U/mg, respectively. As an example of dilution activation, the specific activity for a preparation of cell extracts slowly increased by a factor of 4 after diluting 1000-fold into pH 7.5 buffer at 30 ~ (Fig. 5, open symbols). Heat treatment (60 ~ for 60 min) resulted in a doubling of the specific activity for this same sample (compare the zero time points for the open and closed symbols in

414

1600-

(A) S.u. L.f. U.u. P.m. P.v. H.p. K.a.

M M M M M M M

R R N E E K E

L L L L L L L

L T S T T T T

P

(P) E

I

O

L P P P P

J.b.

M

K

L

S

P

R R R K R R

I K K L K V

Q D D D D E

E 1200

E E E E E E

"9>-

800

o o

400

bO

(B) S.u.

M

L

(S) K

D

I

(R) N

L.f. U.u. P.m. P.v. H.p. K.a.

M L M M L M

V V I I V I

P P P P P P

G G G G G G

A E E E E

I I I L Y

N R R F H

F V V L V

J.b.

R

I

P

G

E

I

L

C

(K) M D M

D

D

E

S S S S

R R R R

K Q Q K

N A A E

S

R

Q

A

H

R

K

E

0

0

210

410

610

810

1 ;0

1 89

1 ;0

Time (min) Fig. 4. Thermal activation of S. ureae urease. A sample of partially purified urease (700 U/mg) from a stationary phase culture was incubated in CED buffer (pH 9.5) at 60 ~ (o), 50 ~ (m), 37 ~ (A), or 30 ~ (o) for the indicated times and urease activity was determined at 30 ~ in the standard assay buffer

(c) 30-

s.u.

(s) F

L.f. U.u. P.m. P.v. H.p. K.a.

M M M M M

S F K K K S

F K T T K N

J.b.

N

T

F

0-

F

Fig. 3 A - C Amino terminal sequences of the S. ureae ureae subunits and comparison to other urease sequences. The amino terminal sequences of the smallest (A), middle (B), and largest (C) subunits of S. ureae urease are compared with urease sequences from Lactobacillus fermentum (Kakimoto et al. 1990), Ureaplasma urealyticum (Blanchard 1990), Proteus mirabilis (Jones and Mobley 1989), Proteus vulgaris (M6rsdorf and Kaltwasser 1990), Helieobaeter pylori (Clayton et al. 1990), Klebsiella aerogenes (Mulrooney and Hausinger 1990), and jack bean (Takishima et al. 1988). Residues in parentheses were only tentatively established. The middle subunit of the U. urealytieum urease has an amino terminal extension compared to the other sequences; the sequence provided corresponds to residues ~ 16-23. The two small subunits of H. pylori urease are apparently fused; the sequences shown correspond to residues ~ 1-9 and ~ 106-113. The amino terminal methionine is removed from the large subunit of the mature K. aerogenes urease (Mobley and Hausinger 1989). Jack bean urease possesses a single subunit; the sequences shown correspond to residues =~ 1-9, ~ 132-139, and # 271-278

Fig. 5), a n d d i l u t i o n o f the t h e r m a l l y a c t i v a t e d s a m p l e f u r t h e r i n c r e a s e d the specific activity (Fig. 5, closed s y m b o l s ) y i e l d i n g n e a r l y the s a m e final v a l u e as the n o n - h e a t t r e a t e d sample. T h e p r o t e i n itself need n o t be d i l u t e d f o r this t y p e o f a c t i v a t i o n ; 24 h dialysis at 30 ~ in C E D b u f f e r ( p H 9.5) o f the s a m e cell extracts (using a m e m b r a n e w i t h M~ = 12000 cutoff) led to o v e r 3-fold a c t i v a t i o n of u r e a s e activity.

>~ 4~

20-

{3 0 0 9 O_ U3

10-

160

z;0

3;0

s;0

6;0

Time (min) Fig. 5. Dilution activation of S. ureae urease. Crude cell extracts from cells harvested in stationary phase were diluted 1000-fold into 100 mM HEPES, 1 mM EDTA buffer (pH 7.5) and monitored for urease activity (9 The same experiment was carried out after first heat treating the cell extracts at 60 ~ for 60 min (e)

Discussion General p r o p e r t i e s o f S p o r o s a r c i n a ureae urease

T h e a b s e n c e o f a c t i v i t y in s p e n t cell m e d i u m a n d the presence o f a c t i v i t y in the s u p e r n a t a n t f r a c t i o n o f d i s r u p t e d cells i n d i c a t e d t h a t S. ureae u r e a s e is a c y t o p l a s m i c enzyme. This f i n d i n g c o n t r a d i c t s a r e p o r t cited in a review b y R e i t h e l (1971) w h i c h c l a i m e d t h a t u r e a s e is a n e x o e n z y m e o f this m i c r o o r g a n i s m . A l t h o u g h m a x i m u m activity o f the S. ureae e n z y m e was o b s e r v e d a t p H 7.5, all p u r i f i c a t i o n steps were c a r r i e d o u t at e l e v a t e d p H b e c a u s e o f the l a b i l i t y o f this urease at n e u t r a l p H values. F o r e x a m p l e , i n c u b a t i o n o f the e n z y m e on ice at p H 7.21 led to a loss o f > 50% a c t i v i t y

415 in 1 h and > 90% loss after 20 h. By contrast, the enzyme was extremely stable to elevated pH as shown by complete retention of activity to p H 12. The alkaline stability of S. ureae urease is unique compared to other ureases that have been examined: e.g., the reported stabilities for other ureases include Brevibacterium ammoniagenes, p H 7 - 1 0 (Nakano et al. 1984), Proteus mirabiIis, p H 7 - 1 0 (Breitenbach and Hausinger 1988), Klebsiella aerogenes, pH 5 - 1 0 (Todd and Hausinger 1987), Arthrobacter oxydans, above p H 5 (Schneider and Kaltwasser 1984), Lactobacillusfermentum, pH 3 - 9 (Kakimoto et al. 1990), and Lactobacillus reuteri, p H 3 - 8 (Kakimoto et al. 1989). The specific activity for S. ureae urease ( > 9300 U/mg protein) is significantly higher than that of many other purified ureases (reviewed in Mobley and Hausinger 1989), including the enzyme from Bacillus pasteurii (997 U/rag, Christians and Kaltwasser 1986). Indeed, the only urease reported to possess a higher specific activity is that of Ureaplasma urealyticum which exhibited a value of 16 750-180000 U/rag (Precious et al. 1987; Saada and Kahane 1988; Stemke et al. 1987). S. ureae is taxonomically closely related to B. pasteurii (Pechman et al. 1976), thus, we expected that its urease would possess a homopolymeric structure containing 1 nickel per subunit as reported for the B. pasteurii enzyme (Christians and Kaltwasser 1986). In contrast, we found that three subunits were present and that 2 nickel were present per large subunit, as found in many other bacterial ureases (reviewed in Mobley and Hausinger 1989). The presence of three subunits in S. ureae urease, deduced from the observation of three bands after denaturing polyacrylamide gel electrophoresis, was verified by sequence analysis. The three peptide sequences (Fig. 3) share substantial homology with sequences from other three-subunit ureases as well as with appropriate portions of the two subunit (Helicobacter pylori) or single subunit (jack bean) enzymes. The latter two proteins are likely to have arisen by gene fusion. The estimated subunit ratio (~:fl:7 = 1:1:2) in S. ureae enzyme is distinct from that of any purified urease. For example, the reported ratio of the three subunits for ureases from K. aerogenes (Todd and Hausinger 1987) and Providencia stuartii (Mulrooney et al. 1988) was 1 : 2 : 2 , from L. fermentum (Kakimoto et al. 1990), L. reuteri (Kakimoto et al. 1989), and Streptococcus mitior (Yamazaki et al. 1990) it was 1:2: 1, and from U. urealyticum it was 1 : 1 : 1 (Thirkell et al. 1989). These estimates for subunit ratios are suspect because binding of Coomassie blue dye to a peptide will be influenced by the peptide amino acid composition. However, sequence data indicate extensive homologies among ureases which should result in similar dye binding properties between species. Activation of urease in cells from stationary cultures Urease activity in some stationary phase cultures was depressed, but the total enzyme units in cell extracts or partially purified samples could be elevated by heat treatment, dilution, or dialysis. This behavior has not been described for any other urease. One possible ex-

planation for this phenomenon invokes the presence of a tight-binding inhibitor in these low activity cells. Starting with partially inhibited enzyme, dilution or dialysis may simply lead to inhibitor dissociation concomitant with an increase in activity. Activation energy may be required for dissociation, hence, the enzymeinhibitor complex could remain intact during cell disruption and initial purification. Thermal activation would reflect simple equilibration of the diluted system resulting in increased activity. However, we have been unable to directly demonstrate the presence of the hypothesized inhibitor by adding various fractions back to fully active enzyme, perhaps because the cellular levels of inhibitor are so low. Further studies are needed to test this inhibitor hypothesis and, if it is correct, to identify the structure and determine the role of the endogenous urease inhibitor. Acknowledgements. We thank Cheng Kao for initiating studies of the S. ureae enzyme, and Andrew Plymale, Weizhang Ye, Jackie Wood, and Julie Breitenbach for preliminary studies. This work was supported by CRGO/U.S.D.A. grant 89-37120-4842 and by the Michigan State Umversity Agricultural Experiment Station.

References

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