JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 71, No. 2, 131-133. 1991

Purification and Characterization of Glyoxalase I from Hansenula mrakii

YOSHIHARU INOUE, LINH-THUOC TRAN, KOJI YOSHIKAWA, KQUSAKU MURATA, AND AKIRA KIMURA*

Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan

Received 27 September 1990/Accepted 29 October 1990

Glyoxalase I was purified from Hansenula mrakii IFO 0895 which was resistant to 25 mM methyl.glyoxal. The molecular weight of the purified enzyme was calculated to be 38,000 by both gel-filtration of Sephadex G- 150 and SDS-PAGE. The enzyme was almost specific to methylglyoxal (K®=0.91 mM). The activity of the en- zyme was not inhibited by metal ion chelators such as EDTA, which is a potent inhibitor for gly0xalase Is from other sources.

Recently, we screened yeast which were resistant to the oxidative stress of linoleic acid hydroperoxide and found that Hansenula mrakii IFO 0895 could grow in a medium containing 4 mM linoleic acid hydroperoxide in which any other yeasts tested could not grow (1). H. mrakii IFO 0895 formed glutathione peroxidase inducibly when the cells were cultured in a linoleic acid hydroperoxide-containing medium and the hydroperoxide moiety of the linoleic acid hydroperoxide was reduced to the alcohol moiety (1). This enzyme was then believed to be the first one which attacks linoleic acid hydroperoxide in H. mrakii IFO 0895 to avoid the oxidative stress of linoleic acid hydro- peroxide; however, the compotlnd is degraded non-enzy- matically to yield some aldehydes, ketones and carboxylic acids, and these compounds are also toxic to microbial cells. One of the prevailing aldehydes found in living cells is methylglyoxal (MG), and the compound is able to arrest the growth of various kind of cells (2). A detoxification sys- tem for MG in microbial cells has been studied by us (2), and a budding yeast, Saccharomyces cerevisiae, was found to have different routes for the detoxification of MG, i.e a glyoxalase system and a reduction/oxidation system (2). The former consists of glyoxalase I (EC 4.4.1.5) (3) and glyoxalase II (EC 3.1.2.6) (4), whereas the latter is made up of NADPH-dependent methylglyoxal reductase (5) and NAD-dependent lactaldehyde dehydrogenase (6). The activities for these four enzymes are closely related to the growth of the yeast cells. In the present study, we found that It. mrakii IFO 0895 was hightly tolerant to MG, and we then purified glyoxalase I from the yeast as an aid to investigate the mechanism against oxidative stress in this microbe.

Cells of H. mrakii were cultured in nutrient medium (1% glucose, 0.5o//00 peptone, 0.2o//00 yeast extract, 0.03% KEHPO4, 0.03O//oo KH2PO4, 0.01% MgCl2; pH 5.5) with reciprocal shaking (140 rpm, 5 cm stroke) at 30°C until the stationary phase. A portion of the culture was transferred to a test tube containing 4 ml of SD minimal medium (2%o glucose, 0.67O/oo yeast nitrogen base; pH 5.5) and MG, and the cells were cultured at 30°C with reciprocal shaking. The cell growth was monitored by measuring the turbidity of the culture at 610nm (OD610). As shown in Fig. 1, H. mrakii was able to grow in a medium containing up to

* Corresponding author.

25 mM MG, whereas the cells of Escherichia coil or Pseudomonas putida cannot grow in medium containing 1.0-1.2 mM MG (7), and the growth of S. cerevisiae is com- pletely arrested in 2.0mM MG-containing medium (8), with the cells finally dying. Therefore, H. mrakii is highly resistant to MG. The activities of MG-metabolizing en- zymes (glyoxalase I, glyoxalase II, methylglyoxal reductase) were examined in cells of 11. mrakii which were cultured in 1-1 SD medium containing 10mM MG or 1 mM linoleic acid hydroperoxidase. Preparation of linoleic acid hydro- peroxide was described previously (1). When the 0D610 reached approximately 1.0-1.2, cells were harvested by cen- trifugation (6,000rpm, 10min) and washed twice with 0.85%o NaC1 solution. Cells were resuspended in 3 ml of 10mM potassium phosphate buffer (pH 7.0) containing 0.5 mM phenylmethylsulfonyl fluoride [KPB/PMSF] and disrupted with a Braun homogenizer at 0°C for 2 rain. Homogenates were centrifuged at 25,000×g for 20min at 4°C, and the resultant supernatants were used as the

131

2 0

I.O

0,1

0.05 [ ] [ ] I I I

20 40 60 80 T i m e {h)

Growth curve of H. mrakii IFO 0895 in a methyiglyoxal- Symbols: ©, 0mM; O, 5 mM; A, 10mM; • ,

S 1::3 O.5 0

~o b,

cD

FIG. 1. containing medium. 25mM; [], 50mM.

132 INOUE ET AL. J. FERMENT, BIOENG.,

sources of enzymes. Assay and definition of the unit for glyoxalase I, glyoxalase II and methylglyoxal reductase were described previously (2-4). Protein was measured by the method of Lowry et al. (9). The specific activity of glyoxalase I in H. mrakii cells grown in SD medium without chemicals was 1.48 uni ts / rag-prote in , whereas in cells grown in SD medium containing 10 mM MG or 1 mM linoleic acid hydroperoxide , it was 1.54 and 2.11 uni ts / rag- protein, respectively. These specific activities of glyoxalase I in the cell extracts of H. mrakii were relatively higher than those of S. cerevisiae (0.238 uni ts /mg-prote in) (10), Aspergillus niger (0.026 uni t s /mg-prote in) (11), P. put ida (0.145 uni ts /mg-prote in) (12) and E. coli (0.012 uni t s /mg- protein) (7). Glyoxalase I was slightly activated in cells of H. mrakii exposed to 1 mM linoleic acid hydroproxide . No difference in the activities of glyoxalase II and methyl- glyoxal reductase in H. mrakii was observed under the cultural condit ions tested (data not shown). In order to compare the propert ies of glyoxalase I in H. mrakii with other glyoxalase Is, we purified it as follows. Unless other- wise stated, all purification procedures were done at 0- 4°C.

Cells (35.53 g as wet wt.) f rom a 5-I culture containing 1 mM linoleic acid hydroperoxide were disrupted by Dyno- Mill for 15 rain, and the homogenates were centrifuged as described above. The resultant supernatant was applied onto a DEAE-cel lulose column (4.2 × 43 cm) equil ibrated with K P B / P M S F . Adsorbed proteins were eluted with a linear gradient of KC1 (0 -1 .0M, 1,500ml) in the same buffer, active fractions (0.9-1.5 mS) were mixed, and solid ammonium sulfate was added to the enzyme solut ion to bring 30% saturat ion. The mixture was then loaded onto a Butyl Toyopear l 650 M column (4.1 × 14cm) equil ibrated with K P B / P M S F containing 300/00 ammonium sulfate and adsorbed proteins were eluted with a decreasing gradient of ammonium sulfate (30-00/00,700 ml) in the same buffer. Active fractions (9.0-8.5 mS) were combined and concen- trated by Amicon PM10 membrane and subjected to a Sephadex G-150 column (1.8 × 96 cm). Af ter gel-filtration, active fractions (Ve/Vo =2.16-2 .46) were pooled, concen- trated as above and charged onto a Blue-Sepharose CL-6B column (1.6 × 5.0 cm) equil ibrated with K P B / P M S F . The column was first washed with 6 × volume of the column with the same buffer, and then 2 × volume of the column with the same buffer containing 2 0 0 m M KC1. The ad- sorbed enzyme was eluted with K P B / P M S F containing 500 mM KC1 and 2 mM glutathione, and then the eluted enzyme was dialyzed against the excess volume of K P B / P M S F and the dialysate was used for the characteri- zation of glyoxalase I.

The puri ty of the enzyme after Blue-Sepharose CL-6B column chromatography was examined by polyacrylamide gel electrophoresis in the presence (Fig. 2A) or absence of

(A)

I 2 I

6 8 K

4 5 K

- 29K

- 1 8 K

D - 14K

(B)

Activity(units) o Io 20 50 i i i I

FIG. 2. Polyacrylamide gel electrophoresis of glyoxalase I of H. mrakii IFO 0895. (A) Polyacrylamide gel electrophoresis in the presence of SDS was performed by the method of Laemmli (17). Lane 1: molecular weight markers [from top to bottom: bovine serum albu- min (Mr 68,000), ovalubumin (Mr 43,000), carbonic anhydrase (Mr 29,000), fl-lactglobulin (M, 18,000) and lysozyme (M, 14,300)]. Lane 2: purified glyoxalase I. (B) Approximately 20ztg of purified glyox- alase I was applied onto duplicate disc gels without SDS and elec- trophoresed at 4°C. One part of the gels was stained for protein with Coomassie brilliant blue, and the other was sliced (slice thickness 5 mm). The gels were transferred into 1.5 ml microfuge tubes contain- ing 0.1 ml of KPB/PMSF, mashed gently and kept at 4°C for 2 h. After centrifugation (15,000rpm, 10rain, 4°C), an aliquot of the supernatant was withdrawn and used for the assay of glyoxalase 1.

sodium dodecyl sulfate (SDS) (Fig. 2B). The enzyme migrated as a single protein band with a molecular weight of 38,0000 (Fig. 2A), a value which coincided well with that obta ined by gel-filtration of Sephadex G-150. Blue- Sepharose CL-6B was adopted for the purification of glyoxalase I f rom sheep liver by Uoti la and Koivusalo (13). As shown in Fig. 2B, the activity of glyoxalase I was re- covered from the section of the gel coinciding with the single band stained with Coomassie bril l iant blue, and the react ion product was identified to be S-lactoylglutathione enzymatically by the use of glyoxalase II (4). Therefore, we concluded that glyoxalase I of H. mrakii was purified. Overall purification procedures and some propert ies of the purified enzyme are summarized in Tables 1 and 2. The purified enzyme was specifically active toward MG (Kin = 0 . 9 1 m M ) ; however, other 2-oxoaldehydes (glyoxal, phenylglyoxal, 4,5-dioxovalerate) or other alkyl aldehydes (acetaldehyde, formaldehyde, glycolaldehyde, propional- dehyde) were all inert as substrates. Activators and inhibi- tots were also investigated. As observed in other microbial glyoxalase Is, the activity of the H. mrakii enzyme also was inhibited by Zn 2+ ( 5 5 ~ at 0.5 mM, 7 7 ~ at 2.0 raM). Al- though ferrous sulfate has been reported to activate S. cerevisiae (3) and A. niger (11) enzymes, it showed the

TABLE 1. Purification of glyoxalase I from H. mrakii 1FO 0895

Step Protein Total activity ~ Specific activity Yield (mg) (units) (units/mg-protein) (%) Fold

Cell extract 3420 6680 1.95 100 1.00 DEAE-cellulose 288 4360 15.1 65.3 7.74 Butyl Toyopearl 650 M 20.3 1490 73.4 22.3 37.6 Sephadex G-150 1.95 1150 589 17.2 302 Blue-Sepharose CL-6B 0.27 965 3570 14.4 1830

Activity was assayed in a mixture (1.0 ml) containing 2.0 mM methylglyoxal, 2.0 mM glutathione, 100 mM potassium phosphate buffer (pH 6.0) and enzyme at 25°C. One unit of enzyme activity was defined as the amount of enzyme forming 1 ~mol of S-lactoylglutathione per rain (2).

VOL 71, 1991 NOTES 133

TABLE 2. Properties of glyoxalase I from H. mrakii IFO 0895

Molecular weight a 38,000 Optimal pH 6.0 Stable pH b 7.5-8.5 Optimal temperature 50°C Stable temperature c 4.0-40°C Substrate specificity (K~) Specific to MG (0.91 mM) Inhibitor Zn 2-, Fe 2-

a Molecular weight was calculated by SDS-PAGE and gel-filtration of Sephadex G-150 column chromatography.

b Relative activity was measured after 24 h incubation at 4°C with 100 mM buffer of various pHs.

c Relative activity was measured after 30 min incubation at various temperatures with 100 mM potassium phosphate buffer (pH 7.0).

MG, Methylglyoxal.

oppos i t e effect on the H. mrak i i enzyme, with inh ib i t ion being 46 .5% at 2 . 0 m M . The activit ies o f g lyoxalase Is purif ied f r o m pig e ry throcytes (14), sheep liver (13), S. cerevisiae (3) and A . niger (11) were inhibi ted by e thy lened iamine te t r aace t i c acid ( E D T A ) . O n the cont ra ry , meta l ion chela tors such as E D T A , 1 ,2-chyclohexane- d iamine te t raace t i c acid and 8 -hydroxyqu ino l ine did not show any effects on H. mrak i i enzyme.

Linole ic acid h y d r o p e r o x i d e is degraded non -enzyma t - ically to yield some a ldehydes such as ma loned i a ldehyde (15), whereas glyoxalase I purif ied f r o m H. mrak i i was a lmos t specific to M G , and o ther a ldehydes tested were all inert as substrates . Glyoxa lase I in H. mraki i was sl ightly ac t iva ted when the cells were cul tured in a l inoleic acid h y d r o p e r o x i d e - c o n t a i n i n g m e d i u m , and the specific activ- ity o f g lyoxalase I in the cell extracts o f H. mraki i was 10- 200-fold h igher c o m p a r e d with those o f o the r m ic roo rgan - isms. The high act ivi ty o f g lyoxalase I in the cells o f H. mraki i would enable the yeast to g row in a M G - c o n t a i n i n g m e d i u m . W e also purif ied methy lg lyoxa l reductase f r o m the cells o f H. mrak i i which were cu l tured under the same cond i t ions to e lucidate the me tabo l i c rou te o f the a ldehyde in the yeast (16). Fu r the r inves t igat ions are now in pro- gress on H. mraki i .

REFERENCES

1. lnoue, Y., Iehiryu, T., Yoshikawa, K., Tran, L-T., Murata, K., and Kimura, A.: Induction of glutathione peroxidase by linoleic acid hydroperoxide in Hansenula mrakii. Agric. Biol. Chem., 54, 3289-3293 (1990).

2. Murata, K., Inoue, Y., Rhee, H.-1., and Kimura, A.: 2-Oxoalde- hyde metabolism in microorganisms. Can. J. Microbiol., 35, 423-431 (1989).

3. Murata, K., Saikusa, T., Watanabe, K., Fukuda, Y., Shimosaka,

M., Inoue, Y., and Kimura, A.: Complete purification of glyox- alase I from Saccharomyces cerevisiae. Agric. Biol. Chem., 50, 2381-2383 (1986).

4. Murata, K., Inoue, Y., Watanabe, K., Fukuda, Y., Saikusa, T., Shimosaka, M., and Kimura, A.: Metabolism of 2-oxoalde- hydes in yeasts: purification and characterization of glyoxalase II from Saccharomyces cerevisiae. Agric. Biol. Chem., 50, 135- 142 (1986).

5. Murata, K., Fukuda, Y., Shimosaka, M., Watanabe, K., Saikusa, T., and Kimura, A.: Metabolism of 2-oxoaldehydes in yeasts: purification and characterization of NADPH-dependent methylglyoxal reducing enzyme from Saccharomyces cerevisiae. Eur. J. Biochem., 151, 631-636 (1985).

6. Inoue, Y., Watanabe, K., Shimosaka, M., Saikusa, T., Fukuda, Y., Murata, K., and Kimura, A.: Metabolism of 2-oxoaldehyde in yeasts: purification and characterization of lactaldehyde dehy- drogenase from Saccharomyces cerevisiae. Eur. J. Biochem., 153, 243-247 (1985).

7. Rhee, H-I., Murata, K., and Kimura, A.: Molecular cloning of the glyoxalase I gene in Pseudomonas putida in Escherichia coli. Biochem. Biophys. Res. Commun., 147, 841-838 (1987).

8. Murata, K., Saikusa, T., lnoue, Y., and Kimura, A.: Molecular cloning of a gene enhancing yeast glyoxalase I activity and its effect on yeast cell size. J. Ferment. Technol., 66, 495-500 (1988).

9. Lowry, O.H. , Rosebrough, N.J . , Farr, A.L. , and Randall, R.J . : Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275 (1951).

10. Kosugi, N., Inoue, Y., Rhee, H-I., Murata, K., and Kimura, K.: Production of S-lactoylglutathione by glycerol-adapted Sac- charomyces cerevisiae and genetically engineered Escherichia coli cells. Appl. Microbiol. Biotechnol., 28, 263-267 (1988).

11. Inoue, Y., Rhee, H-I., Watanabe, K., Murata, K., and Kimura, A.: Metabolism of 2-ketoaldehydes in mold: purification and characterization of glyoxalase I from Aspergillus niger. J. Bio- chem. (Tokyo), 102, 583-589 (1987).

12. Rhee, H-I., Murata, K., and Kimura, A.: Purification and charac- terization of glyoxalase I from Pseudomonas putida. Biochem. Biophys. Res. Commun., 141, 993-999 (1986).

13. Uotila, L. and Koivusalo, M.: Purification and properties of glyoxalase I from sheep liver. Eur. J. Biochem., 52, 493-503 (1975).

14. Aronsson, A-C. and Mannervik, B.: Characterization of glyox- alase I purified from pig erythrocytes by affinity chromatography. Biochem. J., 165, 503-509 (1977).

15. Pryor, W. A. and Stanley, J. P.: A suggested mechanism for the production of malonaldehyde during the autoxidation by poly- unsaturated fatty acids. Nonenzymatic production of prostaglan- din endoperoxides during autoxidation. J. Org. Chem., 40, 3615-3617 (1975).

16. Inoue, Y., Tran, L-T., Yoshikawa, K., Murata, K., and Kimura, A.: Purification and characterization of methylglyoxal reductase from Hansenula mrakii. J. Ferment. Bioeng., 71, 134-136 (1991).

17. Laemmli, U. K.: Cleavage of structural proteins during the assem- bly of the head of bacteriophage T4. Nature, 227, 680-685 (1970).