The yeast Pichia pastoris IFP 206 was isolated

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1 h Agric. Biol. Chern., 47 (ll), , Oxidation of Methanol by the Yeast Pichia pastoris. Purification and Properties of the Formate Dehydrogenase1 J. J. Allais, A. Louktibi and J. Baratti* Universite de Provence, Centre National de la Recherche Scientifique, Laboratoire de Chimie Bacterienne, BP 71, Marseille Cedex 9, France Received April 18, 1983 geneity. The formate The protein dehydrogenase showed a from molecular the yeast weight Pichia of pastoris 68,000 daltons IFP 206 and was was purified composed to homo- of two identical subunits. Its amino acid composition was similar to those of other formate dehydrogenases and was characterized by a high content of acidic residues. The N-terminal end of the molecule was probably blocked. The enzyme activity was NAD+ dependent (NADP+ could not replace NAD+). Its optimum temperature was 47 C and the activation energy 10.8 kcal/mol. The enzyme was active from ph 3.5 to 10.5 with a maximum at ph 7.5. The Michaelis constant for NAD+ and formate were respectively 0.27 and 15 mm. The purified enzyme had no S-formylgmtathione hydrolase activity, strongly suggesting that the true substrate was formate. NADH, cyanide and azide were strong inhibitors of the enzyme. The yeast Pichia pastoris IFP 206 was isolated at the Institut Francais du Petrole and used for the production of proteins from methanol.1) Like all the yeasts so far studied,2) the oxidation of methanol is catalyzed by an alcohol oxidase (EC ), a formaldehyde dehydrogenase (EC ), and a formate dehydrogenase3) (EC ). The alcohol oxidase3} and the formaldehyde dehydrogenase4) of P. pastoris were purified in our laboratory and their properties were studied and compared to enzymes from other yeasts. It was shown that the true substrate of the formaldehyde dehydrogenase was S-hydroxymethyl-glutathione and the reaction product was S-formylglutathione. Usually this product is hydrolyzed to glutathione and formate which is the substrate of formate dehydrogenase. A S-formylglutathione hydrolase has been reported in some yeasts5'6'19) while this activity was not separated from the formate dehydrogenase in Hansenula polymorpha1) and Pichia sp.9) The enzyme formate dehydrogenase has been purified and characterized in several methylotrophic yeasts: H. polymorpha,7) Candida boidinii5\ Pichia pastoris NRRL Y-75568) and Kloeckera sp.10) The enzymes from bacteria11'12* and plants13'14'15* ave also been studied. The present work reports the purification of the formate dehydrogenase of. the yeast P. pastoris IFP 206 and describes the structural and catalytic properties of the enzyme. MATERIALS AND METHODS Microorganism. P. pastoris IFP 206 was grown in a methanol-limited chemostat at 34 C, ph 3.5, dilution rate O.nhr"1 at the Institut Francais du du Petrole by Dr. allerini.^ Cells were harvested by filtration, lyophilized, and stored at - 15 C until use. Enzyme purification. The formate dehydrogenase was f This work was supported by a grant from the Delegation Generate a la Recherche Scientifique et Technique N * To whomall correspondence should be addressed.

2 2548 J.J. Allais, A. Louktibi and J. Baratti purified to homogeneity according to published procedures with only minor modifications. In a typical experiment loog of lyophilized cells were suspended in 4liters of a 5 mmpotassium phosphate buffer (KPB) ph 7.5 containing 30 mmp- mercaptoethanol and 0.5% (v/v) glycerol, and allowed to stand overnight at 4. The cell suspension was then disrupted with a Braun homogenizer in the following conditions: 40ml of the cell suspension were mixed with 30g of glass beads and homogenized for 10sec at speed 1 and 30sec at speed 2. Cell debris was eliminated by a 20min centrifugation at 5,500rpm (Sorvall RC 2B, GSA rotor). The clear supernatant was adjusted to a final concentration of 1% streptomycin sulfate and the pellet was eliminated. The dialyzed supernatant was then chromatographed on a DEAE Sephadex (A-50) column (6.3 x 52cm) equilibrated in 5 mm KPB ph 7.5 containing 30 mm^-mercaptoethanol and 0.5% glycerol. The flow rate was 6.5ml/ hr-cm2. The absorbed proteins were eluted with a linear gradient of sodium chloride (2x4.5liters) from 0 to 0.4m in the equilibration buffer. The fractions with formate dehydrogenase activity emerged at a sodium chloride concentration of 0.18m. They were pooled, concentrated by ultra filtration, and dialyzed. The formate dehydrogenase pool was then filtrated through a Sephadex G100 column (4x 105cm) equilibrated in a 5mM KPB ph 7.5 containing 30mM ^-mercaptoethanol and 0.5% glycerol. The flow rate was 5.9 ml/hr-cm2. The active fractions were pooled, concentrated by ultra filtration, and stored at +4 C until use. Enzyme activity. Formate dehydrogenase activity was assayed spectrophotometrically using the method of van Dijken7) modified by Couderc.3) The incubation mixture contained 100mM KPB, ph 7.5, with 0.4mM NAD+ and 62mM formate. Incubation was done at 37 C and the appearance of NADH was followed at 340nm as a function of time. A molar extinction coefficient of 6.22 x 106cm2/mol was taken for NADH. One enzyme unit was the amount of enzyme which reduced 1 mmol of NAD+ per min. Usually three assays with different enzyme concentrations were run simultaneously and linear regression was used for the calculation of enzyme activity. Analytical methods. Proteins were determined by the method of Lowry at al.16) using bovine serum albumin as thestandard. The concentration of pure enzyme preparations were determined spectrophotometrically by their absorption at 280nm using an extinction coefficient of A{ { m=l0.0.5) Amino acid composition, polyacrylamide gel electrophoresis, NH2-terminal amino acid, and ultracentrifugation experiments were done as previously described.3) High performance liquid chromatography (HPLC) was done using a Spherogel-TSK-G 3000SW column (7.5 x 600mm, Beckman). The enzyme solution (5 to 25/4 containing 1 mg/ml of protein) was chromatographed in a 50mM sodium acetate, 0.3m NaCl buffer at ph 7.0 at a flow rate of 3 ml/min. Detection was done spectrophotometrically at 280nm. For molecular weight determination, calibration was done with standard proteins. Relative elution time was calculated by reference to blue dextran (Pharmacia). nzyme purification RESULTS A typical result of a formate dehydrogenase purification is given in Table I. Starting from loog of cells (dry cell weight) 240mg of purified enzyme could be prepared with a purification factor of The following steps of the procedure were in commonwith the purification of formaldehyde dehydrogenase from the yeast4): preparation of cell free extract, streptomycin sulfate precipitation, DEAE Sephadex chromatography, and Sephadex- G100 filtration. The preparation was found homogeneous by polycrylamide gel electrophoresis, analytical ultracentrifugation, and HPLC as shown in Fig. 1. olecular weight and subunit structure able I. Purification of the Formate Dehydrogenase from P. pastoris IFP 206 The coefficient of sedimentation of the protein was determined by ultracentrifugation at

3 p Purification and Properties of Yeast Formate Dehydrogenase 2549 \ e o <i y according to their molecular weights and a linear calibration curve was obtained by plotting the log of molecular weight versus the relative elution time. Using this curve a molecular weight of 70,000 was estimated. This value was in good agreement with the other methods used and showed that HPLC was a useful method for the fast determination of molecular weight. \ The presence of subunits in formate dehydrogenase was shown by polyacrylamide gel electrophoresis in the presence of SDS. A single band was obtained. By comparison ELUTl'ON TJME(min) with a calibration curve made with standard roteins, a molecular weight of 34,000 was estimated for the subunits. Fig. 1. High Performance Liquid Chromatography of Purified Enzyme amount was 20/^g in 15^1 of buffer. For other These results suggest that the formate dehydrogenase molecule is formed of two conditions see Materials and Methods. probably identical subunits. \ T95 \ S-3 \ LU ^ \ CJ \ LLJ \ ct -125.\ RELATJVE RETENTION TIME Fig. 2. Determination of Molecular Weight by Size Exclusion HPLC. Standard proteins were: cytochrome c (12,500); chymo- The molecular weight of the native enzyme was also determined using HPLC. As shown in Fig. 2, the TSK column fractionated proteins tructural properties The amino acid composition of the P. pastoris formate dehydrogenase is given in Table II and compared with that ofa methylotrophic bacteria Bacterium sp12) and that of seeds of the pea, Pisum sativum.13) The amino acid content was not very different for the three enzymes, which were characterized by a high content of acidic residues. The P. pastoris enzyme showed a lower content in methionine, histidine, and cysteine. The minimum molecular weight of the subunits estimated by the method of Delaage17) was 39,000 for the P. pastoris enzyme. This value is in good agreement with the results of SDS-gel electrotrypsinogen (25,000); ovalbumin (45,000); serum albumin (68,000). The arrow indicates the relative elution time of formate dehydrogenase. phoresis. No NH2-terminal amino acid could be detected by the dansylation method, leading to an enzyme concentration of 5.4mg/ml. A valueof 2o,w=4.8 was calculated from the the conclusion that the NH2-terminal is sedimentation pattern which was typical of a probably blocked. A similar conclusion was homogeneous preparation. The molecular drawn for the P. pastoris formaldehyde weight of the native protein was estimated by dehydrogenase.4) the equilibrium technique of Yphantis using a The absorption spectrum of the enzyme partial specific volume of 0.749g/cm3.5) A from 200 to 700nm showed one single peak at alue of 60,000± 1500 was calculated. 280 nm. Effect of temperature In our conditions of assay, the activity of

4 2550 J. J. Allais, A. Louktibi and J. Baratti Table II. Amino Acid Composition of Formate Dehydrogenases ND, not determined ^-x,oo ^_. m n[ *1 \ ^-n ^l loot. k., c",oo f/hy ^;0-25 V 80. V\ >:40- IIf \ TEMPERATURE C 103/T K TEMPERATURE C Fig. 3. Effect of Temperature on the Activity of Formate Dehydrogenases. A, activity temperature profiles; B, Arrhenius plot of data presented in A; C, enzyme stability. The enzyme solution was incubated 10 min at the desired temperature and the remaining activity was assayed at 37 C under standard conditions. #-à". P. pastoris IFP 206 (this work); å -å, Kloeckera sp.10); *-*, C. boidini.5) formate dehydrogenase was maximumat 47 C as shown in Fig. 3A. The profile is also given for comparison for the formate dehydrogenases of Kloeckra sp.10) and C. boidinii.5) Both enzymes showed higher optimal temperatures of 50 and 55 C respectively. An activation energy of 10.8 kcal/mol was calculated from the Arrhenius plot shown in Fig. 3B. From the data presented in Fig. 3A values of 8.7 and ll.l kcal/mol were estimated for the formate dehydrogenase of Kloeckera sp.10) and C. boidinii.5) The thermal stability of the enzyme is shown in Fig. 3C. Fifty per cent of activity was lost after treatment for lomin at

5 7 0 3 / Purification and Properties of Yeast Formate Dehydrogenase 2551 /^2^L å 10 0 I / 1\ >. // o -40 // 20/ / \ å Fig. 4. Effect of ph on the Activity of Formate Dehydrogenase. Activity curve ( $-#): activity was assayed in 100him KPB adjusted to the desired value. Stability curve (B- ): enzyme samples were incubated 15 min at 37 C in 150mM KPB at different ph values and the remaining activity was assayed under standard conditions. 54 C. Similar values were observed for the Kloeckera (55 C) and C. boidinii (58 C) enzymes. ffect of ph From the ph activity curve shown in Fig. 4, an optimum ph of7.5 was observed for the P. pastoris formate dehydrogenase. The enzyme was active in the range of ph 3.5 to A 50% loss in activity was observed at phs 5.3 and The stability curve is also shown in Fig. 4. The formate dehydrogenase activity was stable from phs 4 to 10 and 50% stability was observed at ph 5.2 and 9.9. These values are very close to the one determined for the activity profile and showed that the activity profile was mainly governed by stability rather than changes in ionization of active site groups. Substrate kinetics Formate dehydrogenase showed Michaelis- Menten kinetics for both substrates, NAD+ and formate, as shown in Fig. 5. The concentration of formate and NAD+ were 62 and.4mm respectively. Apparent Km of 15niM and 0.27mM were calculated for formate and NAD+, respectively. The catalytic constant was 1.2sec"1. No activity was detected when PH : /^ '/r /NAD (mm~1) 10/F0RMATE Fig. 5. Effect of Substrate Concentration on the Activity Activity was assayed under standard conditions except that the formate (O-O) or NAD+ (å -å ) concentrations were varied. E <t ^- -; LU à" ^S^ \ 1-04å X! ^ å / : CD <t 0 /. ' TIME (mm) Fig. 6. Effect of Formate Dehydrogenase on S- F ormylglutathi one. S-Formylglutathione was formed from reduced glutathione and formaldehyde in the presence of formaldehyde dehydrogenase4) and its appearance followed at 240nm. The arrow indicates the addition of 10 /A cell-free extract ( ) or 0.04 units of purified formate dehydrogenase (--). him NADP+ was used instead of NAD+. Figure 6 shows the result of an experiment demonstrating that formate rather than S- formylglutathione was the substrate of formate dehydrogenase. ^-formylglutathione was formed as previously described4) and its appearance followed at 240nm. Addition of purified formate dehydrogenase (0.04 unit in 3ml) did not show any effect on the absorbance at 240nm. In the same conditions van Dijken et ai1] observed a fast decrease in the 5-formylglutathione concentration. More re-

6 2552 J. J. Allais, A. Louktibi and J. Baratti Table III. Inhibitors of Enzyme Activity Activity was measured under standard conditions in the presence of the inhibitor at the concentration of 1 mm except for HCO3~ for which the concentration was 0.32m. cently Patel et al.9) reported a similar observation for Pichia sp. with a partially purified enzyme preparation. In sharp contrast the addition of 10/A of P. pastoris cell-free extract was followed by a fast decrease in the absorbance at 240nm, indicating the hydrolyis of S-formylglutathione. These results suggest the presence in cell-free extract of P. pastoris of a S-formylglutathione hydrolase activity different from the formate dehydrogenase activity. Effect of inhibitors on enzyme activity Table III shows the effect of various inhibitors on enzyme activity. Mercury and copper ions were strong inhibitors of formate dehydrogenase, probably by oxidation of essential SH groups. This hypothesis was confirmed by the inhibition by /7-chloromercuribenzoate (PCMB). Other cations had no effect on enzyme activity. Among anions, nitrite and nitrate showed little inhibition while cyanide and azide were strong inhibitors. Among the reaction products, NADH but not bicarbonate was inhibitory. DISCUSSION The enzyme formate dehydrogenase of Pichia pastoris was purified to homogeneity and someof its structural and enzymatic properties were determined and compared with able IV. Comparison of Structural and Catalytic Properties of Formate Dehydrogenases Uotila et alla) Temperature resulting in 50% loss of activity. Estimated from the published data.

7 Purification and Properties of Yeast Formate Dehydrogenase 2553 G SH I I III IV J V / \ kr '+ ^ + sn ^+ ^ + \ / NAD NADH +H NAD NADH +H M FAD+ FADH2 F S-HMG S-FG Ff H2 2 H2O+1/2O2 I : alcohol oxidase M = methanol II: catalase F = formaldehyde III : formaldehyde dehydrogenase S-HMG = 5-hydroxymethylglutathione IV: S-FG Hydrolase S-FG = S-formylglutathione V: formate dehydrogenase Fr = formate G-SH = reduced glutathione Fig. 7. Oxidation of Methanol by the Yeast P. pastoris. the enzymes from two yeasts, C. boidinii5) and Kloeckera sp.,9) one bacterium,12* and pea seeds13) (Table IV). All enzymes have molecular weights in the range 70,000 to 80,000 and are composed of two identical subunits. The optimum temperature, thermal stability, activation energy, and optimum ph are very similar. All of the enzymes are colorless and contain no prosthetic group. The structure of these formate dehydrogenases NAD+dependent is simpler than the corresponding high molecular weight chromophoric iron-sulfur enzymes (EC ) widely found in bacteria.18) The Michaelis constants for NAD+ and formate are remarkably constant for all formate dehydrogenases (EC ) which are characterized by low affinity for formate. This affinity is one hundred times lower than that found for the usual bacterial enzyme: Formate: NAD+ oxydoreductase (EC ). In conclusion, the formate dehydrogenases, the properties of which are summarized in able IV, form a homogeneous group of enzymes with very similar or identical structural and enzymatic properties. These properties have been conserved during evolution of such different organisms as bacteria, yeasts, and plants. From the results presented here and in our preceding work on alcohol oxidase3) and formaldehyde dehydrogenase4) a complete scheme for the oxidation of methanol is given in Fig. 7. Methanol is first oxidized to formaldehyde by an alcohol oxidase containing FAD as a prosthetic group. The electrons are transferred to oxygen and hydrogen peroxide is formed during the reaction. Consequently no energy is available to the cell from this oxidation step. Formaldehyde spontaneously forms hydroxymethyl-glutathione (S-HMG) with reduced glutathione and S-HMG is oxidized to S-formylglutathione (S-FG) by a NAD+-dependent formaldehyde dehydrogenase. One mole ofnadh+h+ is formed in the reaction. S-FG is then hydrolyzed to glutathione and formate by a specific enzyme. This situation seems general and has been demonstrated in the yeasts C. boidinii6) and Kloeckera sp.19) However, the S-FG hydrolase and formate dehydrogenase activity were found associate in H. polymorpha1) and Pichia sp.9) Formate is further oxidized to CO2 by a formate dehydrogenase, and 1 mol of NADH+H+ is formed during the reaction. Then the overall yield of the complete oxidation of 1mol of methanol is 2mol ofnadh+h+.

8 2554 J. J. Allais, A. Louktibi and J. Baratti The scheme of methanol oxidation in P. pastoris IFP 206 presented in Fig. 7 appeared to be general for all yeasts so far studied. The only difference between the strains was the occurrence of a S-formylglutathione hydrolase activity in C. boidinii,6) Kloeckera sp.19) and P. pastoris (this work). The enzyme was purified to homogeneity649) and found completely independent of formate dehydrogenase. Consequently it is clear that, in these yeasts, formate is the true substrate of formate dehydrogenase. In H. polymorpha1) and Pichia sp.9) the hydrolase and dehydrogenase activities were found associated. However, at least in one case,9) the enzyme was only partially purified and both hydrolase and dehydrogenase could have been present. Further investigations will be necessary to fully confirm that formate dehydrogenase of H. polymorpha and Pichia sp. also has a S-formylglutathione hydrolase activity. REFERENCES 1) D. Ballerini, Revue de Vlnstitut Francais du Petrole, 33, 111 (1978). 2) H. Sahm, Adv. Biochem. Eng., 6, 77 (1977). 3) R. Couderc and J. Baratti, Agric. Biol. Chern., 44, 2279 (1980). 4) J. J. Allais, A. Louktibi and J. Baratti, Agric. Biol., Chem. 47, 1509 (1983). 5) H. Schutte, J. Flossdorf, H. Sahm and M. R. Kula, Eur. J. Biochem., 62, 151 (1976). 6) I. Neben, H. Sahm and M. R. Kula, Biochim. Biophys. Acta, 614, 81 (1980). 7) J. P. van Dijken, G. J. Oostra-Demkes, R. Otto and W. Harder, Arch. MicrobioL, 111, 77 (1976). 8) C. T. Hou, R. N. Patel,A. I. LaskinandN. Barnabe, Arch. Biochem. Biophys., 216, 296 (1982). 9) R. N. Patel, C. T. Hou and P. Derelanko, Arch. Biochem. Biophys., 221, 135 (1983). 10) N. Kato, M. Kano, Y. Tani and K. Ogata, Agric.., Biol. 38, 111 Chem (1974). ll) P. A. Johnson andj. R. Quayle, Biochem. J., 93, 281 (1964). 12) A. M. Egorov, T. Avilova, M. Dikov, V. O. Popov, Y. V. Rodionov and I. V. Berezin, Eur. J. Biochem., 99, 569 (1979). 13) T. Ohyama and I. Yamazaki, J. Biochem., 75, 1257 (1974). 14) L. Uotila and M. Koivusalo, Arch. Biochem. Biophys., 196, 33 (1979). 15) D. Peacock and D. Boulter, Biochem. J., 120, 763 (1970). 16) O. H. Lowry, N. J. Rosebrough,A. L. FarrandR. J.. Randall, Chem., 193, /. Biol 265 (1951). 17) M. Delaage, Biochim. Biophys. Acta, 168, 573 (1968). 18) U. Muller, P. Willnow, U. Ruschig and T. Hopner, Biochem., Eur. J. 83, 485 (1978). 19) N. Kato, C. Sakazawa, T. Nishizawa, Y. Tani and H. Yamada, Biochim. Biophys. Acta, 611, 323 (1980).

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