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UNIVERSITY OF ILLINOIS

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THIS IS TO CERTIFY THAT THE THESIS PREPARED UNDER M Y SUPERVISION BY

• ...... ANNA GINTA ZVILIUS.. *

ENTITLED.A SPECTROPHOTOMETRIC ASSAY FOR THE......

METHYLENE-H.MPT OXIDOKEDUCTASE

IS APPROVED BY ME AS FULFILLING THIS PART OF THE REQUIREMENTS FOR THE

DEGREE rw BACHELOR OF SCIENCE IN MICROBIOLOGY

InttnictorJft Charge

Amovn>:

HEAD OF DEPARTMENT OF^iSS^KSSBSL • HMMMIHBMMMBBMMI

aiM4 A SPECTROPHOTOMETRIC ASSAY

FOR fHE

m e t h y l e n e -h 4m p t OXIDOREDUCTASE

ANNA GINTA ZVILIUS

THESIS

for the

DEGREE OF BACHELOR OF SCIENCE

MICROBIOLOGY

College of Liberal Arte and Sciences

University of Illinois

Urbane, Illinois 1985 ACKNOWLEDGMENT

I wish to express my gratitude to a number of people who made this work possible. First, X would like to thank Trish Hartsell for her guidance and support. They were much appreciated.

X am also indebted to Dr. Mark X. Donnelly for his patient advice. X benefitted greatly from his calm and thorough approach to scientific research. Pierre Rouviere and Ken Noll are to be thanked for their advice, friendship, and for making me laugh.

Giovanni Vallini, Jeff Hoyt, Mike Rataj, Victor Gabriel

Tony Dimarco, and Bill Sheridan all provided invaluable friendship.

I also thank my mother, Julia Zvilius, for her endless patience, above and beyond the call of duty, and for the excellent job of typing this thesis.

Finally, X am very grateful to Prof. Ralph S. Wolfe for his guidance and support and for giving me the opportu- nity to work in his laboratory. TABLE OF CONTENTS

page I. INTRODUCTION...... 1

II. MATERIALS AND METHODS ......

A. Organisms and Growth Conditions...... 13 B. Preparation of Cell Extracts and Coenzymes...... C. Ammonium Sulfate Fractionation ...... 14 D. Fractionation with Fart Protein Liquid Chromatography...... 14 E. Assay for the Oxidoreductase...... 15 F. Digestion of BCE with Protease ...... 16 G. Analytical Methods ...... 16 H. Chemicals U s e d ......

III. R E S U L T S ......

A. Ammonium Sulfate Fractionation ...... 17 B. Determination of the Optimal HCHO Concentration for Ammonium Sulfate 70% Supernatant...... C. Determination of Optimal Temperature . . 17 D. Determination of Optimal Buffer Concentration...... E. Determination of Optimal pH ...... 17 F. Digestion of BCE with Protease ...... 27 G. Effect of F420 ...... 27 H. Effect of Other Electron Acceptors . . . 32

IV. CONCLUSION......

V. SUMMARY ......

VI. LITERATURE CITED...... V

ABBREVIATIONS USED

A.S. ammonium sulfate

BCE boiled cell extract

°C centigrade

COM coenzyme M

DTT dithiothreitol

F420 8-hydroxy-5-deazariboflavin FAD flavin adenine dinucleotide

FMN flavin mononucleotide

FPLC fast protein liquid chromatography

HCHO formaldehyde h 4m p t t e t rahy drome th anop t e r i n

HSEtOH 2-mercaptoethanol

KOAc potassium acetate kPA kilopascal

KPi potassium phosphate

MFR methanofuran

Mg(OAc) 2 magnesium acetate

NAD nicotinamide adenine dinucleotide

NADP nicotinamide adenine dinucleotide phosphate

UV ultraviolet 1

I. INTRODUCTION

Oxidoreductase* are ensymes which catalyse reactions involving the interaction of two compounds, resulting in the oxidation of one compound and the reduction of the other* Such an ensyme has been documented as being part of the pathway of COj reduction to CH^ and involves three novel cofactorsi methanofuran (MFR), tetrahydiomethanopterin

(H^MPT), a folate analogue (Figure 2), and coensyme M (COM)•

The methylene-H^MPT oxidoreductase catalyses the interconversion of methylane-H4MPT and m*th«nyl-H4MPT+. An assay

for the reverse reaction of this ensyme has been developed

(5)• Formaldehyde combines chemically with H^MPT to form methylene-H^MPT (Figure 3). Methylene-H^MPT is then ensymatically converted to methenyl-H^MPT* (Figure 3), with the resulting loss of two electronse Since the end product, raethenyl-H^MPT*, absorbs a 335 nm (Figure 4), the progress of the reaction can be monitored spectrophoto- metrically.

Before any extensive purification of the ensyme could be carried out, it would be necessary to find an electron carrier for the two reducing equivalents generated by the oxidation of methylene~HjMPT» Oxidoreductase activity can be found in crude extracts, but as the ensyme is purified, any electron carrier associated with the ensyme might be lost. At a result, ensyme fractions of increasing purity a

Figure 1. The Biochemical Pathway of Methane Production from C02. Taken from Jones, Donnelly, and Wolfe (10). 3

CH 4 C O a

HS-CoM (HC-)MFR CH3-S-C0M H4MPT

H o O H4MPT MFR {H&-)H4MPT HS-CoM (HjC-) H4MPT 2e

2C-)H4MPT Vs-2e

■ - ** Figure 2. The Structure of H^MPT I I i HD- l \

*HD 0 = C OHOHOHO H _ *HO 2h oI - d I - o I - o I - d I ny |4ri ■I H*OD H H 6

Figure 3. Showing the interconversion of H,MPT and

Two of Its Forms, Methylene-H4MPT and Methenyl-H.MPT o HN-R HCHO — . — ...... c h 3 H^^NrNl^CHa H,MPT methylene - H^MPT methenyl - K,M PT 8

Figure 4. uv-visible Spectra of HjMPT (••••), Methylene-

H4MPT ([H2C-]H4MPT) (---- and ----- ), and Methenyl-H4MPT

([HC-]H4MPT ( ■ — ). The reaction mixture containedi

H4MPT, 50 nmol} HCHO, 6.5 ymol» methylene-H4MPT oxidore- ductaae [from 70% (NH4)2S04 cut], 6 pg of protein.

From Escalante-Semarana, et al. (7).

would show decreasing activity without an externally added carrier. The carrier associated with the enzyme in vivo is still unknown. One p< mibility for an electron acceptor is factor F ^ q (Figure 5), a cofactor which was first reported by cheeseman, et al., in 1972 (2). F42q can be reduced at the one and five positions of the

5-deazaisoalloxazine ring (3).

The goal of this thesis is to develop an assay system

for the methylene-H^MPT oxidoreductase• This includes

finding a compound that can function as an electron carrier in vitro for the methylene-H^MPT oxidoreductase. 11

Figure 5. Structure of f 42q CH3 O COO0 o 0 c o d 0 CHg-CH-CH-CH-CHg-O-P-O CH-C-NH-CH-CH2~CH2-C-NH-CH OH OH OH CH2

C H a ' Q c o o 13

II. MATERIALS AMD METHODS

A* Organisms and Growth Conditions

Methanobacterium thermoautotrophicum strain AH

was cultured in a 200-liter fermentor (New Brunswick

Scientific Co., Inc., New Brunswick, N.J.); culture condi-

tions and storage of whole cells have been described (13).

B. Preparation of Cell Extracts and Coenzymes

Rapidly thawed cell slurry at pH 7.1 was passed

through a French pressure cell at 1.4 x 100,000 kPA.

The broken-cell suspension was collected under a stream

of N2 in stainless-steel centrifuge tubes kept on ice.

Each tube was sealed and centrifuged at 31,000 x g and

5 °c for 40 min. The tubes were transferred into an anaerobic chamber that contained an atmosphere of 971 N2 -

31 H2r the supernatant was dispensed into serum vials which were then sealed with butyl rubber stoppers, and the contents were pressurised with N2 prior to storage at 4 °c. BCE was prepared from cell extract of Methano- bacterium thermoautotrophlcum as previously described (8).

Highly purified F420 and H^MPT were prepared as previously described (4,6). 14

C. Ammonium Sulfate Fractionation

A saturated solution of ammonium sulfate (pH 6.8)

was slowly added to the cell extract supernatant to give

a final ammonium sulfate concentration of 70%. The solu-

tion was left stirring gently in an anaerobic chamber

at 4 °C for 12 hours. The solution was then centrifuged

at 35,000 x g and 5 °C for 25 min. in sealed stainless-

steel tubes which were then returned to the chamber. The

supernatant was decanted and respun at 35,000 x g and

5 °C for 25 min. The pellets were resuspended in 20 mM

KPi, pH 6.8, 10 mM HSEtOH, and pooled. The supernatant

and resuspended pellets were then transferred to separate serum vials, sealed with butyl-rubber stoppers, pressurised and stored at 4 °c. A portion of the supernatant was dialysed against buffer containing 20 mM KPi at pH 6.8, with 10 mM HSEtOH, inside an anaerobic chamber at 4 °C for 12 hours.

D. Fractionation with Fast Protein Liquid Chromatography

$h* FPLC oxidoreductase fraction was a gift from

Dr. M. I. Donnelly and was prepared as followa* A sample

*f A%i% m supernatant left first dialysed against buffer 20 an mx «% m 7, 10 mM notion, m olycwol, * HI N|t0ifO)|\ * portion *t thi. proto in wa. loot** m * Nono-0 aniO»4« OHOfctHf eolawi (l ail 0*0 to ohm lutlit itM ter 15

A 38-ml linear gradient from 0 to 2 M KOAc was used to

develop the column, with detection at 280 nm. Rechromato-

graphy using a 10-min. gradient from 0.8 M to 1.2 M KOAc was performed on a fraction containing oxidoreducta&o

activity. A protein peak that eluted from this second

separation and that showed oxidoreductase activity was

collected and subsequently used as a source of the enzyme.

E. Assay for the Oxidoreductase

The standard reaction mixture (0.75 ml) contained*

120 mM KPi, 350 nmol of HCHO/yg protein, 174 nmol of F42q ,

1 mM DTT, H^MPT, and enzyme.

Anoxic solutions of KPi buffer, H20, and F420 were prepared by idding DTT to a concentration of 1 mM, and then sp fing with N2« inside an anaerobic chamber, aliquots of these stow* solutions (150 mM KPi at pH 6.03, 100 mM

HCHO, 1.4 mM H2MFT, and 5 mg F42Q/ 1) and H20 were added to quartz cuvettes. The cuvettes were stoppered with grey butyl-rubber stoppers, removed from the chamber, and placed

in a 51 °c water-bath. After a cuvette had been heated

for 10 min., the initial absorbance at 335 nm was measured.

The reaction was initiated by injecting an enzyme sample through the stopper with a Hamilton syringe. The cuvette was then replaced in the spectrophotometer and the reaction monitored by following the change in absorbance at 335 nm.

The extinction coefficient of H4MPT-(HCO) at 335 nm, 16

21,6 mM~* cm"* was used to calculate reaction rates,

P. Digestion of BCE with Protease

BCE was digested with protease by adding 1 mg of

Pronase and 1 mg of Papain to 1 ml of BCE. The solution was allowed to stand for 6 hours in an anaerobic chamber at 4 °C, and then assayed for oxidoreductase activity, along with untreated BCE.

G. Analytical Methods

Protein in extracts of M. thermoautotrophicum, and oxidoreductase preparation was estimated by the method of Lowry (11), or the Bradford microassay (1). Bovine serum albumin was used as a standard in both cases. Form- aldehyde was quantitated by the procedure of Nash (12).

UV-visible spectroscopy was performed on a Perkin-Elroer

Lambda 3 spectrophotometer equipped with a Perkin-Elmer

3600 data station. FPLC was carried out on a Pharmacia

FPLC system*

N. Chemicals Used

NAD, NADP, FAD, FMN, Pronase, and Papain were obtained from Sigma Chemical Co* 17

III. RESULTS

A. Ammonium Sulfate Fractionation

Cell extract was treated with a 70% solution of ammo-

nium sulfate. Most of the activity was located in the super-

natant, with a purification factor of 4.85 as shown in Table. 1.

The pellet still contained 24% of the original total activity.

B. Determination of the Optimal HCHO Concentration for Ammonium Sulfate 70% Supernatant

Formaldehyde was added to individual assays in amounts

of from 0.5 to 5.0 ymol. The optimal concentration was

found to be 350 mmol HCHO per gram of protein (Figure 6).

Greater concentrations were found to be inhibitory.

C. Determination of the Optimal Temperature

Assays were incubated at temperatures from 25 °c to

10 °c. fhe optimal temperature was found to be 61 °c

(Figure 7).

D. Determination of Optimal Buffer Concentration

Assays were performed in varying concentrations of

KPi. The optimum concentration was found to be 120 mM KPi (Figure 8).

E. Determination of Optimal pH

Assays were run from pH 5.5 to 7.0. The optimum pH was found to be 5.9 (Figure 9). Table 1. Puri fication of the Qmidoreductase

Specific Total Activity Purifi" Total cation Volume Activity Protein (nmol min-1) yield Treatment (ml) (mol min-*) (mg) mg-1) (%) Factor3

1 • Initial Axtract 57.8 174,200 2774 63.5 100 1.00 2. 70% Ammonium sulfate Fractionation pellet 42.8 41,900 1498 28.0 24 0.44

supernatant 149.0 155^900 507 307.8 88 4.85

The assay mixture contained: 4 |M»1 Hcao, 75 jil BCE, 47 mM KPi at pH 6.1, 1 mM DTT.

^Obtained by dividing the specific activity at any point in the purification by the specific activity of the starting material.

00 19

Figure 6. Optimal HCHO Concentration

Reaction mixtures (1.0 ml) contained: 30 yl H^MPT, 8.5 yg protein, 37.5 mM KPi, 1 mM DTT, and 0.5, 1.0, 2.0, 3*0, 4.0, or 5.0 ymol HCHO. umoi H,MPT(=CH~) FORMED min1 mg1

i 1

a f e k iS ^ . i, . t f * —ai. bin-oj*,. .» *-■ *• ‘'i^iCyryvlii,^ ,.>ira!ftft&.-aw> ■ ♦a -ij -w v .1 t - 1 21

Figure 7. Optimal Temperature for Production of Methenyl-H^MPT

Reaction mixtures (1.0 ml) contained! 2 nmol HCHO, 50 yl BCE (0.23 yg/yl protein), 37.5 mM KPi at pH 6.8, and 1 mM DTT. All components except HCHO were added to the cuvettes in an anaerobic chamber. The stoppered cuvettes were removed from the chamber and heated for 10 min. at the given temperature. The initial absorbance at 335 nm was determined, HCHO was injected to start the reaction, and the change in absorbance at 335 nm was recorded.

(»■■— e) end (o— o) represent two different, complete sets of assays.

t . ,

Figure 8. Optimal Buffer Concentration for Production of Methenyl-H.MPT

Reaction mixtures (1.0 ml) containedt 2 nmol HCHO, 8 pi H4MPT, 15 pg protein, 1 mM DTT, and 0, 40, 80, 120, or 160 mM KPi at pH 6.03. 24

(-HO=)lc**fH lom u 25

Figure 9. Optimal pH for Production of Methenyl-H^MPT

Each reaction mixture (1.0 ml) contained! 2 ymol HCHO, 7 Ml H4MPT, and 5.4 Mg protein (from 70% A.S, supernatant), in 120 mM KPi, 1 mM DTT. The temperature was 61 °C.

Buffer (150 mM KPi) was prepared by adjusting the desired pH at 61 °C. umol H4MPT(=CH-) FORMED - 1 -1 min mg

x» X 27

F. Digestion of BCE with Protease

It was found that the effect of increas absorbance at 335 nm could be produced in an assay mixture containing HCHO and BCE, without the addition of enzyme.

In an effort to determine if this effect was produced by oxidoreductase enzyme still present and active in the boiled extract, some BCE was treated with Pronase and

Papain, two non-specific proteases. The treated solution was assayed for oxidoreductase activity and compared to untreated BCE (Table 2). The treated BCE was no longer active. This indicates that some oxidoreductase had not been irreversibly denatured in the cell extract after boiling.

G. Effect of F42o

Factor Fj 2q w *s assayed as a possible electron acceptor for the oxidoreductase. Enzyme fractions of varying purity were used. Addition of partially pure

F420 increaBe<* the rate of reaction in all cases (Table 3).

The effect produced increased along with increasing purity of the enzyme fractions. An initial, chemical rate, as opposed tv an enzymatic rate, was observed upon adding f420 to essay mixtures lacking enzyme. One assay mixture was scanned before Initiating the reaction by addition of formaldehyde and after the reaction had gone to completion. The resulting spectra (figure Id) Table 2. Digestion of BCE with Protease

Specific Activity Fraction (nmol min"* mg~*)

BCE 61.1

Treated BCE* 0.0

aDigested with Pronase and Papain. on Different Enzyme Fractions Table 3. Effect of F420

Rate (nmol/min)

Enzyme Source Fold Increase -P420 +P420

A.S. 701 3300 4600 1.4 supernatant

Dialyzed A.S. 290 1200 4.0 70% supernatant

FPLC fraction* 0.19 8.02 42

Each reaction mixture (0.75 ml) containedi 14 nmol H4MPT, 100 nmol HCHO, 120 mM XFi at pH 6.03, 174 nmol f 420' 1 mM DTT» end 0.4t pg 701 A.8 . supernatant, 2 . 6 pg dialysed 70« A.S. supernatant, or 10 pi FPIC fraction #30.

*This was a gift from Dr. M. I. Donnelly, the procedure by which it was obtained is outlined in Materials and Methode, on p. 14. Figure 10, The UV-visible absorption spectra of assay mixture before and after initiation of reaction

Spectra were obtained of a reaction mixture before (o) and after (o) initiation by addition of HCHO. The arrows placed at 335 nm and 420 nm indicate production of methenyl-H^MPT and reduction of F420' respectively. The spectra were measured in 1 cm x 1 cm anaerobic quartz cuvettes on a Perkin-Elmer Lambda 3 spectrophotometer equipped with a Perkin-Elmer 3600 data station.

showed a decrease in the absorbance at 420 nm (showing reduction of F ^ q ) and an increase in the absorbance at

335 nm (showing production of methenyl-H^MPT).

The effect of increasing the amount of F ^ q a<*^ed t0 the reaction mixture was also tested, along with comparing pure with impure f 42q (Table 4). Increasing the amount of F420 resulted in greater specific activity of the enzyme.

Using pure F ^ q as opposed to material that was only par* tially purified also resulted in increased specific activity.

H. Effect of Other Electron Acceptors

NAD, NADP, FMN, and FAD were assayed as electron acceptors for the oxidoreductase and compared to F ^ q

(Table 5). While f ^ q produced a great increase in specific activity of the ensyme, none of the other compounds had a significant effect. Table 4. Effect of Varying Amounts of F 420

Amount F42oa Specific Activity Fold (nmol) (nmol min" 1 mg-1) Increase

mm* 80 17.9 2940 36.8 174 5280 6 6 .0 174b 7680 96.0

Each assay (0.75 ml) contained (per ug protein)i 11.2 nmol HjMFT, 350 nmol HCHO, in 120 mM KPi (ph 7 at 61 °C) and 1 mM DTT.

aThe concentration of F.-n in a stock solution was estimated using the extinction coefficient of F ^ et pH 7.5 and 420 ran, 41.4 mM” 1 cm” 1 (9). bPure F420. Table 5. Comparative Effect of Different Electron Acceptors

Specific Activity Electron Acceptor (nmol min" 1 mg"1) Fold Increase none 240 — F420a 8500 35.4 FMN 20 0 0 .8

FAD 210 0.9

HAD 220 0.9 NADP 220 0.9

Bach assay (0.75 ml) contained (per 119 protein) 1 11.2 nmol H^MPT, 350 nmol HCHO, and 34.8 nmol electron acceptor (FMN FAD, NAD, or NAOP), in 120 nM KPi at pH 6.03 and 1 M DTT. *

*3 . 6 nmol F^jq were added per 119 protein. 35

IV. CONCLUSION

Studies were made on the methenyl-H^MPT oxidoreductase. Ammonium sulfate fractionation was used as an initial purification technique, and most experiments were performed on the resulting supernatant. The percent ammonium sulfate used (70%) provided a 4.85-fold purification over the initial cell extract, but 248 of the total original activity was still present in the pellet. Using a slightly less concentrated solution might be more advantageous. Another possibility for future purification would be to use heat denaturation. Since the enzyme is still active in cell extract which has been steamed for 12 hours and because the optimal assay temperature was found to be 61 °e, the enzyme is heat-resistant to a certain extent. It is possible, however, that in cell extract, the oxidereductase is protected from denatutetien by other cell constituents. Factor F|2$ **• found to function as an electron acceptor for the oxidoreduetaae if vitro. f 420 may be the electron carrier for this eitSfme in vivo. Other

(NAD, n a d p , f a d , and FNt) had he apparent effect on the assay. Using F ^ , the reaction eeuld be monitored by observing the change in absorbance at 119 am. 36

V. SUMMARY

Factor F42q was found to fund a as an electron acceptor for the oxidoreductase. FAD, FMN, NAD, and NADP were not electron acceptors. The oxidoreductase was found to be active in BCE. Assay conditions were found to be maximized at pH 5.9, 120 mM KPi, 61 °c. The optimal concentration of formaldehyde was found to be 350 \mol HCHO per mg of protein. Ammonium sulfate fractionation was used as a preliminary purification technique. 37

VI. LITERATURE CITED

1. 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.

2. Cheeseman, P., A. Toms-Wood, and R. S. Wolfe. 1972. Isolation and properties of a fluorescent compound, factor 420, from Methanobacterium strain M.o.H. J. Bactarlol. 1127527-5IE I. Birich, L. D. 197B. The structure of coenzyme F420* a novel electron carrier isolated from Hothanobacterlum strain M.o.H. ph.D. Thesis, University or 111moll at Urbans, Illinois.

4 , Bifieh, L. D., Q. D. Vogels, and R. S. Wolfe. 1978. Proposed structure for coenzyme F.2n from Methanobscterium BJ^bebiflfy 17:4563-4593.

5. Beealant«-8*marena, J. c. 1983. in vitro methanogenesis fro* formalJShyle. fh.D. Thesis, diversity of Illinois at (/rbana, Illinois,

«. C.. J, A. Leigh, K, L. Rinehart, Jr., Formaldehyde actiyation fatter, , a ooenzyme of methahoganasie.

f. Ba«sj^«-9awdpana, J, c., K. L. Rinehart, Jr., and f. ff. fblfe, If f 4. Tatrahydromethanopterin. a carbon Of,fiff in methanogenesis. J. Biol. Cham. 259!9*47-9455. . SscalantS-Bemerena, J. C., end R. S. Wolfe. 19C4. 8 Formaldehyde oxidation and amthanogenasia. J. haet. 158: 721-726. 9. Jaenofcfh; R., P. SchOnheit, and R, K. Thauer. 1984. Studied on the biosynthesis of coensysa Fajq In methano- genic bacteria. tech. Hicrobiof. 1371362-365. 10, Jones, If. J., M. X, Donnelly, and R. 8. Wolfe. 1985. Evidence of a aampon pathway of Oarbon dioxide reduction to methane in affhanegens. •t- - 163:126-131. 11. Lowry* H. 1951. protein measurement with the Folin phenol 0.reagent. J. Biol. Cham. 1031265-279. 38

12. Nach, T. 1953. The colorimetric estimation of formaldehyde by means of the Hantsch reaction* Biochem, J . 55t416~421,

13. Zeikus# J. G,# and R. S. Wolfe. 1972. Methanobacterium thermoautotrophicus sp. n.# an anaerobic# autotrophic extreme thermophi1e . J. Bacteriol. 109*707-713.