UNIVERSITI PUTRA MALAYSIA

CHARACTERIZATION OF A SULFURAMINO ACID LYASE FROM CITROBACTER FREUNDII (KP25)

LIM LENG CHOO

FSMB 2003 13

CHARACTERIZATION OF A SULFUR AMINO ACID LYASE FROM CITROBACTER FREUNDII (KP25)

By

LIM LENG CHOO

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of tbe Requirement for the Degree of Master of Science

February 2003

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DEDICATION

"Dedicated to my beloved mom, dad and bro and also to all my friends."

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Abstract of thesis presented to the Senate ofVniversiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science

CHARACTERIZATION OF A SULFUR AMINO ACID LYASE FROM CITROBACTER FREUNDII (KP2S)

By

LIM LENG CHOO

February 2003

Chairman: Professor Hasanah Mohd. Ghazali, Ph.D.

Faculty: Food Science and Biotechnology

L-Methionine y-Iyase (EC 4.4.1.11; LMGL) is a pyridoxal 5'-phosphate

(PLP)-dependent enzyme that catalyzes the direct conversion of L-methionine to a-

ketobutyrate, methanethiol and ammonia by an a,r-elimination reaction. Seventy nine

LMGL-producing microorganisms isolates were screened from six local sources by

the 5'5-dithiobis (2-nitrobenzoic acid) (DTNB) test. The six local sources were soil

samples from around the Faculty of Food Science and Biotechnology and Central

Research Laboratory, Vniversiti Putra Malaysia and Kuantan sea coast, soil and water

samples from hot springs in Vlu Legong, Baling, Kedah and Pedas, Negeri Sembilan

and intestine samples from chicken. A simple and convenient colorimetric screening

method, the DTNB test detects methanethiol, which reduces DTNB contained in an

agar-plate medium to form yellow colour aryl mercaptan (4 thiol-2-nitro-benzoate)

around the colony of a bacterium that is able to produce LMGL. LMGL was detected

from 45 (57%) of the bacterial isolates by 3-methyl-2-benzothiazolone hydrazone

(MBTH) assay. LMGL activity was quantitatively assayed by determining the amount

of a-ketobutyrate produced spectrophotometricalIy at 320 run after derivatization with

MBTH. Twelve relatively high producers of LMGL were identified by Gram stain, 10

4

types of biochemical tests consisting of potassium hydroxide (KOH), catalase,

oxidase, indole, citrate utilization, phenylalanine deaminase and urease tests and triple

sugar iron agar (ISIA), nutrient agar and MacConkey agar reactions, and by using the

Biolog test kits (Biolog, Inc., Hayward, Calif.). Enterobacter nimipressuralis,

Enterobacter intermedius, Pseudomonas pyrrocinia, Ratstonia pickettii and

Citrobacter freundii (C freundii) were found to be new sources for LMGL while the

remaining two were Escherichia coli and Bacillus cereuslthuringiensis. The

methionine-utilizing enzyme was partially purified from C freundii (KP25) isolated

from soil samples of Kuantan sea coast, which contained the highest activity. The

purification scheme, involving dialysis, removal of nucleic acid with

deoxyribonuclease I (DNase I) and ammonium sulfate [(NRthS04] precipitation

resulted in a purification fold of 0.6 with a recoveIY of 22.6% and a specific activity

of 0.02 U/mg, all using methionine as the substrate. It was found that the partially

purified enzyme extract from C freundii (KP2S) catalyzed D-amino acids better than

L-amino acids and also degraded cysteine and its S-substituted derivatives such as

more effectively than methionine and its S-substituted derivatives. Hence, the result

on substrate specificity of the lyase present in the enzyme extract shows the probable

presence of D-cysteine desulfbydrase (EC 4.4.1.15) and the absence of LMGL. Crude

enzyme extract from C. freundii (KP2S) was characterized by using D- and L-cysteine

instead of DL-methionine as the substrates. The temperature and pH optimum of the

crude enzyme extract were 45°C and pH 9.0 in 125 mM glycine-sodium hydroxide

(NaOH) buffer with each D- and L-cysteine as the substrate.

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi kepeduan untuk ijazah Master Sains

PENCIRIAN SATU LYASE ASID AMINO SULFUR DARIPADA CITROBACTER FREUNDII (KP25)

Oleh

LIM LENG CHOO

Februari 2003

Pengerusi: Profesor Hasanah Mohd. Ghazali, Ph.D.

Fakulti: Sains Makanan dan Biotelmologi

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L-Metionin y-Iyase (EC 4.4.1.11; LMGL) adalah seJems enznn yang

bergantung kepada piridoksal 5' -fosfat (PLP) yang memangkin penukaran terns L-

metionin kepada a-ketobutirat, metanaetiol dan amonia melalui tindakbalas

penyingkiran a,y. Tujuh puluh sembilan isolat mikroorganisma penghasil LMGL

telah disaring dari enam sumber tempatan menggunakan ujian 5'5-ditiobis (asid 2-

nitrobenzoik) (DTNB). Enam sumber tempatan tersebut adalah sampel tanah di

sekeliling Fakulti Sains Makanan dan Bioteknologi dan Makmal Penyelidikan Pusat,

Universiti Putra Malaysia dan pantai Kuantan, sampel tanah dan air dari telaga air

panas di Uiu Legong, Baling, Kedah dan Pedas, Negeri Sembilan dan sampel usus

dari ayam. Satu kaedah penyaringan yang berasaskan warna yang mudah, ujian

DTNB mengesan metanaetiol yang menurunkan DTNB yang terkandung di dalam

media plat agar untuk membentuk warna kuning ari1 mercaptan (4 tiol-2-nitro-

benzoat) sekeliling koloni bakterium yang bernpaya menghasilkan LMGL. LMGL

dikesan dari 45 isolat bakteria dengan ujian 3-metil-2-benzotiazolon hidrazon

(MBTH). Aktiviti LMGL ditentukan secara kuantitatif dengan menentukan amaun a-

ketobutirat yang dihasilkan secara spekrofotometrik pada 320 nm setelah bercampur

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dengan MBTH. Dua belas pengeluar LMGL yang tertinggi dikenalpasti melalui ujian

Gram, 10 jenis ujian biokimia yang terdiri daripada ujian potasium hidroksida (KOH),

katalase, oksidase, indol, penggunaan sitrat, diaminase fenilalanin dan urease dan

tindakbalas agar 'triple sugar iron' (TSIA), nutrien dan MacConkey, dan dengan

menggunakan kit ujian Biolog (Biolog, Inc., Hayward, Calif). Enterobacter

nimipressuralis, Enterobacter intermedius, Pseudomonas py"ocinia, Ralstonia

pickett;; dan Citrobacter freundii (c. freundii) dikenalpasti sebagai sumber barn bagi

LMGL manakala selebibnya adalah Escherichia coli dan Bacillus

cereus/thuringiensis. Enzim yang berupaya menggunakan metionin ditulinkan sePara

dari C. freundii (KP25) yang dipencil dari sampel tanah pantai Kuantan kerana

mengandungi aktiviti enzim yang paling tinggi. Skim penulinan yang merangkumi

dialisis, penguraian asid nukleik dengan deoksiribonukleas J (DNase J) dan

pemeringkatan amonium sulfat [(NRt'hS04] mengbasilkan paras penulinan sebanyak

0.6 dengan pendapatan semula sebanyak 22.6% dan aktiviti spesifik sebanyak 0.02

U/mg, kesemuanya dengan menggunakan metionin sebagai substrat. Didapati enzim

separa tulen dari C. freundii (KP25) berupaya memangkin D-asid amino lebih baik

daripada L-asid amino dan juga menguraikan sistein dan terbitan penggantian

sulfurnya lebih berkesan daripada metionin dan terbitan penggantian sulfumya. Maka,

keputusan spesifisiti substrat untuk lyase yang hadir dalam ekstrak enzim

menunjukkan kemungkinan kehadiran D-sistein disulfilhidrase dan ketiadaan LMGL.

Ekstrak enzim kasar C. freundii (KP25) kemudiannya dicirikan dengan menggunakan

D- dan L-sistein dan bukan DL-metionin sebagai substrat. Suhu dan pH optimum

ekstrak enzim kasar adalah 45°C dan pH 9.0 dalam 125 mM penimbal glisin-sodium

hidroksida (NaOH) dengan setiap D- dan L-sistein sebagai substrat.

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ACKNOWLEDGEMENTS

This project is a corporation between Universiti Putra Malaysia (UPM) and

Japan International Corporation Agency (JICA) under the supervision of Prof Dr.

Hasanah Mohd. Ghazali. Thus, firstly, I would like to extend my most sincere

gratitude to UPM, JlCA and my supervisor, Prof Dr. Hasanah Mohd. Ghazali for the

opportunity to work on this project and the assistance they provided me during the

course of this study. I'm truly grateful to Prof Dr. Hasanah Mohd. Ghazali for the

opportunity to work in her well equipped Enzyme Laboratory, for her constant

guidance and advice and also the confidence she has in me.

I would also like to express my special appreciation to both my co­

supervisors, Assoc. Prof Dr. Raha Abdul Rahim and Dr. Lai Oi Ming for their

comments, suggestions and reviews on my project. Acknowledgement is also due to

Dr. Takashi Tamura for his guidance and contributions during his three months stint

in Department of Biotechnology, Faculty of Food Science and Biotechnology, UPM.

Many thanks to all my friends and colleagues for their guidance, advice,

support and encouragement. I'll always appreciate and cherish the friendship we have.

Finally, I thank my family, whose constant love, support and encouragement have

meant so much to me in the course of my study.

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I certify that an Examination Committee met on 20th February 2003 to conduct the final examination of Lim Leng Choo on her Master of Science thesis entitled "Characterization of a Sulfur Amino Acid Lyase from Citrobacter freundii (KP25)" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. The members of the Examination Committee are as follows:

CLEMENTE MICHAEL WONG VUI LING, Ph.D., Lecturer, Department of Biotechnology Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Cbairman)

HASANAH MOHD. GHAZALI, Ph.D., Professor, Department of Biotechnology Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

RAHA ABDUL RAHIM, Ph.D., Associate Professor, Department of Biotechnology Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

LAI 01 MING, Ph.D., Associate Professor, Department of Biotechnology Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

GULAM R SU MAT ALI, Ph.D., Professor�eputy , School of Graduat Studies Universiti Putra Malaysia

Date: 2 0 f"tAf-< 2003

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This thesis submitted to the Senate ofUniversiti Putra Malaysia has been accepted as fulfilment of the requirements for the degree of Master of Science. The members of the Supervisory Committee are as follows:

HASANAH MOHD. GHAZALI, Ph.D., Professor, Department of Biotechnology Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Chairman)

RAHA ABDUL RAHIM, Ph.D., Associate Professor, Department of Biotechnology Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

LAI 01 MING, Ph.D., Associate Professor, Department of Biotechnology Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

AINI IDERIS, Ph.D., ProfessorlDean, School of Graduate Studies Universiti Putra Malaysia

Date: 8 MAY 2003

DEC LARA TION

I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or currently submitted for any other degree at UPM or other institutions.

Lim Leng Choo

Date: '313/ �H3

TABLE OF CONTENTS

Page

DEDICATION ABSTRACT

2 3 5 7 8 10 13 14 16

ABSTRAK ACKNOWLEDGEMENTS APPROVAL SHEETS DECLARATION FORM LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS

CHAPTER

I

II

INTRODUCTION

LITERATURE REVIEW Enzyme Sulfur Amino Acid Lyases

L-Methionine y-Iyase (LMGL) D-Cysteine desulfuydrase L-Cysteine desulfuydrase Homocysteine desulfuydrase Cystathionine lyases

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20 20 21 21 43 44 45 45

III SCREENING, ISOLATION AND IDENTIFICATION 47 OF LMGL-PRODUCING MICROORGANISMS

IV

Introduction 47

Materials 48 Methods 49

Screening ofLMGL-producing Microorganisms 49 Isolation ofLMGL-producing Microorganisms 49 Identification of LMGL-producing Microorganisms 51

Results and Discussion 54 Screening ofLMGL-producing Microorganisms 54 Isolation ofLMGL-producing Microorganisms 58 Identification ofLMGL-producing Microorganisms 62

Summary 66

ISOLA TION AND PARTIAL PURIFICATION OF LMGL Introduction Materials Methods

Isolation ofLMGL Partial Purification ofLMGL

Results and Discussion

67

67 70 70 70 71 73

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Isolation and Partial Purification ofLMGL Summary

73 84

V CHARACTERIZATION OF SULFUR AMINO ACID 86 LYASE FROM CITROBACTER FREUNDII (KP2S) Introduction 86

Materials 87

Methods 88 Characterization of Partially Purified LMGL 88 Characterization of Crude Enzyme Extract from 91 Citrobacter freundii (KP25)

Results and Discussion 92 Characterization of Partially Purified LMGL 92 Characterization of Crude Enzyme Extract from 106 Citrobacter freundii (KP25)

Summary 106

VI SUMMARY, CONCLUSION AND 110 RECOMMENDATIONS Summruy 110 Conclusion and Recommendations 112

BIBLIOGRAPHY APPENDICES VITA

114 127 150

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Table

1

2

3

4

5

6

7

8

9

10

LIST OF TABLES

Results ofDTNB and MBTH tests

Results of Gram stain and biochemical tests for 12 cultures detected with LMGL

Results of Biolog test kits for 12 cultures detected with LMGL

Effect of polypeptone on the production of LMGL by Citrobacter freundii (KP25)

Effect of extraction methods on the activity of LMGL and protein content

Effect of nucleic acid removal by DNase I and streptomycin sulfate on the activity ofLMGL and protein content

P artial purification of LMGL from Citrobacter freundii (KP25)

Substrate specificities of partially purified enzyme extract from Citrobacter freundii (KP25)

Effect of PLP as cofactor on partially purified enzyme extract from Citrobacter freundii (KP25)

Effect of inhibitors on partially purified enzyme extract from Citrobacter freundii (KP25)

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Page

55

63

64

74

76

78

80

98

102

102

Figure

1

2

3

4

5

6

7

8

9

10

11

1 2

1 3

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1 5

LIST OF FIGURES

a,y-E1imination reaction catalyzed by LMGL

Three-dimensional structure ofLMGL

Sequence alignment of LMGL and other PLP-dependent

enzymes of the y-family based on the crystal structures

Amino acid sequence alignment of Trichomonas vaginalis methionine y-lyases, MGLI and MGL2, with two methionine y-Iyases from Pseudomonas pulido (PPMGLI and ppMGL2) and cystathionine y-lyases from yeast (ScCYS3) and human (HsCGL)

Mechanism of a.,y-elimination and y-replacement or -addition reactions catalyzed by LMGL

Mechanism of LMGL-catalyzed incorporation of deuterium (or tritium) into the 0.- and �-positions of L­amino acids

Screening agar

Appearance of yellow and white colonies on screening agar

Specific activity ofLMGL from 45 screening cultures

FPLC chromatogram of LMGL on Fast Desalting HR 10/10 column for dialysis after nucleic acid removal with DNase I

FPLC chromatogram of LMGL on Fast Desalting HR 1011 0 column for desalting after (Nfu)2S04 precipitation

Effect of temperature on the activity of partially purified LMGL from Citrobacter freundii (KP25)

Effect of temperature on the stability of partially purified LMGL from Citrobacter freundii (KP25)

Effect of pH on the activity of partially purified LMGL from Citrobacter freundii (KP25)

Effect of pH on the stability of partially purified LMGL from Citrobacter freundii (KP25)

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Page

22

26

27

30

32

36

56

56

59

81

8 2

93

94

95

96

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16 Stability of partially purified enzyme extract from 104 Citrobacter freundii (KP25)

17 Effect of temperature on the activity of crude enzyme 107 extract from Citrobacter freundii (KP25) with D- and L-cysteine as substrates

18 Effect of pH on the activity of crude enzyme extract from 108 Citrobacter freundii (KP25) with D- and L-cysteine as substrates

19 Standard curve for a.-ketobutyrate 143

20 Standard curve for BSA 144

21 Screening broth 145

22 Culture ofCitrobacter freundii (KP25) in screening broth 146

23 Biolog microplate 147

24 Biolog MicroStation reader 147

25 Biolog turbidimeter 148

26 Hitachi double-beam model U-2000 spectrophotometer 148

27 Pharmacia Fast Protein Liquid Chromatography (FPLC) 149

ATP BCNU

bp BSA

CF3SH CSF2

C.freundii Da

DNA DNase I

dNTP DTNB DTT

EDTA FPLC

g h

H2

H3

H202 H2S HIV kDa

KH2P04 K2HP04

KOH KP Km L

LMGL Lys

M MBTH

mg MgS04

min m1

mM MRP NaCI

NaOH (N�hS04

run No

PCMB PCR Pgp

LIST OF ABBREVIATIONS

Adenine triphosphate 1,3-Bis (2-chloroethyl)-I-nitrosourea Base pair Bovine serum albumin Trifluoromethanethiol Carbonothionic difluoride Citrobacter freundii Dalton Deoxyribonucleic acid Deoxyribonuclease I Deoxynucleoside triphosphate 5,5' -Dithiobis-2-nitrobenzoic acid Dithiothreitol

Ethylenediaminetetraacetic acid Fast Protein Liquid Chromatography Gram Hour Deuterium Tritium Hydrogen peroxide Hydrogen sulfide Human immunodeficiency virus KiloDalton Potassium dihydrogen phosphate Dipotassium hydrogen phosphate Potassium hydroxide Potassium phosphate Michaelis constant Liter L-Methionine y-lyase Lysine Molar 3-Methyl-2-benzothiazolone hydrazone Milligram Magnesium sulfate Minute Milliliter Millimolar Multidrug related protein Sodium chloride Sodium hydroxide Ammonium sulfate Nanometer Number p-Chloromercuribenzoic acid Polymerase chain reaction P-glycoprotein

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pH PLP

PMSF RNA

rpm sec

TCA TMZ Tris

TSIA Vmax �g

,.u JiM

flInol °c %

Hydrog en ion concentration Pyridoxal 5' -phosphate Phenylmethylsulfonyl fluoride Ribonucleic acid Revolutions per minute Second

Trichloroacetic acid Temozolomide Tris(hydroxymethyl)-aminomethane

Triple Sug ar Iron Ag ar Maximal velocity Microgram Microliter Micromolar

Micromol Degree Celsius P ercentag e

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18

CHAPTER I

INTRODUCTION

Life depends on a well-orchestrated series of chemical reactions. Many of

these reactions, however, proceed too slowly on their own to sustain life. Hence

nature has designed catalysts, which are now referred to as enzymes, to greatly

accelerate the rates of these chemical reactions. The catalytic power of enzymes

facilitates life processes in essentially all life-forms from viruses to man.

Sulfur amino acid lyases such as L-methionine y-Iyase, D-cysteine

desulfhydrase, L-cysteine desulfuydrase, homocysteine desulfuydrase, cystathionine

y-Iyase and cystathionine 13-lyase have versatile functions. The multi catalytic

functions of these enzymes enable their application in various scientific fields. The

application of these enzymes include synthesis of various optically active sulfur and

selenium amino acids, determination of sulfur and selenium amino acids, preparation

of deuterium or tritium-labeled L-amino acids, development of cheese flavour, as a

novel anticancer agent and also as a drug target by prodrugs.

In this study, a LMGL producer was isolated from local sources, and the

physical and chemical properties of the partially purified LMGL from it were

examined. Thus, this study was divided into three chapters with the following

objectives:

1 . to screen, isolate and identifY LMGL-producing microorganisms from local

sources

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2. to isolate and partially purify LMGL from the isolated and identified highest

LMGL producer

3. to characterize the partially purified enzyme

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CHAPTER II

LITERATURE REVIEW

Enzymes

Enzymes are biological catalysts that accelerate a chemical reaction without

itself undergoing any net change (Foster, 1980; Campbell, 1995; Zubay, 1995). An

enzyme does not change the final equilibrium position of a reaction, which is

thennodynamically detennined, and only the rate of attainment of equilibrium of a

feasible reaction is increased (Foster, 1980). Enzymes are the most efficient catalysts

known as they can increase the rate of a reaction by a factor of up to 1020 over

uncatalyzed reactions (Campbell, 1995). Nonenzymatic catalysts, in contrast,

typically enhance the rate of reaction by factors of 102 to 104. Enzymes are highly

specific, even to the point of being able to distinguish stereoisomers of a given

compound. Only small quantities, relative to the concentrations of their substrates, are

needed to considerably increase the rate of chemical reactions and the total amount of

substrate transfonned per mass of enzyme is often vel)' large (Foster, 1980).

All enzymes are proteins and, thus, exhibit the chemical and physical

behaviour of proteins (Foster, 1980). Being proteins, enzymes differ considerably

from traditional chemical catalysts such as hydrogen ions, hydroxyl ions, heavy

metals or metal oxides. Whereas these are most effective in organic solvents, at high

temperatures or at extreme pH values, enzymes operate most efficiently under very

mild conditions. Departure from homogeneous, aqueous solutions, physiological pH

21

and temperature rapidly destroys their activities, but under normal condition the rate

increases achieved are rarely matched by their non-protein counterparts.

A systematic scheme for classifying enzymes was adopted in 1972 by the

International Union of Biochemistry (Zubay, 1995). In this scheme, there are six main

classes of enzymes. The six main classes are: (1) oxidoreductases, (2) transferases, (3)

hydrolases, (4) lyases, (5) isomerases and (6) ligases or synthases. Lyases catalyze the

removal of a group to form a double bond or the addition of a group to a double bond.

In catabolic pathways two general types of enzyme may be distinguished,

constitutive enzymes which are always present in fairly constant concentrations and

inducible enzymes whose DNA operons are usually repressed but which in the

presence of activators are derepressed and transcribed (Foster, 1980).

Sulfur Amino Acid Lyases

L-Methionine y-Iyase (LMGL)

L-Methionine y-Iyase (EC 4.4.1.11; LMGL) is a pyridoxal 5'-phosphate

(PLP)-dependent enzyme with multi catalytic functions (Ito et aI., 1976a; Tanaka et

al., 1977; Soda et al., 1983; Nakayama et aI., 1984a; Nakayama et al., 1984b; Esaki

and Soda, 1987b; Lockwood and Coombs, 1991; Dias and Weimer, 1998b; Soda et

al., 1999; Motoshima et ai., 2000). Known also as methionase, methioninase, L­

methionine y-demethiolase and L-methionine methanethiol-Iyase ( deaminating),

22

LMGL catalyzes the direct converSIOn of L-methionine to a-ketobutyrate,

methanethiol and ammonia by an a,y-elimination reaction (Figure 1).

CH3SCH2CH2CH(NH2)COOH + H20 --) CH3CH2COCOOH + CH3SH + NH3

Figure 1: a;y-Elimination reaction catalyzed by LMGL

LMGL catalyzes the a,y-elimination and y-replacement reactions of L­

methionine and its S-substituted derivatives, a,fl-elimination and fl-replacement

reactions of L-cysteine and its derivatives, deamination and y-addition reactions of L­

vinylglycine and L-allylglycine, and deuterium labeling at a and J3 positions of L­

methionine and other straight-chain L-amino acids (Ito et aI., 1976a; Tanaka et ai.,

1977; Soda et al., 1983; Nakayama et 01., 1984a; Nakayama et 01., 1984b; Esaki and

Soda, 1987b; Lockwood and Coombs, 1991; Dias and Weimer, 1998b; Soda et aI.,

1999; Motosbima et 01.,2000). These reactions are applicable to synthesis of various

optically active sulfur and selenium amino acids, preparation of deuterium or tritium­

labeled L-amino acids, and determination of sulfur and selenium amino acids.

However, it does not act on the D-enantiomers, and cannot cleave the Y,o- and �,Y­

carbon-carbon bonds of norleucine and norvaline (Soda et al., 1983; Dias and

Weimer, 1998b).

Sources

Since its discovery in Escherichia coli and Proteus vulgaris by Onitake,

LMGL has been found in various other bacteria (Tanaka et aJ., 1977; Soda et aI.,

23

1983� Lockwood and Coombs, 1991� Dias and Weimer, 1998b). The enzyme was

subsequently demonstrated in extracts of a soil bacterium (Tanaka et al., 1977;

Lockwood and Coombs, 1991), some rumen bacteria, Bacillus species (Tanaka et ai.,

1977; Soda et al., 1983), Clostridium sporogenes (Tanaka et al., 1977; Lockwood and

Coombs, 1991� Dias and Weimer, 1998b; Motoshima et al., 2000; Yoshimura et al.,

2000), Clostridium botulinum, Clostridium difficile (Coombs and Mottram, 2001),

Pseudomonas putida (= ovalis) (Ito et at., 1976a; Tanaka et al., 1977; Soda et ai.,

1983; Nakayama et at., 1984b; Esaki and Soda, 1987b; Inoue et al., 1995; Soda et al.,

1999; Motoshima et al., 2000), Aeromonas species (Nakayama et al., 1984a; Esaki

and Soda, 1987b; Motoshima et al., 2000), Brevibacterium linens (Dias and Weimer,

1998b), Porphyromonas gingivalis (Yoshimura et al., 2000) andPorphyromonas and

Bacteriodes species (Coombs and Mottram, 2001). LMGL has also been detected in

the anaerobic protozoan parasites, Trichomonas vagina!is (Lockwood and Coombs,

1991; McKie et al., 1998; Coombs and Mottram, 2001) and Entamoeba histolytica

(Coombs and Mottram, 2001) but is not believed to be present in yeast, plant or

mammals (Griffith, 1987; Soda, 1987; McKie et aI., 1998; Motoshima et al., 2000).

LMGL has been purified and characterized at the biochemical and molecular

levels from Pseudomonas pulida (Ito et al., 1976a; Tanaka et al., 1977; Nakayama et

al., 1984b; Esaki and Soda, 1987b; Lishko et at., 1993b; Inoue et al., 1995; Hori et

al., 1996; Tan et al., 1997a; Inoue et al., 2000; Motoshima et al., 2000; Sridhar et al.,

2000) and Trichomonas vaginalis (Lockwood and Coombs, 1991; McKie et al.,

1998), purified from Clostridium sporogenes (Tanaka et al., 1977; Lockwood and

Coombs, 1991� Dias and Weimer, 1998b), purified and biochemically characterized

from Aeromonas species (Nakayama et al., 1984a; Esaki and Soda, 1987b) and

24

Brevibacterium linens (Dias and Weimer, 1998b) and characterized at the molecular

level from Porphyromonas gingivalis (Yoshimura et al., 2000). LMGL is a cytosolic

enzyme inducibly fonned by addition ofL-methionine to the culture medium (Tanaka

et al., 1977; Soda et af., 1983; Lockwood and Coombs, 1991; Dias and Weimer,

1998b).

Characteristics

LMGL has a molecular weight of about 149 kDa to 174 kDa (Ito et al., 1976a;

Tanaka et al., 1977; Nakayama et af., 1984a; Nakayama et ai., 1984b; Lockwood and

Coombs, 1991; Dias and Weimer, 1998b) and consists of four subunits with identical

molecular weights of about 41 kDa to 45 kDa each (Nakayama et aI., 1984a;

Nakayama et al., 1984b; Lockwood and Coombs, 1991; Dias and Weimer, 1998b)

except LMGL purified to homogeneity from Pseudomonas pUlida (=ovalis) IFO 3738

which was found to consists of two nonidentical subunits with molecular weights of

40 kDa and 48 kDa (Tanaka et af., 1977). This enzyme shows the typical absorption

spectrum of PLP -dependent enzyme with maxima at 278 run to 280 run and 420 run to

430 nm (Ito et al., 1976a; Tanaka et ai., 1977; Nakayama et aI., 1984a; Nakayama et

al., 1984b; Dias and Weimer, 1998b), and contains four mole of PLP per mole of

enzyme (Tanaka et al., 1977; Nakayama et al., 1984a; Nakayama et al., 1984b).

However, LMGL purified to homogeneity from Pseudomonas putida ATCC 17453

and Pseudomonas pulida (=ovalis) IFO 3738 also shows a shoulder around 330 nm to

345 nm(Itoetal., 1976a; Tanaka,etal., 1977).