Facultative Methylotroph

(1)

Vol. 175, No. 17 JOURNAL OFBACTERIOLOGY, Sept. 1993, p.5617-5627

0021-9193/93/175617-11$02.00/0

Cloning,

Sequencing,

Expression,

and

Regulation

of the Structural

Gene for

the

Copper/Topa

Quinone-Containing

Methylamine

Oxidase from

Arthrobacter

Strain P1,

a

Gram-Positive

Facultative

Methylotroph

XIAOPINGZHANG,1'2 JOHN H. FULLER,1'2ANDWILLIAM S. McINTIREl 2,3*

Molecular

Biology

Division, Department

of

Veterans

Affairs

MedicalCenter, San Francisco,

California

94121,1* andDepartmentof Biochemistry and

Biophysics2

andDepartmentofAnesthesia, 3

Universityof California, San Francisco, California 94143 Received 2 April1993/Accepted 21June 1993

Deoxyoligonucleotides corresponding to amino acid sequences of methylamine oxidase and polyclonal anti-methylamine oxidase antibodies were used toprobeArthrobacterstrainP1plasmidand chromosomal DNA libraries. Two openreading frames,maoxI andmaoxII, which are greater than

99%o

homologous,werecloned fromthe chromosomal library. The deducedaminoacidsequences of the coding regions are identical except for two residues near theC termini. On the otherhand,the5'- and3'-flankingregions of maoxI and maoxIIare

quite different. While either gene could code for methylamine oxidase, the

dissimilarity

in the 5'-flanking regions indicates that the genes are

differently

regulated. It was determined that maoxII alone encodes methylamine oxidase. The

tyrosyl

residue which is converted to topaquinoneinthe matureenzymewaslocated by comparison with ammo acid sequences at the cofactor sitesinothercopper/topa quinone-containingamine oxidases.Transcriptional startsites andpossibleregulatoryelements were identified in the5'regionof maoxI andmaoxII,andstem-loopstructures werefound in the3'-flanking regions. Highlevelsofmethylamineoxidase areproduced when Arthrobacter strain P1 is grown onmethylaminealoneoronglucose plusmethylamine,but growth on LB medium plus methylamine resulted in very lowproduction of the enzyme.ExpressionofmaoxII from its ownpromoter inEscherichiacoligrown on glucose or LB medium with or withoutmethylamine gave thesame levelofproductionofmethylamineoxidase.

Thegram-positive facultative methylotroph Arthrobacter strain P1 can utilize methylamine as the sole carbon and energy source for growth. This substance is oxidized

by

methylamine oxidase, and the resulting formaldehyde is assimilated via the Embden-Meyerhof fructose-bisphos-phate aldose/transaldolase variant of the ribulose monophos-phate cycle (22). Methylamine oxidase is an a2 enzyme containing1 g-atomof

Cu(II)

and1mol ofcovalently bound quinonecofactorper mol of subunit.Methylamineoxidaseis strikingly similar to

copper/quinone-containing

amine oxi-dases in theplant andanimalkingdoms (24). Exampleshave been identified among gram-negative bacteria

(Escherichia

coli and Klebsiellaaerogenes[8,

37]),

gram-positive bacteria

(Arthrobacter

species), yeast and fungi, seedlings and ma-ture terrestrial plants, birds, fish, mollusks, mammals (24), andpossibly marine phytoplankton(28).

When the existenceof theseoxidaseswasfirstrealized in the 1940s, it was suggested that the oxidases contained pyridoxal phosphate as the sole organic cofactor. More recently, pyrroloquinoline quinone, the noncovalently boundprostheticgroupofanumberof bacterial dehydroge-nases(9, 23),wasproposedasthecovalently bound cofactor in the copper amine oxidases. As in the case made for pyridoxal phosphate, the evidence was circumstantial, and no direct structural proofwas forthcoming. This issuewas resolved in 1990, when Klinman and colleagues presented incontrovertiblechemical, physical,andstructural evidence proving that the true cofactor of bovine plasma amine oxidase is 6-hydroxydopa quinone, also known as topa

* Corresponding author.

quinone

(15).

Other,more recentstudies reaffirm this fact(5, 16, 27). This quinone isformed byco- orposttranslational modificationof aspecific tyrosyl residue within the polypep-tide chain

(27).

It is not known whether the phenolic side chainofthis aminoacylgroup ismodified via the interven-tionof anexternalenzyme orwhether the immature oxidase self-catalyzes the requisite oxidation(s) with the predicted participationofthe enzyme-boundCu(II). Although thetopa quinone cofactor is unusual, it is not unique. Tryptophan tryptophylquinone inbacterial methylamine dehydrogenase (25)and thecross-linked cysteinyl-tyrosylgroupingalactose oxidase of Dactylium dendroides (14) are also cofactors formed, in a directfashion, by minor modification of intact aminoacyl side groups. Thus, in the pastseveral years, a newclass of enzymeprostheticgroupshas emerged.

Itseemedfittingand essential tocomplementourcurrent chemical andbiochemical studiesof the structure, function, andbiosynthesis of methylamine oxidasewith modern mo-lecular biological methodologies. Herein, we report the results ofour cloning, sequencing, expression, and regula-tionstudiesofthe structuralgeneforArthrobacterstrainP1 methylamine

oxidase.

Becauseofthe striking similaritiesof thecopper-containing amine oxidases across all phyla, what wegleanfrom our work should have relevance for allthese enzymes.

MATERIALSAND METHODS

Materials. All

re§triction

endonucleases and DNA-modi-fying enzymes were obtained from New England Biolabs, Beverly, Mass.; Boehringer Mannheim, Indianapolis, Ind.; or GIBCO BRL, Gaithersburg, Md. Xgtll, E. coli Y1090 5617

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5618 ZHANG ET AL.

(rK-

MK+), and E. coli HB101 were purchased from Promega Corp., Madison, Wis. Sodium dodecyl sulfate (SDS), agarose, and polyacrylamide were obtained from Bio-Rad Corp., Richmond,Calif.Formaldehydeandsucrose were from Fisher Scientific, Pittsburgh, Pa. a-35S-dATP and

[t32P]ATP

were from DuPont-New England Nuclear, Boston, Mass., and Amersham Life Sciences, Arlington Heights, Ill. Methylamine, 3-phenylethylamine, andcholine hydrochlorides;chicken egg whitelysozyme,grade 1; amino acids; andantibiotics werepurchasedfrom SigmaChemical Corp., St. Louis, Mo. If available, "molecular biology grade" salts and buffers were obtained from International

Biotechnologies,

Inc., New Haven, Conn. Allother chemi-cals were ofreagent grade quality or better.

Bacterialstrains,plasmids,andgrowth conditions. Arthro-bacter strainP1wasgrownat30°C in a minimalmedium(19) containing 0.6%

(wt/vol)

methylamine hydrochloride as a nitrogen source and the sole source of carbon; thevitamin mixture wasomitted.Thisorganism was also grown on other compounds forexpression studies, as described inResults. E. coli Y1090 (rK- MK) was used as the host strain for Xgtll.E. coli HB101 was used asthe host strain for some library construction and cloning experiments (34) with the plasmidspBR322andpGEM-7Zf(+) (Promega).For expres-sion studies, E. coliHB101 was grown at 37°Ceither inthe minimal medium used forArthrobacter strain P1, supple-mented with 0.3%

(wt/vol)

glucose and/or 0.3% (wt/vol) methylamine hydrochloride, 1,ug ofthiamine

ml-',

40 ,g of L-leucine

ml-',

and 40 ,ug of L-proline

ml-',

or in Luria-Bertani (LB) medium. Plasmid-bearing strains of E. coli grown on rich medium were cultured in the presence of ampicillin at 100

p,g

ml-.

Southern blot

analysis.

Twenty-microgramsamples of Ar-throbacterstrain P1 chromosomalDNA were digestedwith different restriction endonucleases and size fractionatedby electrophoresison a0.8%

(wtlvol)

agarosegel. Thegel was blotted with a Hybond-N+ membrane according to an Amershamprotocol (34), anddetectionwaswith nonradio-active DNA probes. Oligonucleotide probes were labeled with the3'-oligotailing system fromAmersham. Restriction DNA fragments were labeled by using an Amersham ECL nucleotidedetectionlabeling kit. Hybridization andwashing were carried outaccording tothe manufacturer'sprotocols. Protein isolation and amino acid sequence analysis. Meth-ylamine oxidase was purified fromArthrobacter strain P1 grown on 0.6%

(wt/vol)

methylamine(22). N-terminal amino acidsequenceanalysis ofmethylamine oxidasewas accom-plished at the Sequencing Facility at the University of California, Davis. Sequence data for other methylamine oxidase peptideswere supplied byDavid M. Dooley, Am-herst College,Amherst,Mass. Polyclonalantibodies against purified methylamine oxidase were raised in rabbits and werepurified by standard methods(11).

Constructionandscreening oflibraries. Plasmid and chro-mosomal DNAs from methylamine-grown Arthrobacter strainP1 werepurified by CsCldensity gradient centrifuga-tion(30). Librarieswere constructedby partialdigestion of DNA with the frequently cuttingrestriction enzymes

AluI,

BstUI, HaeI, RsaI, and ThaI, all of which produce blunt ends (26). The digested DNA was pooled and size fraction-ated bysucrose density gradient (10 to40%,

wt/vol)

centrif-ugation to obtainfragmentsof 4 to 9kbp. These wereligated toan

EcoRI-NotI

adaptor(Pharmacia LKB Biotechnology, Piscataway, N.J.), and the adaptor ends were phosphory-lated with T4polynucleotidekinase. Insert DNA was ligated with dephosphorylated

EcoRI-generated

Agtll arms,

pack-aged

in vitroby using the Packagene System from Promega, and transfected intoE. coli Y1090 for immunoscreening of plaques for expressed fusion protein(13).

On the basis of amino acid sequences of portions of methylamineoxidase, two degenerate oligodeoxynucleotide mixtures were synthesized at the Department of Veterans Affairs Medical Center, San Francisco Molecular Core Fa-cility. The sequencesofthe oligonucleotides are 5'-GA(T/C) ATGGA(A/G)TA(T/C)CCNGA-3' and

5'-ATGCA(T/C)TT

(T/C)GA(T/C)TT(T/C)(C/A)G-3'.

Southern blot analysis of Arthrobacter strain P1 chromosomal DNAwas carried out with these oligonucleotide probes. Two bands of approxi-mately 2.1 and 3.1 kbp were identified from the digestion with BamHI. On the basis of this result, two enriched plasmid libraries were constructed (27a). Two DNA frag-ments were isolated from Arthrobacter strain P1 chromo-somal DNA digested with BamHI, and these fragmentswere ligated in separate reactions into the BamHI site of pBR322. TherecombinantDNAwas transformed into E. coli HB101. Colonies were transferred to an Amersham Hybond-N+ membrane. Plasmid amplification, colony transfer, and de-naturation were performedaccording to the manufacturer's instructions. Librarieswere screened with-y-32P-end-labeled oligonucleotide probes at 45°C (34). The membranes were washed thoroughly with 5x SSC(1x SSCcontains 150 mM NaCl and 15 mM sodium

citrate)-0.1%

(wt/vol)SDSat room temperature. DNA fragments from positive clones were isolated and cloned into the pGEM-7Zf(+) vector for se-quencing.

DNA sequence analysis. Plasmids with progressive unidi-rectional deletionsfrom each end of the two DNAfragments were constructed by exonuclease III digestion with the Erase-a-Base Systemfrom Promega (12). DNA sequencing was done by the dideoxynucleotide chain termination method (35), using Sequenase version 2.0 and TAQuence version 2.0 DNA sequencing kits (United States Biochemi-cal,Cleveland, Ohio). Sequencing compilations,open read-ing frame identification and translation, restriction map construction, sequence comparisons, and Clustal analysis wereperformed with the PCGENEgroup ofprograms from IntelliGenetics, Inc., Mountain View, Calif.

RNA isolation and Northern (RNA) blotting. Arthrobacter strain P1 was grown on minimal mediumorLBmedium at 30°C to anoptical densityof 1.0 at 600 nm. Lysozyme was added to a final concentration of 0.5 mg ml-1, and the cultures wereincubatedat30°Cforanother hour. Cellswere harvested bycentrifugation, and the pelletwasresuspended in 2packed-cellvolumes of waterand then in4packed-cell volumes of RNAzol B fromBiotecxLaboratories, Inc. Cells were ruptured by sonication for 3 min (50 W, 3-mm

tip).

RNA wasprecipitatedwith anequal volume ofisopropanol, resuspended in double-distilled water, and reprecipitated with 0.1volume of 3 Msodium acetate and 2.5 volumes of ethanol. Twenty micrograms of RNA was denatured and size fractionated on a 1.0% (wt/vol) agarose

gel

containing

2.2 M formaldehyde (34). After transfer to a Hybond-N+ membrane, RNAfragmentswere detected

by

hybridization withnonradioactiveDNAprobeslabeledwith anAmersham ECL nucleotide detection labeling kit. Probe

labeling,

hy-bridization, and washing were carried out as for Southern blotanalyses.

Primer extension assay. Primer extension

analysis

of tran-scriptional initiation sites was accomplished as

previously

described (7). Five micrograms of RNA fromArthrobacter strain P1 and 1 to 3 ng of

y-32P-end-labeled oligonucleotide

primer were used in each reaction. Tomap exact

transcrip-J. BACTERIOL.

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METHYLAMINE OXIDASE FROMARTHROBACTER STRAIN P1 5619 0

A

_ >- ,F

FE

° - E E =: . . a. .A C4 --wIL w E a3.

..

.. .c .

.z4

in,

B

o.* EE-0 s %I a a a VI AI

FIG. 1. Restriction map, sequencing scheme, andsubcloning strategyformaoxI(A) andmaoxII (B). All ofthe deletion cloneswere

constructedbydigestingthe entire DNAfragmentwith severalrestrictionendonucleases, isolatingsmallfragments,andligatingthem intothe

pGEM-7Zf(+)vectorandunidirectionally truncating ligated fragmentswithexonuclease III. Thehorizontalarrowsindicate thelengthsand directions of individualsequencing analyses runningfromtheT7orthe SP6primerbindingsites. Bothstrandsweresequencedcompletely.

IdenticalregionsinmaoxIandmaoxIIarealignedinaccord with the restriction endonuclease sites locatedatthesamelongitudinal position.

Theheavybars ineach cloneidentifytheopenreadingframes formaoxIandmaoxII.

tionalstartsites,sequencingreactionswereperformedwith

thecorrespondingDNAand thesameprimerthatwasused for theprimerextensionreaction.

Immunoblot analysis. Western blot (immunoblot) detec-tion ofproteinwascarriedoutasdescribedpreviously (30a). Cellswereharvested when theopticaldensityofthe culture at600nmreached 1to1.5. The E. coliHB101cellpelletwas

lysedinSDSsamplebuffer. TheArthrobacter strain P1 cell pelletwas resuspended in sample buffer and sonicated (50 W, 3-mmtip)for30s.The cellextractwaselectrophoresed on an 8% (wt/vol)polyacrylamide gel containing 0.1% (wt/ vol)SDS, andproteinswere electrophoretically transferred

toanitrocellulose membrane(Schleicher&Schuell, Keene, N.H.). The membrane was immunostained by incubation

with polyclonal antibodies against Arthrobacter strain P1 methylamineoxidase and then withanti-rabbitalkaline phos-phatase conjugate. Nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate were used for color detection

of the oxidase (protoBlot Western Blot AP system; Pro-mega).

Nucleotide sequence accession numbers. The nucleotide

sequences for maoxI and maoxII presented in this report have beendepositedintheGenBankdata baseunder

acces-sionnumbers L12983 andL12990,respectively.

RESULTS

Cloningandsequencing of the Arthrobacter strain P1 meth-ylamine oxidasegene. Inorder to clonethe genecodingfor Arthrobacter strain P1 methylamine oxidase, chromosomal libraries were constructed by using both phage Xgtll and

plasmid pBR322. Screening of the chromosomal phage li-brarywith antibodies resulted in fivepositive clones.Four of thesewereconfirmedby hybridizationwithnonradioactively labeled oligonucleotides designed to correspond to known amino acid sequences of the enzyme. Screening of the enriched plasmid libraries with the oligonucleotide probes yielded twopositiveclones.Restriction endonuclease

map-ping indicatedthattheseclones segregated intotwogroups,

designated I and II. TheDNA sequencesof clones within each group overlap each other. For sequencing, a 2.8-kbp

DNA fragment from group I and a 2.2-kbp fragment from

group II were used to produce deletion clones. Figure 1A and B show restrictionmaps and sequencing strategies for two open reading frames designated maoxI and maoxII, respectively. As shown in Fig. 1, both strands of the two DNA fragments were sequenced independently and

com-pletely.BothmaoxI and maoxII consist of1,944nucleotides,

which translates intopolypeptides of 648 amino acids with deduced molecular weights of 72,728 and 72,805,

respec-l a I I r I%f I . I., m .

km

-.

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5620 ZHANG ET AL.

gaattcaccattggcgatgatgactcactgccaatggaacccctgcggttcctcagcacgcagatccaggatgacattgtcctcctcgatatccgcgacaacaccaagtggcttgatgcc

ggcgtggtcagcttcgcagcggactggtccttcagtttcgacgtcggcatgagetttctggaaatccacggcccggtgccccgggtcaaggaagaaaacatcatccagcgtgccgaacag

ttcctcatgcgcctccagccgggcgagcaattccgccgcaccaactggaccatgaccgtgggtcgccgcttggacacctccaccgagacctatcccgaatggggaccggaccgcggcacc

* * * * $ * * * $ $ * *

atcgccacggatcccgagatgccggacaagctccacctccgcgtcgaagtccagcacttggtccgccttccgcacaccggcaccctgctgttcctgatccgcagctacctcctaccactc

attcaaaaaaggcctcgcataaaccttttgagtggcgtagttttttcacacaga

-43

region -35 region

-10

region

A1

aaggacatcgcccaggttccggcctggcgcgagaggttcggcaacatcctggccgaactgccccaagatatggccgattacaagggcatcaccaattaccggaaggcagcctccgaatgg

ronZaornntnartaratararPratnnerteernnnantearenerentntartennrttrararaetnteenannnrenntntnonrezznnro+1

VA-~~*u9"b v*uVuUvu *"W*wU*UU99 ""9ib;9U"X*99"999*L*Lg, *LUWL*~*Wayua LyL*Wayyvy *yL~*yvyIaayy *vtALL LU

120

240 360 480

6

600 iorl

aoxI

ctgtc_gccggctaaacaacacataaaaracaaaaaacaagattagtcGTGAC

ATGCTGATCTGAG

GTGGGTGM

CGTTGGATCTTGTCTGTG

720 alloxII

ccgtttacaacagggatccacgtcccttc_c___ccaggaa

aga

gttATGACMGAATGCTGAATCTGAGGCTTTGGTGGGTG

MCGCACCCGTTGGATCCGTTGTCGCGTGTG

246 MaoxI

S/D

ValThrLeuAsnAlaGluSerGluAlaLeuValGlyValSerHisProLeuAspProLeuSerArgVal

23

MaoxII

METThrLeuAsnAlaGluSerGluAlaLeuValGlyValSerHisProLeuAspProLeuSerArgVal

23

I I

IaoxIII

GAGATTGCGCGTGCGGTGGCGATCTTGAAG

CCTGCTGC

GTCGTTCCGGMATCAGTGTGGAGTTGCGTGAGCCGTCCAAGGATGA

MCGCC

TTGCGGTG

840/366

MaoxI/II

Gl

uIleAlaArgAlaValAlaIleLeuLysGluGlyProAlaAlaAlaGluSerPheArgPheIleSerValGluLeuArgG1uProSerLysAspAspLeuArgAlaGlyValAlaVaI

63/63

aaoxI/II

GCCCGTGAGGCTGACGCTGTGTTGGTTGATCGTGCGCAC CGTTCG

AGGCTGTTGTTGATCTTGAGCGGGACGGTGGATTCGTGAGCTGTTGGCCGAGAACATCCAGCCG

960/486

MaoxI/II

AlaArgGluAlaAspAlaVal LeuValAspArgAlaGI nAlaArgSerPheGluAlaValValAspLeuGl uAlaGlyThrValAspSerTrpLysLeuLeuAlaGluAsnIleGlnPro 103/103

aoxl/II

CCGTTCATGTTGGATGAGTTCG

ATGGAGGACGCTGCAAGCCGTCATCGCGGCGTTGUACTGGCCTGACCMCCTGGACCTGGTCTGMMACTGG

1080/606

Maoxl/II

ProPheMETLeuAspGluPheAlaGluCysGIuAspAlaCysArgLysAspProGluVal

IleAlaAlaLeuAlaLysArgGlyLeuThrAsnLeuAspLeuValCysPheGl

uProTrp

143/143

aoxI/II

TCCGTGGGGTAAMCCGGTGAGACAAC1200/CGMGATGCGTGCGCTGGTGTTCGTCGTGACG

TGATGATTUCCGTACGC

CCCGAT

CTTCATTGTMC

1200/726

Maoxl/II

SerValGlyTyrPheGlyGluAspAsnGluGlyArgArgLeuMETArgAlaLeuValPheValArgAspGluAlaAspAspSerProTyrAlaHisProIleGluAsnPheIleValPhe

183/183

aoxl/II

TACGA

TGAACGCCGGCAAGGTGGTCCGTCTCGMGACGACCGGCCATCCGGTGCTTTCCGCGGGGGTACTACCTGCCCAAGTACGTCGGTGMGCCGCACGGAMTGAGG

1320/846

MaoxI/II

TyrAspLeuAsnAlaGlyLysValValArgLeuGluAspAspGlnAlaIleProValProSerAlaArgGlyAsnTyrLeuProLysTyrValGlyGluAlaArgThrAspLeuLysPro

223/223

I

aoxI/IIH

TTG1/CATCACCCAGCCCGAA966TCTTCACGGTCACGGGTMCCACGTCACGTGGGCTGACTGGTCTTCCGGGTCGGGTTCACCCCGCGTGAGGGCTGGTGCTGCACAGCTC

1440/966

Maoxl/II

LeuAsnIleThrGlnProGl

uGlyAlaSerPheThrValThrGlyAsnHisValThrTrpAlaAspTrpSerPheArgValGlyPheThrProArgGluGlyLeuVal

LeuHisGlnLeu

263/263

aoxl/II

AAGTTCAAGGACCA150TGGACCGTCCGGTGATCAACCGTGCTTCGCTCTCGGATGGTCGTCC

CTACGGTGACACGGCC CGGTCTTCGACTCGGGC

1560/1086

MaoxI/II

LysPheLysAspGlnGlyValAspArgProVal

IleAsnArgAlaSerLeuSerGluMETValValProTyrGlyAspThrAlaProValGlnAlaLysLysAsnAlaPheAspSerGly

303/303

I

maoxI/II

GAGTACAACATCGGCAACATGGCC2CTCCCTGACTGGGTTGTGACTGCCTGGGTGAGATCAAGTACTTCGACGGTCATTCCGTGGATTCCCAC

U

CGTGGACATCAGAAC

1680/1206

MaoxI/II

GluTyrAsnIleGlyAsn3ETAlaAsnSerLeuThrLeuGlyCysAspCysLeuGlyGluIleLysTyrPheAspG4yHisSerValAspSerHisGlyAsnProTrpThrIleGluAsn

43/343

aoxI/II

GCGATCTGCATGCACGAAGAAGACGACTCGATCCTGTGGAAGCACTTCGACTTCCGCG

CCGAGACACGCCGGTCCC

AACTCGTGAMCCTTCATCUCGGTCGC

1800/1326

MaoxI/II

AlaIleCysMETHi

sGluGluAspAspSerIleLeuTrpLysHisPheAspPheArgGluGlyThrAlaGluThrArgArgSerArgLsLeuVal

IleSerPheIleAlaThrValAla

383/383

aaoxI/II

4CTACGAGTACGCGTTCTACTGGCACCTGTTCCTCGACGGGTCATTGAGTTCTGGTCAAGGCCACGGCATCCMUACCGCCGCAACTGC

TGAG

CCCGTATGGC

1920/1446

Maox/I/I

AsnTyrGluTyrAlaPheTyrTrpHiisLeuPheLeuAspGlySerIleGluPheLeuVal

LysAlaThrGlylleLeuSerThrAlaGlyGlnLeuProGlyGluLysAsnProTyrGly

423/423

ttt

AaoxI/II2

CAGTCGTTGAA2

5CGGCCTCTA6CCCATCCACCAACACATGTTCM0CGTC

TGGACTTCGAACTCGACGGGGTU

CGCCGTCTAMGTGCTGGAATAC

2040/1566

MaoxI/II

GI

nSerLeuAsnAsnAspGlyLeuTyrAlaProIleHisGlnHisNETPheAsnValArgMETAspPheGluLeuAspGlyVal LysAsnAlaValTyrGluValAsMETGluTyrPro

463/463

I/aoxI/II

GAGCACAACCCCACCGGCACCGCGTTCATGCTGGACCGmGcTCGAACCGAGCAGAAAGCATCCGCAAAACGAACGAGCAAGCACCGTTCT

ATCGCGACCAA

2160/1686

Maoxl/II

Gl

uHisAsnProThrGlyThrAlaPheMETAlaValAspArgLeuLeuGluThrGluGlnLysAlalleArgLysThrAsnGl

uAlaLysHisArgPheTrpLysIleAlaAsnHisGlu

503/503

IldOX

Iaoxl

iaoxI

iaoxl

aaoxII

aaoxI

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METHYLAMINE OXIDASE FROM ARTHROBACTER STRAIN P1 5621

Iaox/IITCMG

CCTCGTCAACG

TCGCCTACGGCTCATCCACAACGGCATCAACTC

G

CGAC

TACGTCTUACGCG

TTC

CGGAACAATC

2280/1806

MaoxI/II SerLysAsnLeuValAsnGl

uProValAlaTyrArgLeuIleProThrAsnGlyIleGlnLeuAlaAlaArgAspAspAlaTyrValSerLysArgAlaGl nPheAlaArgAsnAsnLeu 543/543

* I * * I * * I $ $ I *

saox/IITGGGTCACG

TACGACCGCACGA

CGCTTCGCWUG

ATACCCCAACCMGCCAUG

CCGACGACGGCTGCACATCTGGACC

CCGCAACATCGTCGATACC 2400/1926

Maoxl/II

TrpValThrAlaTyrAspArgThrGluArgPheAlaAlaGlyGluTyrProAsnGlnAlaThrGlyAlaAspAspGlyLeuHisIleTrpThrGlnLysAspArgAsnIleValAspThr

583/583

aoxI/II

GA20TCGTGGTCTGGTACCCTTCGGCATGCACCACGTCGTCGCCTCGAAGACTGGCCCGTGATGCUCGCCAAAACATCGGCTTCATGCTCGAAUCCACGGCTTMCMCCMMC

2520/2046

Maoxl/II

AspLeuValValTrpTyrThrPheGlynETHisHisValValArgLeuGluAspTrpProValMETProArgGlnAsnIleGlyPhelETLeuGluProHisGlyPhePheAsnGInAsn

623/623

* I * * I * * I * * 1 *

CCCACCCTCMACCTCCCCACCAGCACCAGCAC

ACCCAAACGGTGAAGCTGACACCTGCTWCAGWAMClGATMccagggccaacaaggactggcgcggaatccaaaccaccgcgc

CCCACCCTC4AU

UACCT

AACC\CACGT4GCGCCTCGCCCGCGAGgggttttcgcgccgctcatcaaccagagttcggtccccCgtg

ProThrLeuAsnLeuProThrSerThrSerThrThrGl

nThrGlyGluAlaAspThrCysCysHisAsnGlyLys***

---ProThrLeuAsnLeuProThrSerThrSerThrThrGlnThrGlyGl uAlaAspThrCysCysHisThrAspLys$

cagtccatcctgggcgcctcctccaggggctctccgtaaacccgggcgggtgaagacccgcccctgaccccggtccccggtgcgacctccagcagtaccgggaaccgaacctttccctaa

ctgctgtcagtgccggggaccgattccaccttcttagtcctcatata

2640

2166

648

648 2760

2213

* * * * *~~

aaagacttcggtccccggtgctgttggtagccgtgccggggaccggatccacttaaaaa

2819

FIG. 2. Nucleotide andaminoacidsequencesofmaoxIandmaoxII(indicatedatthestartofeachline). Regionswithidentical nucleotide

or aminoacidsequencesareindicatedbymaoxI/IIorMaoXI/MaoXII, respectively.The numbersatthe end of each line refertonucleotide

oramino acidpositionsin thesequences.Thenucleotides ofmaoxIarenumberedstartingwith +1atthe far 5' end. Thenucleotides ofmaoxII

arenumbered with the firsttranscriptional initiation site (cytidine)as +1. The nucleotides in the5'-flanking regionofmaoxIIare given negative numbers.Theasterisks markeveiy10thnucleotide, startingwithnucleotide+1of themaoxIsequence,while theverticallines mark every10th aminoacylresidue in thesequence.Thetranscriptioninitiationsites ofmaoxIIaremarked withtriangles.Theputativepromoter

elements in the -43, -35, and -10 regionsofmaoxIIare soindicated under thecorresponding sequences. S/Drepresents theputative Shine-Dalgarno sequences. Previouslydetermined amino acid sequences are indicated by double underlining. The topa quinone site is indicatedbyvertical arrowsat position 385. The synthesized oligonucleotideused for theprimer extension assay isshown withsingle underlining, justtothe3' side of thestartof thecoding regions.Thetranslationalstopsitesarerepresented by***.Thestem-loopstructures

in3'-flanking regionsareindicated with horizontalarrows.

tively (Fig. 2). The known amino acid sequences of three

peptides from Arthrobacter strain P1methylamine oxidase correspond exactlytoregionsofMaoxI and MaoxII marked in Fig. 2. These identities suggest that either maoxI or

maoaxII could code for methylamine oxidase. The N-terminal amino acid of methylamine oxidase is leucine, which is encoded by TTG. For maoxII, a methionine codon was

found nine codonsupstreamfrom the leucinecodon;

how-ever,formaoxI, there isnomethioninecodon foundnearby andinframein thisregion of its nucleotidesequence.The 3' end of a Shine-Dalgarno (36) sequence, AGGAGT, was

identified 33 bp upstream from the TTG codon for both

genes.AGTGvalinecodon which followed5bases after the

end of the AGGAGT sequence could be employed as a

translational initiation codon inmaoxI(Fig. 2). Two stem-loopstructureswerefound downstreamof the translational stopcodon formaoxI. The first stem-loopstructure,located 14 bp downstream from the stop codon, runs from base 2612 to base 2646 and has a 12-base pairing stem and an

11-base loop (-30.6 kcal [ca. -128 kJJ mol-V) (Fig. 2). The second stem-loop structure occurs frombases 2769 to 2805, has a stem of 10bp and a 17-base loop (-29.8 kcal

[ca. -125 kJ] mol-1), and is followed by five adenine residues(Fig. 2). Incontrast,there isonlyasingle stem-loop

structure in the 3'-adjoining region of maoxII. It is 29 bp downstream fromthe translational stopcodon, spansbases

2154to 2189, and hasan11-basepairingstemanda14-base

loop (-32.0 kcal [ca. -134

kJJ

mol-1) (Fig. 2). These stem-loop structures presumably serve as transcriptional

terminators.

Southern blot analysis of Arthrobacter strain P1 chromo-somal DNA.Chromosomal DNA isolated from Arthrobacter strain P1wasdigested with the restrictionenzymesEcoRI,

HindIII,

KjpnI,

BamHI, andApaI, inseparateexperiments. A Southern blot was probed with a 1.5-kbp BamHI-SmaI

restrictionfragment from maoaxI (Fig. 1) which containspart of thecoding region that is identical for thetwomaoxgenes.

Asshown inFig. 3,twocross-hybridization bands of differ-entsizes androughly equal intensitieswerefound in all the

digests. Since the DNA sequence of the probe does not contain any restriction site recognition sequences of the

enzymes used for digestion, the Southern blot analysis indicates thatArthrobacter strain P1 has two homologous

genes.Thus, eitherone orboth could code formethylamine oxidase.

maoxII encodes methylamine oxidase. Total RNA was

isolated from Arthrobacter strain P1 grown on minimal

mediumsupplemented with methylamineasthe sole carbon

andenergy source.Northern blotanalysiswascarriedoutby using two small DNA fragments as probes; in order to preventanypossibility of cross-hybridization, the fragments were isolated from maoxI and maoaxII in regions where no

significant homology between the two genes was found.

These twoprobesspan +373to +609 inmaoxI and -51 to

ixxI

maoxII

MaoxI

MaoxII

iaoxI

udaoxl

iaoxi

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5622 ZHANG ET AL. .> 3 0 tL 23.1kbp-U 9.4kbp 6.Skbp-4.3kbp 2.3kbp -2.0Okbp 1 2 34 5

FIG. 3. Southern blotanalysisof Arthrobacter strain P1 chromo-somal DNA. The chromosomal DNAwas digested with the indi-cated restriction endonucleases. The blot was probed with the nonradioactivelylabeled1.5-kbpBamHI-SmaI restrictionfragment frommaoxI(Fig. 1), which contains thepart of thecoding region that is identical inmaoxIandnaotxIL

+139 in maoxII(Fig. 2). As illustrated in Fig. 4, oneband

was identified with theprobefrommaaxIIbutnotwith the probefrommaoxI(lanes1and2).This band shows thesame sizeasthatprobedwith the1.5-kbpBamHI-SmaI restriction

fragment from maoxI, a fragment containing the identical

coding region of thetwo genes (Fig. 4, lane3). This result indicates that methylamineoxidaseis encodedbymaoxII.

Transcriptional analysisofmaoxII.Tolocate the

transcrip-tion startsite, total RNA isolated frommethylamine-grown

Arthrobacter strain P1 wasanalyzed bytheprimerextension method. A 27-mer oligonucleotide complementary to the

2 3

FIG. 4. Northern blot analysis with probes specific for nmaotxI

and maaxII. Total RNA was isolated from methylamine-grown Arthrobacter strain P1 cells. Twenty micrograms of RNA was

loaded in each lane. The RNA in each lanewashybridizedwitha

differentnonradioactivelylabeled DNAfragment. The probe used for lane 1wasspecificformaoxII,spanningfrom -51to+139. The probeused for lane 2contains sequence between +373 and +609,

specificformaoxI(Fig. 2).There isnosignificant homologybetween thesetwoprobes. Lane 3wasprobedwitha1.5-kbpBamHI-Saml

restrictionfragment, which containsasequence that is identical in

maoxIandmaoxII.Thepositivebandis indicated withan affow.

0 \

~~~A-T

* \

~~~A-1>

1 2

345 5'

FIG. 5. Localization of themaoxlltranscription initiation site by primer extensionanalysis. RNA isolated from methylamine-grown Arthrobacter strain P1 cellswasused forprimer extension assay. Lane 5carries theprimer extension product. Lanes 1, 2, 3, and4are

sequenceanalyses ofmaaxIIcarriedoutwith thesameprimer used forprimer extensionassay.Thesequenceof theregion is shownon theright for clarity. The transcriptional initiation sitesareindicated witharrows.

sequence 5'-GGATCCGTTGTCGCGTGTGGAGATTGC-3' (Fig. 2) was synthesized and used for a primer. The three transcriptionalstartsites of maotxH were found at acytidine and two adenine residues located 176, 175, and 173 bp upstream from the translational start site(Fig.2 and5).The

5

4

3

2

1 5432

FIG. 6. NorthernblotanalysisofRNAisolated from

Arhrobac-terstrainP1 grownondifferent substrates. Tlwentymicrogramsof RNAisolated fromcellsgrowninthepresence of 0.3%methylamine (lane 1), 0.3%

13-phenylethylamine

(lane 2), 0.3% choline chloride

(lane 3),or 0.2% glucose (lane 4) or in LB medium (lane 5)was

loaded. Theblotwashybridizedwith thenonradioactively labeled BamHI-SamlrestrictionfragmentofmnaoxI.Thesizeof thepositive

bandisindicatedatthe right.

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8 7 6 5 4 3 2 1 glucose+ + + + + + + +

LBmedium methylamin. +

FIG. 7. Immunoblot analysisofproteins isolated from Arthro-bacter strain P1 grown on different substrates. Lanes: 1, LB medium; 2,LBmediumplus0.3%methylamine; 3,LB mediumplus

0.6%methylamine; 4, glucose; 5, glucose plus 0.3% methylamine; 6, glucose plus 0.6% methylamine; 7, 0.6% methylamine. The same

amountof cellextractwasloaded in eachlane. Lane 8 carries 50ng

ofpurified methylamineoxidase fromArthrobacterstrain P1. Im-munostainingwasperformed with antibodies against methylamine

oxidase.

putative -43 (AAAAAA), -35 (TCGCAT), and -10 (TAGTTI)regions, whicharepossibleregulatory elements, werefoundupstreamfrom thetranscriptionalstartsite(Fig. 2). Another Northern blot analysiswascarriedoutwithtotal RNAprepared frommethylamine-grownArthrobacterstrain P1.The1.5-kbpBamHI-SmaI restriction DNA fragmentwas

usedastheprobe. As illustratedinFig. 6,onebandatabout 2.2 kbwasobserved. This result indicates that thetranscript

ofmaoxII is monocistronic.

Induction ofmethylamine oxidase

(maarxi)

geneexpression.

Methylamine oxidase is producedinverylarge quantities in

methylamine-grown Arthrobacter strain P1 cells (22). To study the induction of methylamine oxidase gene

expres-sion, total RNA was isolated from P1 cells grown under different culture conditions. A Northern blot was probed with the 1.5-kbp BamHI-SmaI restriction DNA fragment (Fig. 1), andapositive band of the expected 2.2-kb size for

themacxIItranscriptwas detected for the RNA of

methyl-amine-grown cells (Fig. 6, lane 1). A second band at1.3 kb

was alsoseen. It is assumedthat it results from(nuclease?) degradation of the 2.2-kb transcript. The formation of a

smallermaocxIItranscriptorcross-hybridization of the probe

with another gene transcript (not macxl) cannot be ruled out. No significant amount of transcript was seen for the

cellsgrown on,B-phenylethylamine,choline,glucose,orLB

medium (Fig. 6, lanes 2 to 5, respectively). Immunoblot analysis was carried out with antibodies raised against purifiedmethylamine oxidase of Arthrobacter strain P1. As shown inFig. 7, methylamineasthesole carbon andenergy sourceisableto inducehigh levels of methylamine oxidase (lane 7). However, LBmediumsupplemented with methyl-aminedoesnotinducehigh levelsoftheenzyme(lanes 1to 3). A high level ofenzyme wasalsodetected from the cells grownonminimal mediumsupplemented with both glucose

andmethylamine (Fig. 7, lanes 5 and 6).

Expression ofmaoxland maoxlI in E. coli. The 2.8-kbp

DNA fragment containing matcxI and the 2.5-kbp DNA fragment containing maoaxIIwere subcloned in the pGEM-7Zf(+) vector and transformed into E. coli HB101. Both fragments contain putative promoter regions, entire coding regions, and 3'-flanking regions. Proteinwas extracted and

subjected to immunoblot analysis with the polyclonal anti-bodies against methylamine oxidase from Arthrobacter

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

FIG. 8. Immunoblot analysis ofmethylamine oxidase from E. coli andArthrobacter strain P1. E. coli and P1weregrownunder

different culture conditions as indicated. Protein samples were

obtained from E. colicarrying either the unmodifiedvector(lanes1, 2, 9,and10),thevectorcontaining the 2.8-kbp DNA fragment from maoxI(lanes 3, 4, 11, and 12),orthevectorcontaining the2.5-kbp DNAfragmentfrom maoxII(lanes5, 6, 13, and 14). Proteinsamples isolated from Arthrobacter strain P1wereloaded in lanes 7, 8, 15, and16. Thesameamountofcellextractwasloaded in each lane.

Lane17 contains 50ngofpurifiedmethylamine oxidase of Arthro-bacter strain P1. Immunostainingwas performed with antibodies

against methylamine oxidase.

strainP1.AsillustratedinFig. 8,oneband ispresentin cell extracts of E. coli harboring eithermaoxIormaoxHl. The

bands represent proteins with the same subunit molecular

weight

as

displayed by

methylamine oxidase purified from ArthrobacterstrainP1 (Fig. 8,lanes 3to6, 8, 11to 14, and 17).No corresponding bandwasobservedfor the cellextract of E. colicarrying the unmodifiedvector(Fig. 8, lanes 1,2,

9,

and

10).

Additionally,E. coli cells harboring maoaxIand maocxIIweregrown onLB mediumcontainingmethylamine

or on minimal medium supplemented with glucose and

methylamine (Fig. 8,lanes 3to6 and11to14). Thepresence

ofmethylamine does not elevate the level of methylamine oxidase production by E. coli. Compare this with the results obtained with Arthrobacter strain P1 (Fig. 8, lanes 7, 8, 15, and16)grownonmethylamine in minimal medium

contain-ing glucose

or onLB medium.

DISCUSSION

Analysis

and comparison of the nucleotide sequences and

translated amino acidsequencesofmaoxland maoxII. Inour

endeavor to clone and sequence the structural gene for

methylamine oxidase from Arthrobacter strain P1,we

hap-pened to identify two highly homologous open reading

frames that we designated macxI and macxHl. While it seemed that either of these genes could code for

methyl-amine oxidase, a Northern blot analysis of RNA isolated from cellsgrownonmethylamineasthe sole carbonsource

indicated that methylamine oxidase is encoded only by maaxII. Another Northern blot analysis showed that

tran-scriptsofmaaxIIweredetected only from the cellsgrownon

methylamine, not from the cells grown on other tested

carbon sources (Fig. 6). This result suggests that only methylamine caninducemaaxIIexpression, and induction

possibly occurs by elevating transcription initiation or by

increasingmRNAstability.

The nucleotide sequences ofmaoxI and maoxIIare

pro-vided inFig. 2. The sequences aregreaterthan99% identi-cal. There isnosignificanthomologyupstreamof the trans-lationalstartsites. Thedifferences in the putativepromoter regions are probably required for differential regulation of

+4+4+ + ++

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5624 ZHANG ET AL.

Lentil

---kf

2 MaoxII

mt---

2 Hansenula

ME---

2 K(1ebsiella

mangi

kfsprktalalavavvcawqspvfaHGSEAHMVPLDKTLQEFGADVQWDDYAQMFTLIKDGAYVKVKPGAKTAIVNGKSLELPVPVVMKEGKAWV

100

0 6oX

0 0

0000

00 o0oo

0 O0

00

0 OX

Lentil

alfsvltllsfhavf_sFTPLHTHPLDPITKEEFLAV_TIV1NKYPISNNKLAFHYIGVDDPEKDLVLKYETSPTLISI_PRKIFVVAIINSTH--EIlI

100 MaoxII

---1

naeseaLVGVSHPLDPLSRVEIARAVAILKEG-PAAAESFRFISVELREPSKDDL---RAGVA-VAREADAVLVDRAQARSFEAVV

84 Hansenula

---RLRQIASQATAASAAPARPAHPLDPLSTAEIKAATNTVK-S-YFAGKKISFNTVTLREPARKAYIQWKEQGGPL-PPRLAYYVI

LEAGKPGVKEGLV

96 Kiebsiella

SDTFINDVF1SGLD4TFQVEKRPHPLNSLSAAEISKAVTIVKAA-PEF9PNTRFTEISLHEPDKAAVWAFALQGTPVDAPRTADVVlDGKH--VIEAVV

149

00 *oo

X

0ooo

o

o0

o

o0

o oo

0 of00o

0 0

00oo0

U

00O

0 0

X000 0 0 0 0 0

0

00 00400

0 1000000OXOOO00OO

Lentil

DLTIKSIVSDNIHNGYGFPVLSAAEQFLAIDLPLKYPPFIAS---VNKRGL-NISEIVCSSFTWFGE----EKNSRTVR-VDCFMKESTVNIYVRPIT

191 MaoxII

DLEA-GTVDSKLLAENIQPPF78LDEFAECEDACRKDPEVIAA--LAKRGLTNLDLVCFEPWSVGYFGEDN---EGRRLlRALVFVRDEADDSPYAHPIE

178 HIansenula

DlASlSVIETRAL--ETVQPIlTVEDlCSTEEVIRNDPAVIEQCVlSGIPANEMHKVYCDPWTIGYDERWG9---TGKRL0QALVYYRSDEDDS0YSHPLD

190 Kilebsiella

DL2NKKILSWPI--KGAHG9VllDDFVSVQNIINTSSEFAE--VLKKHGITDPGKVVTTPLTVGFFDGKDGLQQDARllKVVSTYLDTGDGNYWAHPIE

293 UX

0 0

0 00 0

000 X

O0

0

0 00 0

00000 0

Of

O0X

0X000

XIX

0

10 OX

00

0

0 O0

0000X00000

0 00X000IX

000 X

Lentil

GITIVADLDLMKIVEYHDRDTEAVPTA-ENTEY---QVSKQSPPFGPKQHSLTSHQP8GPGFQINGTSVSWAN7KFHIGFDVRAGIVISLASIYDLEKHK

287 MaoxlI

NFIVFYDlNAGKVVRlE--DD2AIPVPSARGNYlPKY2----

VGEARTDLKPLNITQPEGASFTVTGNHVTWADWSFRVGFTPREGLVLHQLK-FK-DQGV

270 Hansenula

-FCPIVDTEEKKVIFI DI

PNRRRKVSKHKHANFYPKHMIEKVGAMRPEAPPINVTQPEGVSFKMTGNVMEWSNFKFHIGFNYREGIVLSDVS-YN-DHGN

288 K(iebsiella

NLVAVVDLEAKKIIKIE--EGPVIPVPMEPRPY---DGRDRNAPAVKPLEITEPEGKNYTITGDTIHWQNWDFHLRLNSRVGPILSTVT-YN-DNGT

383

0 0XOlOO

00 0 0

0 X

Xool0060XX000Xl00000000

o0000f

OXO

oX

0 00 X0

I 0

00

00 0000

00000000

000 0

0 Lentil

SRRVLYKGYISELFVPY3DPTEEFYFKTFFDSGEFGFGLSTVSLIPNRDCPPHA3FIDTYIHSADGTPIFlENAICVFEQYGNIMWRHTETGIPNESIEE

387 MaoxII

DRPVINRASLSEMVVPYGDTAPVQAKKNAFDSGEYNIGNMANSLTLGCDCLGEIKYFDGHSVDSHGNPWTIENAICMHEEDDSI

LWKHFDFR-E--GTAE

367 Hansenula

VRPIFHRISLSEMIVPYGSPEFPHQRKHALDIGEYGAGYMTNPLSLGCDCKGVIHYLDAHFSDRAGDPITVKNAVCIHEEDDGLLFKHSDFR-DNFATSL

387 Kilebsiella KRQV4YEGSLGG7IVPYGDPDVGWYFKAYLDSGDYG9GTlTSPIVRGKDAPSNAVllDETIADYTGKPTTIPGAVAIFERYAGPEYKHLE7G-K---PNV

479 O0XWX00WX0

0

0 of

I0000X

IX

I

000

00 00 000000000

0000

00

I

.

V

X0000000oo00o

oo

0

00 00o00ooo0

00

0 Lentil

SRTEVDlAIRTVVTVGNYDNVLDWEFKTSGW4KPSIALSGIlEIKGTNIK

----8HKEIKEEIHGKLVSANSIGIYHDHFYIYYLDFDIDGTQNSFEKTS

483 MaoxII

TRRSRKLVISFIATVANYEYAFYWHLFLDGSIEFLVKATGI

---

LST--AGQLPGEKNPYGQSLNNDGLYAPIHQHMFNVRMDFELDGVKNAVYEVD

459 Hansenula

VTRATKLVVSQIFTAANYEYCLYWVFMQDGAIRLDIRLTGI

---

LNTYILGD-DEEAGPWGTRVYPN-VNAHNHQHLFSLRIDPRIDGDGNSAAACD

479 Kiebsiel

la

STERRELVYVRWISTVGNYDYI

FDWVFHDNGTIGIDAGATGIQAVKGVLAKTMHDPSAKEDTRYGT- LIDHNIVGTTHQHIYNFRLDLDVDGENNTLVAND

578 OXOOXW00000 0000

0 of

to

0 X

0000

0

000

0 fo

10

0

0 X

X O

0X

0

00 OO

X

O

00

0

000 Lentil

LKTVRIVDEVQEKSYWTT-ETQTAKTESDAKITIGLAPAEL----VVVNPNIKTAVGNEVGYRLIPAIPAHPLLTEDD-YPQI---RGAFTNYNVWVT

572 MaoxII

ME---YPEHNPT---GTAFMAVDRLLETEQKAIRKTNEAKHRFWKI-ANHESKNLVNEPVAYRLIP-TNGIQLAARDDAYVSK---RAQFARNNLWVT

546 Hansenula

AKSSPYPLGSPENMYGNAFYSEKTTFKTVKDSLTNYESATGRSWDIFNPNKVNPYSGKPPSYKLVS-TQCPPLLAKEGSLVAK---RAPWASHSVNVV

577 Kiebsiella PEVKPNTAGGPR---7TSTQVNQYTIDSEQKAAEKFDPGTIR---LLSNTSKENRGNPVSYQIIPYAGGTHPAATGAKFAPDEWIYHRLSFNDKQLWVT

672 O

O

of

000

0 XXOO

00

00 0XO0

XX

O

0 OX

0

0 0000

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00000

of

Q---IIELKNGL---VDFMLI---:--AYDRTERFAAGEYPNQATGAD-DGLHIWTQKDRNIVD-TDLVVWYTFGMHHVVRLEDWPVMPRQNIGFMLEPHGFFNQNPTLNLPTSTSTTQTGE

----PYKDNRLYPSGDHVPQWSGDGVRGMREWIGDGSENIDNTDILFFHTFGITHFPAPEDFPLMPAEPITLMLRPRHFFTENPGLDIQPSYAMTTSEAKRAVH

RYHDTERYPEGKYPNRSAHD--TGLGQYAKDDESLTNHDDVV-WITTGTTHVARAEEWPIMPTEWALALLKPWNFFDETPTL---GEKK---100 0OF0 000

0

00 O0000XO00

0 lOolW0

ol 0WX

00 ---ADTCCHTDK

KETKDKTSRLAFEGSCC--GK

---K

-K~~~~~ 587 635 673

751

648 692 756

FIG. 9. Comparisonof theaminoacidsequencesof severalcopper/topaquinone-containingamine oxidases. Allsequenceswerederived by translation of the cloned gene sequences of the respective proteins. Sequences: MaoxII, methylamine oxidase from gram-positive Arthrobacter strain P1; Lentil, lentil (L. culinaris) seedling diamine oxidase (33);Hansenula, methylamine oxidase from the yeastH.

polymorpha (6); Kiebsiella, tyramineoxidase fromgram-negativeKaerogenes(37). Symbols: 0,conservedamino acid ineachsequence;

0,conservativechangeamongthesequences;

T,

conservedhistidylresidueineachsequence;

I

,topaquinonein thematureproteins.The

symbols above eachgroupresult fromaClustalanalysisofall foursequences.Thesymbolsbelowresult fromaClustalanalysisofthe lower threesequencesfrom themicroorganisms.Thelowercase,underlined letters indicatepresequencesorputativepresequences,whicharenot presentin thematureproteins.

expressionof the two genes.Nucleotidesequences nearthe C-terminus codons and downstream from the stop codons arealso different forthe twogenes, with theexception that the sequences of the stem part of the maoxII stem-loop structureis conserved in the second stem-loopstructure of maoxI(Fig.2). Thededuced aminoacidsequencesofmaoxI and maoxII are

identical,

exceptthatAsn-646and

Gly-647

in MaoxI are

replaced

byThr-646 andAsp-647in MaoxII

(Fig.

2).

The high homology in the coding regions of these two genes suggests that thegenes originated relatively recently from an ancestor gene by duplication. This raises some questions. Why are there two so

closely

related genes presentinArthrobacter strainP1? IsmaaxIinducible? Ifso, whichalkylamine isrequired for

induction,

and willP1 grow on this amine?

(Recall

that methylamine does not induce maoxIexpression

[Fig. 4].)

Are thereanyfunctional differ-encesbetween the twogene products?

Similar duplication ofthe structuralgenes inE. coli has beenreported.Forexample,E. coliK-12 has twostructural genes (argF and

argl)

for ornithine carbamoyl-transferase (3, 18, 38). High homologywasobservedforargF andargI (78.1%atthe nucleotidelevel, 86%atthe aminoacidlevel). The two gene products associate to form four functional catalytic trimers, designated FFF, FFI, FII, and III. The FFFand IIIisozymes exhibit nearly identical kinetic

param-TABLE 1. Arthrobacter promoter sequences Sequence Promoter

-35region -10region Reference 6-Hydroxy-D-nicotine oxidase TTGACA TATCAAT 4,21

(A. oxidans)

D-Xylose(D-glucose)isomerase TTGACA TATAGTT 20 (Arthrobacter strainNRRL

B3728

ermA(Arthrobacter sp.) TCGGAC TATCCT 32 Methylamine oxidase TCGCAT TAGT'T This study

(MaoxII)(Arthrobacter

strainP1)

eters but differ in physical characteristics such as heat

stability. Translation elongation factor EF-Tu of E. coli is encodedbytwostructuralgenes,tufA and

tuJB

(2, 39). The

nucleotide sequences of these two genes are also highly

homologous. The amino acidsequences ofthese twogene

products are identical exceptfor several C-terminal amino acids. Since the two EF-Tu genes are functionally and

structurallyindistinguishable (10, 29), itwas suggestedthat

theadditionaltufgeneisrequiredtosupplyextraEF-Tu for

emergency requirements. The regulatory mechanisms

re-sponsible for the induction of maoxI and maoxII expression

areunderstudy. Whether therearefunctional and structural

differences between theproducts of thesetwogenesremains

tobeseen.

One adenine-rich sequence (AAAAAA) was centered at -43 formaoxII(Fig. 2). Thissequencehasbeen reportedto be present in the promoter region of the 6-hydroxy-D-nicotine oxidase gene of Arthrobacteroxidans (4). It has

beenproposed that this consensus sequenceis involved in the function of certain promoters (17). The -35 region TCGCAT andapotential -10 region TAGTIT (Fig. 2)are

separated by a usual space of 17 nucleotides (31). The comparison of these two putative promoter elements of maaxII with those found ingenesfromvariousArthrobacter

species shows modesthomology in the -35 region and the -10region (Table 1).

Comparison of amino acid sequences ofcopper-containing

quinoproteins. The amino acid sequence adjacent to the MaoxII cofactor site was identified by comparison of the translated sequence ofmaoxII with sequences of known

cofactor sites for other copper/topa quinone-containing amine oxidases (16, 27). The methylamine oxidase tyrosyl residue, which is convertedto topa quinone, is located at residue 376 from the N-terminal leucine in the mature protein. Table 2 presents a comparison of amino acid

se-quencesofvariousoxidasesatthecofactorregions of each. Figure 9 provides a comparison of the complete amino

acidsequencesoffourcopper/topa quinone-containing oxi-dases. The tyramine oxidase from K aerogenes was not originally identified as an amine oxidase of this type (37). However, our comparison of the sequence of this protein

Lentil

MaoxII

Hansenula

K(iebsie/la

MaoxII

Hlansenula

Kiebsie7/a

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5626 ZHANG ET AL.

TABLE 2. Amino acidsequences adjacent to the cofactor sites of several amine oxidases

Source (enzyme) Sequencea Reference

Bovine plasma (monoamine oxidase)b -VYSIMLXoYVXDMVE YPNQAIE- 27

Porcine serum (monoamineoxidase)b -SYSTMLNgDYvxDMIEHP- 16

H.polymorpha (monoamineoxidase)c

-QIFTAAmYCLYWV!XlQDQAIR-

6

Kaerogenes(monoamine oxidase)c -WISTVGMYIFDWVEHDNQTIG- 37

MaoxII (monoamineoxidase)c

-FIATVAMYAFYWHLFLDaSIE-

This study

Porcine kidney (diamineoxidase)b

-(T/D)SJTTIiDYIXDFIEYY-

16

Pea seedling(diamine oxidase)b -VGNgDNIAXD- 16

Lentil seedling(diamineoxidase)c -TVYJTVGMXDNVLDWEFKTS.QW1K- 33

a Boldface q indicates the positions of topaquinonein thesequences. Boldface Y indicates that tyrosine codons were found in these positions. An X indicates anunknown residue. T/D in parentheses for the porcine kidney sequence indicates that both D and T were detected in this position. Double underlining indicates conservedresidues, while single underlining indicatesconservative differences among the sequences.

bSequences determined by protein sequencing of isolated cofactor peptides.

cSequences translated from clonedDNAsequences.

with that ofmethylamine oxidase made it clear to us that it mustbe soclassified. Obviously, all these proteins are very similar, particularly those isolated from the gram-positive bacterium Arthrobacter strain P1, the gram-negative bacte-rium K aerogenes, and theyeastHansenulapolymorpha. Thesethree enzymes are monoamine oxidizers. By Clustal analysis, the threesequences are14.7% identical,and30.2% of the other positions differ byconservative substitutions. Relatively few gaps are introduced to maintain optimal overlap.Theenzymefrom lentil(Lensculinaris) isadiamine oxidase. Assuch, it could have somewhat different proper-tiesand structure.When all fouroxidases areincluded in the Clustalanalysis, the identity is 6.8% andconservative sub-stitutionsoccur atthe21.9% level. Several regionsarehighly homologous, notably the sequence surrounding the topa quinone site (residue 385 inthe MaoxII

sequence).

On the basis ofnumerous lines ofinvestigation, it has been pro-posed that the enzyme-bound

Cu(II)

has three axial histidine ligands.Anearlierreport(1) suggested that histidyl residues atpositions 26, 264, and 375 in the lentil seedling diamine oxidaseandpositions 23, 267, and376 in the H.

polymorpha

enzyme

(Fig.

9) are Cu(II) ligands. These correspond to positions 15 (His), 249 (Arg), and 358

(His)

of MaoxII. However, histidyl residues atthe second of thesepositions are not conserved in all the sequences shown in Fig. 9. Rather, thehistidyl residues alignedwith those atpositions 15, 358, 436,and 438 of the MaoxII sequence areconserved in all four sequences inFig.9. Thus, atleastthreeof these are the most likely candidates for the

histidyl

ligands of

Cu(II)

in each enzyme. The molecular

weights

calculated from the sequences inFig. 9are80,647, 77,533, 64,362, and 71,858for the

Klebsiella,

yeast,

andlentilseedlingenzymes andMaoxII,

respectively.

The valuesdeterminedby tradi-tional methods are

80,000

forthe

Klebsiella

enzyme,

78,000

fortheplant enzyme, and80,000to 82,000forMaoxII

(22,

24,37). Obviously, themethod used to measure this param-eter for these oxidases provides high estimates in some cases. Low values derived from the nucleotide sequences may be due to the carbohydratecontent of mature eukary-oticproteins (24).

maoxIand maoxIIexpressioninE.coli.Thegeneproducts expressedin E.colifrom maoxI and maotxIIexhibitthesame size as that ofpurified methylamineoxidase fromArthrobac-terstrain P1 (Fig.8). Although it ispossiblethat transcrip-tion is started from the plasmid promoter outside the in-serted fragment, it is more likely that the expression is controlledbythe Arthrobacter promoters, since the2.5-kbp DNAfragment,includingthe promoterregion, entirecoding

region, and 3'-flanking region of maoxII, was inserted into the plasmid in the orientation opposite to that of the lac operator.Surprisingly, methylaminedoes not induce maotxH expressionin E. coli(Fig. 8).Atentativeexplanation is that E.coliHB101doesnothavethetranscriptionalfactor which stimulates the induction of macxII expression by methyl-amine.

ACKNOWLEDGMENTS

This researchwasfundedbya Department of Veterans Affairs Medical Center Merit ReviewGrant, by Program Project Grant HL 16251 from the National Institutes of Health, and by an Academic SenateGrant from theUniversity ofCalifornia,San Francisco.

We thank Jaeho Kim and WalterWeyler for numerous helpful suggestions and discussions, andwethank David M. Dooley forthe sequences ofpeptides from methylamine oxidase and for continued interest and support.

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