Information on EC 1.4.9.1 - methylamine dehydrogenase (amicyanin)

-

-

?

1,6-diaminohexane + acceptor + H2O

?

-

acceptor: phenazine ethosulfate/2,6-dichlorophenolindophenol

654866, 656086

-

?

1,7-diaminoheptane + acceptor + H2O

?

-

acceptor: phenazine ethosulfate/2,6-dichlorophenolindophenol

654866, 656086

-

?

1,8-diaminooctane + H2O + acceptor

?

1-aminopentane + acceptor + H2O

pentanal + NH3 + reduced acceptor

-

acceptor: phenazine ethosulfate/2,6-dichlorophenolindophenol

654866, 656086

-

?

12-aminododecane + acceptor + H2O

dodecanal + NH3 + reduced acceptor

-

-

?

12-aminododecanoic acid + acceptor + H2O

?

-

-

r

2-phenylethylamine + 2 H2O + 2 acceptor

2-phenylacetic acid + NH3 + 2 reduced acceptor

-

primary amine

-

?

2-phenylethylamine + acceptor + H2O

2-phenylacetaldehyde + NH3 + reduced acceptor

-

acceptor: phenazine ethosulfate/2,6-dichlorophenolindophenol

654676

-

?

benzylamine + acceptor + H2O

benzaldehyde + NH3 + reduced acceptor

benzylamine + H2O + ferricyanide

benzaldehyde + NH3 + reduced ferricyanide

-

-

?

butylamine + acceptor + H2O

butanal + NH3 + reduced acceptor

dithionite + amicyanin + H2O

?

-

two-electron-reduced MADH is obtained by exposing the enzyme either to a 3fold molar excess of methylamine or to 2 mM dithionite

-

?

ethylamine + acceptor + H2O

acetaldehyde + NH3 + reduced acceptor

histamine + acceptor + H2O

? + NH3 + reduced acceptor

-

396587

-

?

methylamine + acceptor + H2O

formaldehyde + NH3 + reduced acceptor

methylamine + acceptor + H2O

methanal + NH3 + reduced acceptor

methylamine + amicyanin + H2O

formaldehyde + NH3 + reduced amicyanin

-

685087, 688702

-

?

methylamine + amicyanin + H2O

formaldehyde + reduced amicyanin + NH3

methylamine + H2O + 2 amicyanin

formaldehyde + NH3 + 2 reduced amicyanin

methylamine + H2O + 2,6-dichloroindophenol

formaldehyde + NH3 + reduced 2,6-dichloroindophenol

-

-

r

methylamine + H2O + 2,6-dichloroindophenol + phenazine ethosulfate

formaldehyde + NH3 + reduced phenazine ethosulfate + ?

-

-

r

methylamine + H2O + amicyanin

formaldehyde + ammonia + reduced amicyanin

methylamine + H2O + amicyanin

formaldehyde + NH3 + reduced amicyanin

-

-

?

methylamine + H2O + cytochrome c-550

formaldehyde + NH3 + reduced cytochrome c-550

-

-

r

methylamine + H2O + K3Fe(CN)6

formaldehyde + NH3 + reduced K3Fe(CN)6

-

-

r

n-butylamine + H2O + ferricyanide

butanal + NH3 + reduced ferricyanide

-

-

?

n-hexylamine + acceptor + H2O

hexanal + NH3 + reduced acceptor

n-nonylamine + acceptor + H2O

nonanal + NH3 + reduced acceptor

-

-

?

n-pentylamine + acceptor + H2O

pentanal + NH3 + reduced acceptor

-

-

?

phenylethylamine + acceptor + H2O

phenylacetaldehyde + NH3 + reduced acceptor

propylamine + acceptor + H2O

propionaldehyde + NH3 + reduced acceptor

RCH2NH2 + acceptor + H2O

RCHO + NH3 + reduced acceptor

13 entries

tryptamine + acceptor + H2O

?

-

686104

-

?

additional information

?

-

16 entries

2-phenylethylamine + 2 H2O + 2 acceptor

2-phenylacetic acid + NH3 + 2 reduced acceptor

-

primary amine

-

?

methylamine + amicyanin + H2O

formaldehyde + reduced amicyanin + NH3

-

amicyanin is the in vivo electron acceptor

396582

-

?

methylamine + H2O + 2 amicyanin

formaldehyde + NH3 + 2 reduced amicyanin

methylamine + H2O + amicyanin

formaldehyde + NH3 + reduced amicyanin

-

-

?

additional information

?

-

cysteine tryptophylquinone

heme c

8 entries

pyrroloquinoline quinone

-

covalently bound

quinoid cofactor

-

alpha subunit contains unknown quinoid cofactor

-

quinone

-

contains a quinone similar to but not identical with the prosthetic group of EC 1.1.99.8

396579

tryptophan tryptophylquinone

Ca2+

Co2+

Fe

-

enzyme contains two heme groups

KCl

-

the rate of transfer of the first electron from methylamine-reduced MADH to amicyanin is increased by monovalent cations

Mn2+

Na+

Zn2+

-

additional information

8-hydroxyquinoline

-

slight

396576

amicyanin mutant M98K

-

mutant acts as a competitive inhibitor in the reaction of native amicyanin with methylamine dehydrogenase indicating that the M98K mutation has not affected the affinity for its natural electron donor. The crystal structure of M98K amicyanin reveals an overall structure very similar to native amicyanin but the type I binding site is occupied by zinc instead of copper

715814

-

Borohydride

-

396580

Cuprizone

cyclopropylamine

-

mechanism-based inhibitor. The resulting inactivation is accompanied by the formation of a covalent cross-link between the alpha and beta subunits of the enzyme. No cross-linking is seen with mutant enzymes alphaF55A or alphaF55I mutant enzymes. With mutant enzyme alphaF55E cross-linking of subunits is observed

655553

hydrazine

-

hydroxylamine

iodoacetate

-

isoniazid

KCN

-

n-Butyraldehyde

-

N-ethylmaleimide

-

Neocuproine

-

slight

p-chloromercuribenzoate

p-Nitrophenylhydrazine

-

phenylhydrazine

Quinacrine

Quinine

-

slight

Semicarbazide

additional information

-

immobilized enzyme: little change in sensitivity to inhibition

396573

-

amicyanin

-

741759, 742302, 742527

-

benzylamine

-

activates sQH-AmDH

Dithionite

-

rapidly activates sQH-AmDH, activation process involves a reduction process

dithiothreitol

-

rapidly activates sQH-AmDH, activation process involves a reduction process

MauG

-

n-butylamine

-

activates sQH-AmDH

additional information

the gene which encodes MauG is located in the methylamine utilization (mau) gene cluster of several gram negative bacteria. The mau cluster contains the two structural genes for MADH as well as accessory proteins that are required for MADH biogenesis

741759

-

0.003

1,12-diaminododecane

-

0.021 - 0.72

1,6-diaminohexane

0.007 - 0.38

1,7-Diaminoheptane

0.004 - 2.5

1-aminopentane

0.033

12-aminododecanoic acid

-

0.12

2,6-dichloroindophenol

-

0.007 - 0.87

Butylamine

0.042

cytochrome c-550

-

0.019 - 9.2

ethylamine

0.78

K3Fe(CN)6

-

0.004 - 22.4

methylamine

16 entries

0.014

phenazine ethosulfate

-

0.006 - 1.3

Propylamine

3.4 - 43

1,6-diaminohexane

3.4 - 32

1,7-Diaminoheptane

3.4 - 20

1-aminopentane

1.5

2,6-dichloroindophenol

-

3.1

2-Phenylethylamine

-

pH 7.5, 30°C

654676

0.77 - 74

amicyanin

-

3.8 - 22

benzylamine

4.2 - 34

Butylamine

7 entries

2.3

cytochrome c-550

-

4.5 - 24

ethylamine

15

K3Fe(CN)6

-

0.14 - 77

methylamine

18 entries

7.1

phenazine ethosulfate

-

26

phenethylamine

-

3.1 - 27

Propylamine

additional information

-

turnover numbers for deuterated substrates

654676

-

13.5

-

3.5

-

3.86

-

3.9

-

4.67

-

additional information

7

-

1,12-diaminododecane

7.4

-

methylamine

7.5

8

9

-

putrescine

additional information

-

30

40

-

1,12-diaminododecane

Hyphomicrobium zavarzinii

ZV580

Hyphomicrobium zavarzinii ZV580

-

ZV580

-

AM1

-

Methylorubrum extorquens ATCC 14718 / DSM 1338 / JCM 2805 / NCIMB 9133 / AM1

AM1

-

Mycobacterium convolutum

inducible enzyme

396578

-

Paracoccus denitrificans IFO 12442

7 entries

-

-

-

396582, 396583, 685296

-

AM1

396573, 396579, 396580

-

K95

-

-

evolution

the qhp genes are very widely distributed, not only in many Gram-negative species but also in a few Gram-positive bacteria, bacterial distribution of qhp and associated genes, overview. The subunits constituting QHNDH are encoded by ORF1 (alpha subunit), ORF4 (beta subunit), and ORF3 (gamma subunit). Of the other genes in the operon, ORF2 encodes an [Fe-S] cluster and S-adenosylmethionine (SAM)-binding protein, a member of the radical SAM superfamily, and ORF5 encodes a protein of approximately 22.5 kDa belonging to subfamily S8A of peptidase family S8 (the subtilisin family) with the conserved Asp/His/Ser catalytic triad characteristic of this subfamily

741905

malfunction

the genes mauF and mauE are membrane proteins with no homology to characterized proteins, and are thought to be involved in transport of MADH subunits into the periplasm. Knocking out either gene leads to no detectable beta-subunit in the periplasm, and an unusual beta-subunit leader sequence is consistent with it being trafficked by a specific transporter. The loss of mauF and mauE additionally leads to a drastic reduction in alpha-subunit. The third gene, mauD, is homologous to disulfide isomerases, and is likely specific to the MADH beta-subunit, which has six disulfides. In the absence of mauD, periplasmic alpha-subunit levels are close to normal, but again there is no detectable beta-subunit implying that the disulfides are key to beta-subunit stability. When the final required gene, mauG, is knocked out, there are normal levels of MADH alpha- and beta-subunit in the periplasm, but no methylamine dehydrogenase activity is present. This has focused attention on the mauG gene product as a likely participant in TTQ biosynthesis

physiological function

additional information

9 entries

Paracoccus denitrificans PD1222

4y5r, download, 3D-view

SCOPe (4y5r)

CATH (4y5r)

A1BB97

100000

101700

-

calculated from amino acid sequence

112000

-

gel filtration

396576

114000

-

119000

Hyphomicrobium zavarzinii

12000

-

two bands appear in an SDS-PAGE: 12 kDa and 42 kDa

133000

-

ultracentrifugation

396580

138000

-

complex of methylamine dehydrogenase and amicyanin, gel filtration

13960

-

L subunit, MALDI

13970

-

L subunit, calculated from sequence

14000

-

2 * 14000 + 2 * 41000, X-ray crystallography

14200

-

SDS-PAGE, beta subunit

36500

38500

Mycobacterium convolutum

-

gel filtration

396578

39000

-

1 * 60000 + 1 * 39000, SDS-PAGE

39234

-

1 * 53917 + 1 * 39234 + 1 * 8597, calculated from amino acid sequence

39360

-

calculated for beta-subunit, containing a 26 aa sequence which seems to correspond to a signal peptide that might be also involved in the correct translocation from the bacterial cytoplasm to the periplasmic space.

41000

-

2 * 14000 + 2 * 41000, X-ray crystallography

41160

-

H subunit, calculated from sequence

41170

-

H subunit, MALDI

42000

43300

-

SDS-PAGE, alpha subunit

47000

-

1 * 47000, SDS-PAGE

53917

-

1 * 53917 + 1 * 39234 + 1 * 8597, calculated from amino acid sequence

54230

-

calculated for alpha-subunit, existence in this protein of two consensus sequences (CXXCH) identifying as binding pockets for the two haem groups

56000

-

gel filtration

58000

-

1 * 58000 + 1 * 42000, SDS-PAGE

396576

59500

60000

-

1 * 60000 + 1 * 39000, SDS-PAGE

8486

-

small subunit, MALDI-TOF mass spectrometry

8579

-

calculated gamma-subunit

8597

-

1 * 53917 + 1 * 39234 + 1 * 8597, calculated from amino acid sequence

95300

-

sedimentation equilibrium

dimer

heterotetramer

heterotrimer

monomer

tetramer

trimer

-

alpha,beta,gamma, X-ray crystallography

396588

additional information

proteolytic modification

additional information

one week in 19% PEG4000, 70 mM acetate buffer, pH 4.1

-

as MADH/amicyanin binary complex

-

688702

crystals are grown in presence of 100 mM sodium citrate, pH 5.6, 18% PEG 4000 and 21% t-butyl alcohol. Complex of enzyme with the inhibitor phenylhydrazine determined at 1.7 A resolution

-

654076

in complex with electron transfer protein amicyanin and with amicyanin mutant P52G. Model of electron transfer reaction between enzyme and amicyanin

-

672193

macroseeding using a 9-to-1 mixture of monobasic sodium (3 M) and dibasic potassium (3 M) phosphate solutions as precipitant

-

685087

MauG in complex with pre-methylamine dehydrogenase, hanging drop vapor diffusion method, using 23-25% (w/v) PEG 8000, 0.1 M sodium acetate, 0.1 M MES pH 6.4, at 20°C

724308

MauG in complex with preMADH, X-ray diffraction structure determination and analysis at 2.1 resolution

MauG-pre-enzyme complex, hanging drop vapor diffusion method, using 0.1 M sodium acetate, 0.1 M MES pH 6.4, 22-26% PEG 8000

molecular dynamics simulations of the complex of redox proteins methylamine dehydrogenase and amicyanin to generate configurations over a duration of 40 ns in conjunction with an electron trnasfer pathway analysis. In the wild-type complex, the most frequently occurring molecular configurations afford superior electronic coupling due to the consistent presence of a water molecule hydrogen-bonded between the donor and acceptor sites. The water bridge function of nearby solvent-organizing residues by limiting the exchange of water molecules between the sterically constrained electron transfer region and the more turbulent surrounding bulk. When the water bridge is affected by a mutation, bulk solvent molecules disrupt it, resulting in reduced electronic coupling

-

716750

preMADH complexed with MauG, X-ray diffraction structure determination and analysis at 2.1 A resolution

-

713510

purified preenzyme pre-MADH in complex with activator mutant Q103N MauG, X-ray diffraction structure determination and analysis

741893

sitting drop method, crystal structure of alphaF55A in complex with its electron acceptors, amicyanin and cytochrome c-551i. Little difference in the overal structure is seen, relative to the native complex. There are significant changes in the solvent content of the active site and substrate channel. Crystal structure of alphaF55A with phenylhydrazine covalently bound to tryptophan tryptophylquinone in the active site

-

654628

MADH-amicyanin binary complex, hanging drop vapour diffusion method, in 28-29.5% PEG8000, 0.2 M Li2SO4, and 0.1 M phosphate (pH 6.5)

-

the crystal structure of the complex of MADH and amicyanin is determined to 2.5 A using the hanging-drop method. Enzyme is a heterotetramer consisting of two heavy chains and two light chains. The heavy chain of MADH folds into a characteristic, seven-blade beta-propeller domain and contains an N-terminal extension (residues 1-80) that wraps around the neighboring light chain, fixing it to the tetrameric enzyme. The light subunit consists mainly of loop regions with only four beta-strands, stabilized by a total of six disulfide bridges, containing the active site of the enzyme, a tryptophan tryptophylquinone moiety formed by Trp57 covalently linked to Trp108

-

hanging drop vapour diffusion method with 18% (w/v) polyethylene glycol 2000 monomethylether and 50 mM NiCl2

-

-

-

alphaF55A

alphaF55I

betaD32N

-

preparation contains a major species with six disulfides but no oxygen incorporated into betaTrp57 and a minor species with both oxygens incorporated, which is active. 1000fold increase in KM-value for methylamine

656282

betaD76N

-

mutant enzyme is completely inactive

656282

betaI107N

betaI107V

F55A

-

mutation of the alpha subunit

396586, 396587

F55E

-

mutation of the alpha subunit

396586

T122A

-

the presence of Thr122 has a deleterious effect on the proton transfer step that is proposed to determine the rate of the reaction, the substitution of Thr122 by Ala does not significantly modify the preference of the proton by atom OD2 of Asp76

686067

K14E

-

mutation of cytochrome c-550

K14Q

-

mutation of cytochrome c-550

additional information

2.6 - 10.6

-

75 min

396574

6 - 11

-

2 weeks, room temperature

396571

50

-

10 min

65

-

3 min, 97% loss of activity

396577

80

-

15 min, stable, inactivated above

396574

84

-

30 min, 40% loss of activity (immobilized enzyme), about 85% loss of activity (soluble enzyme)

396573

dialysis against diethyldithiocarbamate, stable

-

396580

freezing and thawing, stable

-

immobilized enzyme

-

no difference in temperature-stability and pH-stability at 1 mg protein/ml and 0.0005 mg/ml

-

-15°C, 1 mg/ml protein concentration

-

396574

-

DEAE column chromatography

-

Hyphomicrobium zavarzinii

enzyme is isolated to near purity by a sequence of hydroxyapatite and anion exchange chromatography

-

HiLoad Superdex 200 gel filtration

-

685218

ion-exchange chromatography with DEAE-Toyopearl resin

-

nickel-affinity column chromatography

Superdex 75 gel filtration

-

using a DEAE column

-

expressed in Escherichia coli

-

expressed in Escherichia coli BL21 cells

-

expressed in Rhodobacter sphaeroides

Expression in Escherichia coli, the production of knock out-mutants unable to catabolize 2-phenylethylamine is carried out by mutagenesis.

-

genes qhpA, qhpB, and qhpC encoding the alpha, beta, and gamma subunits of QHNDH, respectively. The structural genes encoding the three QHNDH subunits constitute an operon harboring six apparent open reading frames (ORFs) that are transcribed in a coordinated manner upon addition of amines to the culture medium. qhpADCBEF genes constitute a hexacistronic operon. DNA and amino acid sequence determination and analysis, and reverse transcription PCR expression analysis. Regulation of qhp genes, overview

741905

heterologous expression of cytochrome c-550 in Escherichia coli

-

heterologous expression of mutated alpha subunit in Rhodobacter sphaeroides

-

396586

MADH is a heterotetramer consisting of two alpha subunits and two beta subunits which are encoded by mauB and mauA, respectively

741759

PreMADH can be generated by expression of recombinant MADH in the background of a mauG deletion

-

713510

recombinant expression of pre-MADH in Rhodobacter sphaeroides

742527

the methylamine utilization (mau) gene cluster contains 11genes, two of which encode the 42.5 kDa alpha-(mauB) and the 14.2 kDa beta-subunits (mauA) of MADH, recombinant co-overexpression of genes mauAB in Rhodobacter sphaeroides in the presence of mauDEFG, the four genes required for MADH maturation results in an alpha2beta2 MADH protein that has no catalytic activity, because the protein contains all six beta-subunit disulfides, but has a partially synthesized TTQ cofactor with only a single -OH group added to the indole of betaTrp57 (betaTrp57-OH)

an AraC family transcriptional regulator, ancoded by gene qhpR, activates expression of the qhp operon in response to the addition of n-butylamine to the culture medium

741905

analysis