FEMS MicrobiologyLetters 15 (1982) 79-82
Published by ElsevierBiomedical Press
79
Properties and subunit structure of methylamine dehydrogenase
from Thiobacillus A2 and Methylophilus methylotrophus
G.W. Haywood, N.S. Janschke, P.J. Large a n d J.M. Wallis
Department of Biochemistry, Universityof Hull, Hull, HU6 7RX, U.K.
Received and accepted20 April 1982
1. I N T R O D U C T I O N
In many methylotrophic bacteria which can
grow on methylamine as sole carbon source, the
first step in the metabolism of the growth substrate is the oxidation of methylamine to formaldehyde and ammonia via a nicotinamide
nucleotide-independent methylamine dehydrogenase (EC 1.4.99.3) [1-3] (Eqn. 1).
CH3NH3 ~ + H 2 0 + Dye ~ H C H O + NH~+ Reduced dye
(1)
This enzyme has been found in a wide range of
bacteria [1 ], including organisms using the ribulose
bisphosphate, hexulose phosphate and serine pathways for the biosynthesis of cell material.
The enzymes from Pseudomonas AM1 [2-4]
and Pseudomonas J [5-7] have been intensively
studied. Pseudomonas AM1 uses the serine pathway of carbon assimilation. Much less is known
about the methylamine dehydrogenases from
Thiobacillus A2 [8,9], an organism which uses the
ribulose bisphosphate cycle for carbon assimilation [9] and Methylophilus methylotrophus [1O,11 ],
an organism which uses the hexulose phosphate
pathway for the synthesis of cell material. Since
these organisms differ taxonomically and physiologically from one another, it was of interest to
determine whether their methylamine dehydrogenases showed significant differences.
The present paper reports the purification of
the methylamine dehydrogenases of Thiobacillus
A2 and M. methylotrophus and demonstrates that
apart from significant differences in heat stability
and electrophoretic mobility, the subunit composition and substrate specificity of the two enzymes
are very similar to those of Pseudomonas AM 1.
2. MATERIALS A N D M E T H O D S
2.1. Materials
5-Aminopentylagarose was obtained from
Sigma, hydroxyapatite (Bio-Gel HTP) from BioRad. Wurster's blue was p r e p a r e d from
N, N, N', N'-tetramethyl-p-phenylenediamine and
the radical cation of 2,2'-azino-di-(3-ethylbenzthiazoline sulphonate) (ABTS) from ABTS by
bromine oxidation as described in [12]. Other
materials were obtained from Sigma or Fisons.
2.2. Growth and harvesting of the organisms and
preparation of cell-free extracts
Methylophilus methylotrophus NCIB 10515 and
Thiobacillus A2 (obtained from Dr. J.P. van Dijken, Microbiological Laboratory, Delft University
of Technology, Delft, The Netherlands) were grown
on 0.5% (w/v) methylamine hydrochloride as described previously [11]. Extracts were prepared in
a French pressure cell [11].
0378-1097/82/0000-0000/$02.75 © 1982 Federation of European MicrobiologicalSocieties
80
2.3. Enzyme assays
Methylamine dehydrogenase was assayed as described by Eady and Large [2].
2.4. Chemical estimations
Protein was determined by the method of Bradford [ 13].
2.5. Polyacrylamide gel electrophoresis
This was done at p H 8.3 using the gel system of
Davis [14] without spacer gels. Sodium dodecyl
sulphate-polyacrylamide gels were prepared and
used as described previously [15] using the Boehringer Cornbithek 161365 kit of calibration proteins.
2.6. Purification of the enzymes
Methylamine dehydrogenase from Pseudomonas
AM1 was prepared as described by Boulton and
Large [16].
Methylamine dehydrogenase from Thiobacillus
A2 was purified as follows. Cell-free extracts were
heated for 20 min at 75°C with careful stirring,
and the brown greasy precipitate was centrifuged
off (30 rain at 50000 × g, 4°C). The supernatant
was applied to a column (10 cm X 2,5 cm diam.) of
5-aminopentylagarose equilibrated in 20 mM
potassium phosphate p H 7.0, and the column was
washed with the same buffer. The enzyme was
eluted with a linear gradient of 20-300 m M potassium phosphate p H 7.0 in 300 ml. The peak of
activity emerged at a phosphate concentration of
110 raM. Fractions containing more than 50% of
the maximum activity were combined and concentrated in an Arnicon Model 52 concentration
cell using PM10 Diaflo membranes. A typical
purification is shown in Table 1. The 18-fold purified material showed only very slight traces of
impurities on polyacrylamide gel electrophoresis.
Methylarnine dehydrogenase from M. methylotrophus was purified as follows. Cell-free extracts
were heated for 20 min at 70°C before centrifugation as above. The supernatant was diluted with
water to 5 m M and applied to a column (10 cm ×
2.5 cm diam.) of 5-aminopentylagarose equilibrated in 5 rnM potassium phosphate p H 7.0.
After washing with the same buffer the column
was eluted with a linear gradient of 5-100 rnM
potassium phosphate p H 7.0 in 200 ml. Fractions
containing more than 50% of the maximum activity were combined and diluted to 5 rnM phosphate
concentration with distilled water. They were then
applied to a hydroxylapatite column (10 cm × 1 cm
diam.) and washed with the same buffer. The
column was then eluted with a linear gradient of
5-300 m M potassium phosphate in 200 ml. The
peak of enzyme activity was at 235 rnM. Fractions
with more than 50% activity were combined and
concentrated. The 50-fold purified material was
homogeneous on polyacrylamide gels.
Table 1
Purification of methylamine dehydrogenase from Thiobacillus A2
Step
Volume
(ml)
Protein
concentration
(mg/ml)
Total
units
Specific activity
(units/rag protein)
Yield
(%)
(1) Crude extract
(2) Supernatant
after heat treatment
(3) Combined eluates from
column after concentration
64
24.5
93.4
0.060
100
60
7.5
61.2
0.136
65.5
2.3
11
4.63
56.3
1.103
60.2
18.3
Purification
l
81
3. RESULTS
3.1. Heat stability of methylamine dehydrogenase in
crude extracts
The stability of methylamine dehydrogenase activity in crude extracts of Thiobacillus A2 was
closely similar to that of Pseudomonas AM1 [2],
while the enzyme from M. methylotrophus was
significantly less stable at 80°C, although it would
still withstand 20 min at 70°C (Fig. 1) with only a
10% loss of activity.
strates for the Thiobacillus and M. methylotrophus
enzymes included 2-bromoethylamine, 2-chloroethylamine, fl-aminopropionitrile, 2-methoxyethylamine and fl-alanine methyl ester.
The enzyme from Thiobacillus A2 failed to reduce 2,6-dichlorophenolindophenol, N A D ÷ ,
N A D P ÷ or ferricyanide. The following compounds were active as electron acceptors (apparent
K m values in parentheses): phenazine methosulphate (173 #M), radical cation of ABTS (14.5
p.M), Wurster's blue (98 /~M) and horse heart
cytochrome c (14 ffM). It should be noted that
these are all one-electron acceptors.
3.2. Specificity for electron donors" and acceptors
3.3. Effect of inhibitors
The amines oxidized by both enzymes resembled very closely those active with the enzymes
from Pseudomonas AM1 [2] and Pseudomonas J
[5]. Additional compounds which were active sub-
100 L.A
--
v
~.
8O
The enzyme from Thiobacillus A2 was sensitive
to the same inhibitors as the enzyme from Pseudomonas AM1 [2]. Semicarbazide (33 /IM) caused
98% inhibition after 15 min preincubation With the
enzyme, KCN (33 ~M) 3%, aminoacetonitrile (330
ffM) 30% inhibition, isoniazid (667 /~M) 55%,
trans-2-phenylcyclopropylamine (667/zM) 20% and
cuprizone (280 ffM) 100%.
6O
3. 4. Electrophoretic mobility of the purified enzymes
40
>I--"
The relative mobilities of the two enzymes were
compared with that of the enzyme from Pseudomonas AM1 in polyacrylamide gels using the buffer
system of Davis [14]. The values observed
(bromophenol b l u e = 1.0) were: enzyme from
Pseudomonas AM1 (two active bands) 0.26 and
20
>Z
J
8
_< 6
z__ 4
Table 2
Relative molecular masses of the subunits of methylamine
dehydrogenase from taxonomically different bacteria
2
1
I
I
I
I
I
1
5
10
15
2Q
25
30
TIME
Organism
(rnin)
Fig. l. Temperature stability of the methylamine dehydrogenases of Thiobacillus A2 and Methylophilus methylotrophus.
1 ml of a crude French press extract of either organism (protein
concentration 14 m g / m l ) was heated at the indicated temperature in a thin-walled tube in a water-bath. At the times indicated, samples (0.1 ml) were removed and stored in ice until
assayed. Extract from Thiobacillus A2 at 70°C (O), and 80°C
(A), Extract from M. methylotrophus at 70°C (O); 80°C (A).
Pseudomonas AM 1
Pseudomonas J
Thiobacillus A2
Methylophilus methylotrophus
103. M r
Ref.
Large
subunit
Small
subunit
40
40
49
42.7
13
13
14.1
15.9
[4]
[6]
This
work
82
0.28, from Thiobacillus A 2 0.71 a n d from M. methy lotrophus O. 12.
3.5. Subunit structure of the purified enzymes
On sodium dodecyl sulphate-polyacrylamide
gels, b o t h enzymes showed the presence of two
p r o t e i n b a n d s , an intensely staining large polyp e p t i d e a n d a less intensely staining small polyp e p t i d e ( T a b l e 2). These o b s e r v a t i o n s are qualitatively similar to those m a d e with the enzymes from
Pseudomonas A M 1 [4] a n d Pseudomonas J [6].
cently discovered blue c o p p e r p r o t e i n a m i c y a n i n ,
which is believed to link the m e t h y l a m i n e dehyd r o g e n a s e with c y t o c h r o m e c [18]. T h e need for
this i n t e r a c t i o n with the r e s p i r a t o r y chain could
p o s s i b l y e x p l a i n why the p r o p e r t i e s of methyla m i n e d e h y d r o g e n a s e are so similar in b a c t e r i a
f r o m widely differing p h y s i o l o g i c a l a n d t a x o n o m i c
groups.
ACKNOWLEDGEMENTS
W e t h a n k J.P. van Dijken, J.A. D u i n e a n d J.
F r a n k , Jzn. for v a l u a b l e discussions in the early
stages of this work.
4. D I S C U S S I O N
A l t h o u g h the m e t h y l a m i n e d e h y d r o g e n a s e from
M. methylotrophus is significantly less stable t h a n
the enzymes f r o m Thiobacillus A 2 or Pseudomonas
A M 1 it will nonetheless w i t h s t a n d 30 m i n at 70°C.
T h u s the e n z y m e f r o m Pseudomonas J s t a n d s out
as r e m a r k a b l y less stable than the o t h e r enzymes
[5]. O n e is t e m p t e d to correlate the s u b u n i t structure of the e n z y m e with this t h e r m o s t a b i l i t y . Shirai
et al. [4] have shown that the light subunits from
the m e t h y l a m i n e d e h y d r o g e n a s e s f r o m Pseudomonas A M 1 a n d Pseudomonas J are virtually identical in their a m i n o acid c o m p o s i t i o n a n d heat
stability, while there are significant differences in
a m i n o acid c o m p o s i t i o n b e t w e e n the h e a v y subunits, suggesting that this is the cause of the relative t h e r m o l a b i l i t y of the Pseudomonas J methyla m i n e d e h y d r o g e n a s e in c o m p a r i s o n with that of
Pseudomonas A M 1 . E x t e n d i n g this conclusion to
the p r e s e n t results, it seems p r o b a b l e that the
difference in M r ( a n d thus also of c o m p o s i t i o n )
b e t w e e n the enzymes f r o m M. methylotrophus a n d
Thiobaeillus A 2 m a y e x p l a i n the greater h e a t stab i l i t y of the latter. T h e difference in p r i m a r y structure w o u l d also explain the different electrop h o r e t i c mobilities of the intact enzymes (Section
3.4.). It also seems r e a s o n a b l e to infer that the
light subunits p r o b a b l y differ m u c h less from one
a n o t h e r , which m i g h t b e expected since they are
believed to be the b i n d i n g site for the pyrroloquinoline quinone (methoxatin) chromophore
[1,4,17]. It seems p o s s i b l e also that the light subunit m a y be the site of i n t e r a c t i o n with the re-
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