K. DECKER, V. D. DAI, H. MÖHLER, AND M. BRÜHMÜLLER
1072
D- and L-6-Hydroxynicotine Oxidase, Enantiozymes of Arthrobacter
oxidans
K . DECKER, V . D . D A I , H . M Ö H L E R , a n d M . BRÜHMÜLLER
Biochemisches Institut der Universität Freiburg, 78 Freiburg i. Br., West-Germany
( Z . Naturforsch. 27 b , 1072—1073 [1972] ; received May 10, 1972)
D- and L-hydroxynicotine oxidase were obtained in homogeneous form and compared with
regard to molecular weight, subunit structure, reaction mechanism, and specificity. The most
striking differences between these enantiozymes are the type of coenzyme binding and the reactivity towards artificial two-electron acceptors.
structure, reactivity towards two-electron acceptors,
FAD-content, and coenzyme binding, the D-specific
enzyme having one mole of covalently bound F A D
per mole of protein. F A D as the prosthetic group of
D-6-hydroxynicotine oxidase was identified by spectroscopy and AMP-determination, that of the L-specific enzyme by paper chromatography of the dissociated coenzyme and by reactivation of the apoenzyme by FAD 3 .
The flavoproteins, D- and L-6-hydroxynicotine
oxidase, are induced when Arthrobacter oxidans is
grown in the presence of D- and L-nicotine, respectively 2 . Both enzymes are absolutely stereospecific, the enantiomeric substrate acting as a competitive inhibitor. They f o r m the same product,
[6 -hydroxypyridyl (3) ] - ( y - N - methylaminopropyl) ketone ( " k e t o n e " ) , from either D- or L-6-hydroxynicotine by an apparently identical mechanism 3 .
The term enantiozymes is proposed for pairs of
enzymes having these properties.
Oxygen appears to be the physiological electron
acceptor for both enzymes. One-electron acceptors
such as cytochrom c and ferricyanide as well as
methylene blue and 2.6-dichloro-phenol-indophenol
do not reoxidize the reduced L-6-hydroxynicotine
The D- and L-6-hydroxynicotine oxidases were
obtained in homogeneous form. As summarized in
the Table they differ in molecular weight, subunit
L-HNO
HO
Ö2X
CH 3
HO
6 -hydroxynicotine
N
r y
CHI
H20
O-HNO
02
Table.
6-hydroxynicotine
oxidase
mol. W .
D-
53000
L- (ref. 4)
93000
f/fo
f y c
HO
D-
/
6-hydroxy-A/-methylmyosmine
N
n
HN
0
I
CH 3
"Ketone"
+ H202
D- and L-6-Hydroxynicotine oxidase from Arthrobacter oxidans.
polypeptide
chains/
mole
FAD/
mole
F A D bound
(absorption
maxima)
turnover number
moles/min/mole
1.26
1
1
covalently
(272, 355,
450, S 4 7 5 )
1190
( p H 9 . 2 ; 30 °C)
5 • 10-5 M
1.22
2
2
noncovalently
(273, 370,
443, S 4 6 3 )
1760
( p H 7 . 5 ; 29 °C)
2•IO-5 M
KM
(substrate)
reactivity t o w a r d s
electron acceptors
1 e-
2 e-acc.
—
- f (stimul.)
+
— (inhib.)
+
Requests for reprints should be sent to Prof. Dr. K. DECKER. Biochemisches Institut der Universität, D-7800
Hermann-Herder-Straße 7.
Freiburg,
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02
8- ( A - 3 - H I S T I D Y L - ) F L A V I N
IN
D-6-HYDROXYNICOTINE
oxidase 3 . Moreover, methylene blue was shown to
be a strong inhibitor of the overall reaction. Oneelectron acceptors are also ineffective with the Dspecific enzyme; however, methylene blue and 2.6dichlorophenol-indophenol are able to reoxidize the
reduced D-6-hydroxynicotine oxidase. In the presence of these dyes the rates of the overall process
are higher than those with oxygen.
Anaerobic titrations of both enantiozymes with
their respective substrates produced a steady tran1
2
K . DECKER and H. BLEEG, Biochim. biophysica Acta [Amsterdam] 1 0 5 , 3 1 3 [ 1 9 6 5 ] .
M . GLOGER
and
K . DECKER,
Z.
Naturforsch.
2 4 b,
1016
1073
OXIDASE
sition f r o m the oxidized to the reduced f o r m . Spectral evidence of a semiquinoid intermediate stage
could not be obtained. This was seen, however, after
illumination of the enzymes in the presence of
EDTA.
It remains to be elucidated whether and to what
extent reactivity towards artificial two-electron acceptors of D- and L-6-hydroxynicotine oxidase and
the type of coenzyme binding are a consequence of
the respective quaternary structure of these proteins.
3
4
K. DECKER and V. D. DAI, Europ. J. Biochem. 3, 132 [1967].
V. D. DAI, K . DECKER, and H. SUND, Europ. J. Biochem. 4 ,
95
[1968].
[1969].
Covalently Bound Flavin in D-6-Hydroxynicotine Oxidase
from Arthrobacter oxidans
M . BRÜHMÜLLER, H . M Ö H L E R , a n d K . D E C K E R
Biochemisches Institut der Universität, Freiburg i. Br., West-Germany
( Z . Naturforsch. 27 b , 1073—1074 [1972] ; received May 10, 1972)
Flavoprotein, Covalently bound F A D , 8a- (A 3 -histidyl) -riboflavin
D-6-hydroxynicotine oxidase contains 1 mole of F A D covalently bound to one mole of enzyme.
T o identify the covalent linkage between F A D and protein, an amino acid derivative of riboflavin
(HNO-flavin) was isolated and purified. It was obtained from flavin peptides by hydrolysis with
6 N H C l at 95 ° C or with aminopeptidase M. The riboflavin derivative had the spectral characteristics
of 8a-substituted flavins. It showed a pH-dependence of fluorescence with a p K of 4.65 and 86%
quenching at p H 7. In thin layer chromatography it was identical with 8a-(A-3-histidyl)-riboflavin.
Hydrolysis of HNO-flavin in 6 N HCl at 125 ° C liberated 1 mole of histidine per mole of flavin
as shown by amino acid analysis. Since F A D is the coenzyme of D-6-hydroxynicotine oxidase, these
results are taken as evidence that this enzyme contains 8a-(A-3-histidyl)-flavin-adenine-dinucleotide
in the active center.
Unusual Abbreviations:
HNO-flavin, riboflavin attached to
one amino acid of D-6-hydroxynicotine oxidase; His-riboflavin, 8a-histidyl-riboflavin.
D-6-Hydroxynicotine oxidase f r o m
oxidans,
purified
enzyme
yielded
a
fluorescent
product,
migrating differently than F A D in paper chromatography and high voltage electrophoresis. These re-
Arthrobacter
sults were taken as evidence f o r the presence of a
catalyzing the oxidative cleavage of D-6-
covalently bound flavin in D-6-hydroxynicotine oxi-
hydroxynicotine
to
(6-hydroxypyridyl-(3)
methyaminopropyl)-ketone,
)-(y-N-
contains one mole
of
dase.
T o identify the covalent linkage between
FAD
F A D per mole of enzyme. Methods known to rever-
and the apoprotein, an amino acid derivative of
sibly dissociate the flavin coenzyme f r o m the apo-
riboflavin
protein failed to release the coenzyme. Treatment
peptides either b y hydrolysis in 6 N HCl at 95 ° C
(HNO-flavin)
was isolated f r o m
flavin
of the enzyme with 5 % trichloroacetic acid resulted
in vacuo f o r 12 h
in a supernatant without any absorbance in the
taining prolinase. The aminopeptidase digest was
1
or with aminopeptidase M con-
visible range whereas the precipitate displayed the
then incubated in 10% trichloroacetic acid in order
typical
to hydrolyze the flavin dinucleotide to the mono-
flavin
spectrum. Tryptic digestion of
the
Requests for reprints should be sent to Prof. Dr. K. DECKER,
Biochemisches Institut der Universität, D-7800
Freiburg,
Hermann-Herder-Str. 7.
nucleotide and subsequently at p H 5 . 7 with acid
phosphatase f o r dephosphorylation. The
resulting
HNO-flavin was purified b y preparative thin layer
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