D. Becker et al., 2-COMPONENT FLAVIN-DEPENDENT PYRROLE-2-CARBOXYLATE MONOOXYGENASE FROMRHODOCOCCUS SP, European journal of biochemistry, 249(3), 1997, pp. 739-747
Pyrrole-2-carboxylate can serve as the sole source of carbon, nitrogen
, and energy for a strain tentatively identified to belong to the genu
s Rhodococcus. An NADH-dependent oxygenase activity was detected in ce
ll extracts that initiated the degradation of the substrate. During pu
rification of the enzyme, this activity was separated into two protein
components which were both purified to apparent homogeneity. A small
monomeric 18.7-kDa protein designated as reductase, catalyzed in vitro
the NADH and FAD-dependent reduction of cytochrome c and had an NADH-
oxidase activity. The second component, a 54-kDa protein with a trimer
ic native structure had no enzymatic activity by itself, but exhibited
a pyrrole-2-carboxylate-dependent oxygen consumption when it was comp
lemented with the reductase component, FAD, acid NADH. This indicated
that the large protein referred to as oxygenase was responsible for th
e oxygen-dependent hydroxylation of the substrate. The rate of an unco
upled NADH oxidation without hydroxylation of the substrate was found
to be strongly dependent on the molar ratio of both components. The un
coupling was nearly completely suppressed by a 5-7-fold molar excess o
f the oxygenase component. The small protein was N-terminally blocked.
It was thus proteolytically digested and four of the resulting peptid
es were sequenced comprising 47 amino acids. The sequences of these fr
agments were similar to the sequences reported for the small component
of different two-component flavin monooxygenases. Furthermore, the N-
terminus of the oxygenase component showed high sequence similarity to
the second, usually large subunit of these enzymes and to two single-
component flavin monooxygenases. Thus, the enzyme from Rhodococcus sp.
designated as pyrrole-2-carboxylate monooxygenase belongs to the rece
ntly discovered new class of two-component flavin aromatic monooxygena
ses. Some of the basic properties of both components were determined a
nd their interaction during catalysis was investigated.