(Received for publication, August 23, 1994; and in revised form, December 14, 1994)
From the
Aromatic amine dehydrogenase (AADH) and methylamine dehydrogenase (MADH) are the only two enzymes known to use the cofactor tryptophan tryptophylquinone (TTQ). Each catalyzes oxidative deamination of a distinct class of primary amines. A detailed comparison of their circular dichroic spectra indicates that both proteins share a similar fold with their TTQ cofactors residing in similar environments and that this may be a useful diagnostic probe for TTQ enzymes. Alcaligenes faecalis cells induced to express AADH also express a large amount of the blue copper protein, azurin. Oxidized azurin is rapidly reduced by a catalytic amount of AADH in the presence of the substrate, tyramine. Three A. faecalis cytochromes-c and three other cytochromes-c were tested for electron transfer activity with AADH. Azurin markedly facilitated electron transfer from AADH to each cytochrome. This suggests that AADH and azurin may form an electron transfer complex with a c-type cytochrome, analogous to the crystallographically determined MADH-amicyanin-cytochrome c-551i complex (Chen, L., Durley, R. C. E., Matthews, F. S., and Davidson, V. L.(1994) Science 264, 86-90). The similarities of MADH and AADH plus the demonstration of azurin and multiple cytochromes as functional electron-transfer partners suggest that both TTQ-bearing enzymes share common mechanisms for oxidative deamination and subsequent electron transfer.
A recent advance toward understanding the diversity of redox
mechanisms used in nature is the discovery that amino acids can be
modified for special catalytic purposes to act as novel cofactors. In a
few cases, there is sufficient structural information to unequivocally
define the cofactor and its attachment to the surrounding protein. An
example is the cofactor, tryptophan tryptophylquinone (TTQ), ()which is formed by the covalent attachment of one
tryptophan indole ring to a second tryptophan ring with one ring
oxidized to an orthoquinone (Fig. 1). This structure was deduced
by McIntire et al.(1991) as the cofactor for methylamine
dehydrogenase (MADH). How TTQ is formed is still unknown, but it has
been shown that the two tryptophans are encoded as normal amino acid
residues as determined by the DNA sequence of the MADH gene
(Chistoserdov et al., 1990). Recently, we have determined that
a related enzyme, aromatic amine dehydrogenase (AADH), also uses the
TTQ cofactor and is the second known example of a TTQ enzyme
(Govindaraj et al., 1994). AADH is expressed by the soil
bacterium, Alcaligenes faecalis, which allows it to break down
aromatic amine substrates for use as a source of carbon and energy.
These substrates are mainly
-phenylethylamines in which the phenyl
ring is hydroxylated as in dopamine and tyramine, for example (Iwaki et al., 1983).
Figure 1: The structure of TTQ.
Great strides have been made to elucidate
the structure of MADH and the TTQ cofactor. Crystal structures have
been reported for MADH from Thiobacillus versutus (Vellieux et al., 1986; 1989) and Paracoccus denitrificans (Chen et al., 1991, 1992b). Both structures confirm the
tetrameric geometry with the
cofactor included in the smaller subunit (
). The larger subunit
(
) displays the 7-fold, circular repetition of small
-sheets
that was first seen in the structure of influenza neuraminidase
(Varghese et al., 1983; Bossart-Whitaker et al.,
1993) and more recently in galactose oxidase (Ito et al.,
1991) and methanol dehydrogenase (Xia et al., 1992).
Both
MADH and AADH catalyze oxidative deamination. In MADH, it has been
suggested that the amine moiety of the substrate displaces a quinone
oxygen to form an iminoquinone intermediate as part of the mechanism
(Backes et al., 1991; Brooks et al., 1993). The
participation of TTQ is supported by the crystal structures that show
the cofactor near the surface of the subunit (near a cleft
between the
and
subunits, which would allow an amine
substrate to approach the cofactor). Furthermore, hydrazine inhibitors
have been used to show that carbon atom C-6 of TTQ is the probable site
of reaction (Huizinga et al., 1992). A crystal structure for
the binary complex between P. denitrificans MADH and its
electron receptor, amicyanin, reveals the copper atom of amicyanin
within 10 Å of the TTQ cofactor of MADH (Chen et al.,
1992a). Most recently, the structure of a ternary complex of MADH,
amicyanin, and cytochrome c-551i has been solved and suggests
the geometry for an electron transfer complex involving MADH,
amicyanin, and cytochrome c-551i (Chen et al., 1993;
1994).
This wealth of structural information for MADH prompts the questions (i) does AADH have a similar structure to MADH and (ii) does AADH also transfer electrons to a copper protein and/or a cytochrome as part of its catalytic mechanism? To address these questions, we have measured the circular dichroic spectra of AADH and MADH and performed a detailed analysis to determine the similarities of their structures. In addition, we have isolated an azurin and three c-type cytochromes from A. faecalis and tested their ability to function as electron acceptors for the AADH-catalyzed reaction. The ability of AADH and azurin to react with cytochrome c from other sources was also examined. The results of these experiments show that AADH and MADH share many properties and suggest that they may have evolved from a common ancestor.
Figure 2: SDS-PAGE of the large cytochrome from A. faecalis (lane 2), aromatic amine dehydrogenase (lane 3), and azurin (lane 4) analyzed on a 4-20% gradient gel. In lane2, the large cytochrome band at 38 kDa stained positively for heme; a faint band just below it did not. The molecular weight standards (Bio-Rad) in lane1 are phosphorylase B, 97,400; bovine serum albumin, 66,200; ovalbumin, 42,699; bovine carbonic anhydrase, 31,000; soybean trypsin inhibitor, 21,500; and hen egg white lysozyme, 14,400.
Figure 3:
Comparison of oxidized (solid
line) and reduced (dotted line) forms of AADH (reduced by
the addition of tyramine) (A), azurin (reduced with sodium
dithionite) (B), and large cytochrome (reduced with sodium
dithionite) (C). All of these proteins were isolated from the
same growth of A. faecalis cells (Institute for Fermentation,
Osaka, 14479) induced to express AADH with -phenylethylamine.
Spectra of the reduced forms of azurin and the cytochrome are identical
to ones resulting from AADH-catalyzed oxidation of
tyramine.
The electron transfer pathway for oxidative deamination involving the TTQ enzyme methylamine dehydrogenase is well understood biochemically, and the proteins that probably link this pathway to cytochrome oxidase have been elucidated with crystal structures (Chen et al., 1994). In contrast, the electron transfer partners for aromatic amine dehydrogenase were previously unknown, and possible structural similarities between AADH and MADH were poorly understood. We describe here proteins from A. faecalis that appear to parallel the electron transfer pathway from MADH to c-type cytochromes in P. denitrificans and present circular dichroic spectra that suggest strongly that the overall fold of AADH is like that of MADH and in particular that the environments of both TTQ cofactors are similar.
Figure 4:
Circular dichroic spectra of AADH and
azurin from A. faecalis compared with MADH and amicyanin from P. denitrificans. A, far UV spectra of AADH and MADH; B, far UV spectra of azurin and amicyanin. C, near
UV-visible spectra for oxidized and reduced MADH; D, near
UV-visible spectra for oxidized and reduced AADH. The insets of C and D show the oxidized minus reduced
difference spectra for MADH and AADH, respectively. The ellipticity,
(mean residue weight), is in units of degree
cm
/dmol.
We also compared the CD spectra of both proteins in the visible and near UV regions to assess the similarity of aromatic groups, including the TTQ cofactor (Fig. 4, C and D). The spectra of the oxidized proteins are very similar, and strong positive ellipticities were observed around 450, 290, and 262 nm. The band at 450 nm corresponds to the absorption peak (Fig. 3A) of the oxidized quinone moiety of the TTQ chromophore. Reduction of the dehydrogenases with dithionite in both cases caused loss of all three major bands with the appearance of positive bands at 333 and 290 nm (MADH) and 335 and 294 nm (AADH). These changes mirrored those in the UV-visible absorbance upon reduction (Fig. 3). A negative CD band at 272 nm (AADH) and 277 nm (MADH) was also observed. The signals at 272-277 and 290 nm are presumably derived from asymmetrically oriented tyrosine and tryptophan residues, respectively (Strickland, 1974). The oxidized minus reduced CD difference spectrum was nearly identical for both proteins (Fig. 4, C and D, inserts).
The near UV-visible CD spectral region of the dehydrogenases is clearly dominated in the oxidized proteins by the TTQ chromophore. The band positions and intensities are comparable enough to suggest that the orientation of TTQ is similar in both proteins. Denaturation of the oxidized proteins with 4 M guanidine HCl resulted in loss of all CD bands, except a weak negative one around 277-275 nm (data not shown). This band appears to correspond to the band seen in the reduced protein, although the intensity of the former is only about 15% of that in the native reduced proteins. From the above results, MADH and AADH have similar far UV-CD spectra consistent with related secondary structures. In the aromatic and visible regions, they also have similar CD spectra in the oxidized, reduced, and denatured states, suggesting homologous tertiary structures.
The
functional role of azurin was investigated by testing its ability to
receive electrons from AADH. Incubation of oxidized azurin with either
oxidized AADH or tyramine alone caused no change in the redox state of
azurin. However, when 5.5 µM azurin was incubated with
0.033 µM AADH in 0.1 M potassium phosphate
buffer, pH 7.5, addition of 50 µM tyramine caused complete
reduction of azurin as indicated by the complete loss of absorbance at
625 nm. Under these non-saturating conditions, the initial rate of
azurin reduction was 960 min.
A. faecalis has been reported to express both an azurin and a pseudoazurin,
which participate in various electron transfer pathways. For example,
azurin has been identified as the electron acceptor for the
molybdenum-containing enzyme, arsenite oxidase (Anderson et
al., 1992), while pseudoazurin acts as an electron donor for
nitrite reductase (Kakutani et al., 1981). Curiously, it was
observed that pseudoazurin in an aerobic solution containing ascorbate
or cysteine but lacking nitrite caused inactivation of nitrite
reductase. A suicide mechanism involving the production of peroxide has
been proposed (Kakutani et al., 1981). It should be noted that
no copper protein other than azurin was observed during the
purification of proteins from the -phenylethylamine-grown cells of A. faecalis.
The protein was analyzed by SDS-PAGE; the gel was lightly stained with Coomassie Blue and subsequently analyzed for heme with dimethoxybenzidine (Francis and Becker, 1984). The band at 38 kDa stained positively (green), indicating the presence of covalently bound heme. Control experiments with the same stain were positive for horse heart cytochrome c and negative for cytochrome c peroxidase, which has a noncovalently bound heme (data not shown).
The oxidized form of the cytochrome has a
prominent absorption peak at 408 nm and a broad band centered around
550 nm (Fig. 3). It could be reduced by the addition of a few
grains of solid sodium dithionite, which shifted the soret peak to 418
nm and induced the appearance of two peaks at 523 and 553 nm. These
spectral properties and the covalent attachment of a heme are
characteristic of c-type cytochromes. A cytochrome with
similar spectral properties was observed in the study of arsenite
oxidase from A. faecalis (Anderson et al., 1992).
Whether this is the same protein cannot be determined at present since
the cytochrome described in that study was not extensively purified. ()Amino terminal peptide sequencing of the first 20 residues
showed no homology to any known cytochrome. (
)
A sample of the cytochrome was used to test its ability to receive electrons from AADH. When the cytochrome was incubated with tyramine and AADH alone, no change in the spectrum occurred. When azurin was included in the same reaction mixture, but prior to adding the cytochrome, the spectrum quickly changed to be identical to that of the reduced cytochrome (Fig. 3). However, when azurin was added after the cytochrome had been incubated with AADH, the spectrum of the cytochrome slowly changed, showing the appearance of the 523- and 553-nm peaks, but the soret peak shifted only slightly from 408 to 410 nm. In both cases, when the reduction of the cytochrome appeared complete, a minimal amount of the oxidizing agent potassium ferricyanide was added, which caused full reoxidation of the cytochrome as judged by its spectrum. Subsequently, full reduction could be achieved by adding a few grains of dithionite.
Three well characterized cytochromes from other sources, horse heart cytochrome c and cytochromes c-550 and c-551i from P. denitrificans, were also tested for electron transfer ability (Table 2). With two of these cytochromes, very little reaction occurred when each was incubated with AADH and tyramine. On addition of azurin, the rates increased approximately 50-fold for horse heart cytochrome c and 100-fold for cytochrome c-550. Cytochrome c-551i showed significant electron transfer ability with AADH in the absence of azurin but also exhibited a 4-fold stimulation in rate when azurin was present. Thus, with each of the c-type cytochromes that were examined, azurin was demonstrated to mediate electron transfer from AADH, and with most of the cytochromes, azurin was absolutely required to observe any significant tyramine-dependent cytochrome reduction by AADH.
Electron transfer interactions between blue copper proteins and cytochromes have long been recognized as some of the many complex redox pathways manifested by denitrifying bacteria. In P. denitrificans, amicyanin may transfer an electron to cytochrome c-550 or c-551i after reduction by MADH (Husain and Davidson, 1986). These cytochromes presumably transfer electrons to cytochrome oxidase. A similar system appears to operate in Alcaligenes. As shown here, azurin is capable of mediating electron transfer from AADH to three endogenous cytochromes. Cytochromes from P. denitrificans were also able to accept electrons from azurin in the AADH-catalyzed reduction of tyramine. We note that a variety of non-physiological combinations of azurins with cytochromes c have already been studied as model systems for investigating structural aspects of electron transfer processes (Wherland and Pecht, 1978). The new information presented here will provide a basis for future studies of interactions between this azurin and its physiological as well as non-physiological redox partners.
In
addition, the current experiments show that AADH uses the blue copper
protein, azurin, to mediate electron transfer to cytochromes homologous
to the role of amicyanin in P. denitrificans. These structural
and functional similarities suggest that AADH and MADH may have evolved
from a common ancestor to yield enzymes with distinct substrate
specificities. Information for understanding this specificity is
suggested by a new high resolution structure of MADH. It
shows that the methylamine binding pocket is small, which helps to
explain why MADH does not accept large substrates like tyramine. AADH
probably has a different binding pocket geometry designed to accept
larger substrates, and it will be interesting to see how this binding
pocket selects against methylamine as a substrate. A final speculation
is prompted by the recently determined crystal structure of a ternary
complex between MADH, amicyanin, and cytochrome c-551i (Chen et al., 1994). Assuming that AADH would form a similar complex
with azurin and a cytochrome, how would the known difference in
structure between azurin and amicyanin affect it? The most significant
difference is that azurin has two helices near the copper binding site
missing in amicyanin. The position of these helices would not appear to
affect the interaction with AADH but would more likely affect the
interaction with a cytochrome. Hopefully, more direct information will
come from crystal structures of AADH and its complex with azurin and
from solution studies of the electron transfer reaction between AADH
and azurin, which we are currently pursuing.