Nov 27, 1989 - Yates for comments on an earlier version of this manuscript. Michael E. BAKER. Department of Medicine, M-023,. University of California, San ...
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Biochem. J. (1990) 267, 839-841 (Printed in Great Britain)
n Bl$EHtLETTERS
JuRSLL Sequence similarity between Pseudononas dihydrodiol dehydrogenase, part of the gene cluster that metabolizes polychlorinated biphenyls, and dehydrogenases involved in metabolism of ribitol and glucitol and synthesis of antibiotics and 17fl-oestradiol, testosterone and corficosterone There has been intense interest in characterizing the gene clusters found in Pseudomonas pseudoalcaligenes and Pseudomonas putida because they enable these organisms to metabolize completely various aromatic hydrocarbons such as biphenyl, polychlorinated biphenyl (PCB), toluene, and naphthalene [1-4] or convert them to less toxic forms that can be safely dispersed in the environment [5,6]. The genes for these enzymes have been cloned from P. pseudoalcaligenes (1] and P. putida [2,3], and they consist of several proteins that catalyse various steps in the oxidation of aromatic hydrocarbons to simpler molecules that can be used by the micro-organism as a source of carbon and energy. We reported [7,8] that 17,6-hydroxysteroid dehydrogenase [9], an enzyme important in the synthesis of 17fl-oestradiol and testosterone, is homologous to actIII, an enzyme important in antibiotic synthesis in Streptomyces coelicolor [10,11]. Moreover, these two proteins share a common ancestor with rat corticosteroid 1 I/J-hydroxysteroid dehydrogenase [8,12], Escherichia coli glucitol-6-phosphate dehydrogenase [13] and Klebsiella aerogenes ribitol dehydrogenase [14], enzymes that are used for metabolizing polyols as a source of carbon and energy, as well as to Rhizobia proteins that are part of the gene cluster that induces root nodulation in Rhizobia's host [15-17]. Recently, we searched the database for other members of this protein superfamily and uncovered a novel similarity between P. pseudoalcaligenes and P. putida dihydrodiol dehydrogenase and actIII, 17,J-hydroxysteroid dehydrogenase, and other members of their protein superfamily. Later, as this manuscript was being completed, Zylstra & Gibson published the complete sequence of P. putida dihydrodiol dehydrogenase [3]. Their comparison of this sequence with P. pseudoalcaligenes dihydrodiol dehydrogenase indicated that the N-terminus of P. pseudoalcaligenes dihydrodiol dehydrogenase began at position 34 in the sequence determined by Furukawa et al. [1]. Thus, we adjusted the numbering for the residues in P. pseudoalcaligenes dihydrodiol dehydrogenase in the alignment with actIlI and 17/1hydroxysteroid dehydrogenase and other members of this superfamily to conform to the numbering of Zylstra & Gibson [3]. In addition, we did a comparison of P. putida dihydrodiol dehydrogenase, which has about 65 % sequence similarly to P. pseudoalcaligenes dihydrodiol dehydrogenase, with proteins in the actIII/17f7-hydroxysteroid dehydrogenase superfamily. The alignment of residues 6-259 of S. coelicolor actIlI and Vol. 267
residues 5-254 of P. pseudoalcaligenes dihydrodiol dehydrogenase is shown in Fig. 1. Out of 244 possible matches there are 70 (28.5 %) identities and 46 (19%) conservative replacements. The ALIGN [18] score is 11.I standard deviations higher than that obtained with 1000 comparisons of randomized sequences of these proteins. The probability (P) of getting such a score by chance is 6 x 10.29 Table 1 summarizes the comparison scores for P. pseudoalcaligenes and P. putida dihydrodiol dehydrogenase with human 17,8-hydroxysteroid dehydrogenase [9], Escherichia coli glucitol-6-phosphate dehydrogenase [13], Klebsiella aerogenes ribitol dehydrogenase [14], Rhizobium meliloti nodG protein [15,16], Bradyrhizobium japonicum fixR protein [16], Eubacterium bile acid 7-dehydroxylase [19], Bacillus megaterium glucose dehydrogenase [20] and mouse adipocyte p27 protein
[21].
The region of similarity between P. pseudoalcaligenes dihydrodiol dehydrogenase and actIII comprises 250 residues, which is 92% of the 273 residues of the dihydrodiol dehydrogenase and 95 % of the 261 residues of actIl. With the exception of human 17fl-hydroxysteroid dehydrogenase, which contains 327 residues, the lengths of most of the other dehydrogenases listed in Table 1 vary from 244 residues (mouse adipocyte p27 protein) to 278 residues (fixR). In several proteins, the segments that are similar to dihydrodiol dehydrogenase are greater than 200 residues. These include glucitol-6-phosphate dehydrogenase 257-residue segment, bile acid 7-dehydroxylase 217-residue segment and nodG 228-residue segment, which constitute over 60% of these proteins. The other proteins have segments of at least 165 residues that are similar to dihydrodiol dehydrogenase. The proteins with the lowest ALIGN comparison scores are human 17fl-hydroxysteroid dehydrogenase and rat 1 1/1-hydroxysteroid dehydrogenase. The latter protein has ALIGN scores of 6.3 S.D. and 6.1 S.D. with the dihydrodiol dehydrogenases of P. putida and P. pseudoalcaligenes, respectively. Steroid affinity labeling of 17,l-hydroxysteroid dehydrogenase has identified residues 204-223 as being part of the steroid-binding domain [22]. Thus, the similarity between 17,1hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase decreases near the steroid-binding domain. Most of the proteins listed in Table 1 are oxidoreductases. This similarity in enzyme function to the two Pseudomonas dihydrodiol dehydrogenases, combined with the strong ALIGN comparison scores, several of which are over 10 standard deviations, suggest that the dihydrodiol dehydrogenases share a common ancestor with the proteins in Table 1. Metabolism of toluene and PCBs begins with conversion of these compounds to a dihydrodiol by a dioxygenase. For example, toluene is oxidized to cis-toluene dihydrodiol through the addition of molecular oxygen to the aromatic nucleus. This compound is then reduced by the dihydrodiol dehydrogenase to 3-methylcatechol, using NADI as the cofactor. The similarities reported here suggest a lik-ely location of the NADI binding site. This information comes from Yamada & Saier's comparison [13] of the sequences of E. coli glucitol-6-phosphate dehydrogenase
BJ Letters
840 G E A V L I T G G A S G L G R A L V D R F S E V A L V T G A T S G I G L E I A R R L * SG R G * * E * * L * T G
Dihydrodiol ActIII
S 6
Dihydrodiol ActIlI
V A E A - K V A V L D K S A E R L A E L E T G K E G L R V F V C A R G E E G L R T T L K * E L V V E
Dihydrodiol ActIII
D V R S D L G D N V L G I V G . . E L R E A G V E A D G R T C D V R S * D V R S G L
Dihydrodiol ActIII
K Q A A S R C V A R F G K I D T L I P N A G E A L V A A V V E R Y G P V D V L V N N A G * D V N A G R * G L *
Dihydrodiol ActIlI
I -
Dihydrodiol ActIII
A F D E V F H I N VK G Y I N L T G V F R V T KV F
Dihydrodiol ActIII
L V A S R G N V I F T I S L E R G T G R I V N I A S Jt * * S L G
-
L E D Q V P E I
. . -S L D A W D Y S T A L V D L P E E R P G G G A T A E L A D E L W L D V V E T * * * AE A TAEL
H A V K A L P A Q V L K A G G M * K A
N A G F Y P N G G T G G K Q G V V H
G
Dihydrodiol ActIII
G P L Y T A A K Q A I V G L V R E L A F E L A A P Y S A S K H G V V G F T K A L G L E L *VG * Y * A K L E L
Dihydrodiol ActIII
A P Y - V R V N G V G P G G M N S D M R G P A R T G I T V N A V C P G F V E T P M A A S V V N A p G M *
Dihydrodiol ActIII
S S L G M G S K - A I S T V P L A D M L K S V R E H Y S D I W E V v S S T E E A F D R I T A * D T
Dihydrodiol
V L P I
ActlIl
R V P I * P. I
Dibydrodiol
R G D A A P A S G A L V N Y D G G L G 2S4 P G A A A V T A Q A L N V C - G G L G 259 A L A A G G G L G
ActIII
G R M P E V E E Y T G A Y V F F A T G R Y V Q P S E V A E M V A Y L I G E G R
Fig. 1. Alignment of Pseudomonas pseudoalcaigenes dihydrodiol dehydrogenase with Streptomyces coelicolor actHl protein Identities are noted in the space below the sequences; an asterisk (*) denotes conservative replacements. Out of 244 possible matches there are 70 (28.5 %) identities and 46 (19 %) conservative replacements. An ALIGN analysis of these sequences, with a gap penalty of 8, yields a score that is I .1 standard deviations higher than that obtained with 1000 comparisons of randomized sequences of these segments. The probability of getting such a score by chance is 6 x 10-29.
and K. aerogenes ribitol dehydrogenase with a consensus sequence of nucleotide binding domains from dehydrogenases, developed by Wierenga et al. [23], from which they concluded that the N-terminal 30 residues of these two dehydrogenases contain the nucleotide binding domain. Coleman et al. [19] and Jornvall et al. [24] reached a similar conclusion for the location of the nucleotide binding domain in bile acid 7-hydroxylase and for glucose dehydrogenase. It seems likely that the N-terminal
part of dihydrodiol ing domain.
dehydrogenase contains the nucleotide bind-
The similarities reported here indicate that oxidoreductases for group of substrates that includes: metabolism of
a diverse
polyols (e.g. ribitol, glucitol), aromatic hydrocarbons (toluene,
naphthalene, polychlorinated biphenyls), over a dozen Streptomyces antibiotics, and steroids (e.g. 17,8-oestradiol, testosterone, corticosterone) are derived from a common ancestor. Moreover, the organisms containing these enzymes appear to use them for different biological functions. They can be used to supply a source of carbon and energy for cell growth (e.g. glucitol-6-phosphate dehydrogenase, ribitol dehydrogenase) or they can be used to synthesize molecules that act as intercellular signals (e.g. actIII, 17fl-hydroxysteroid dehydrogenase, 11/,hydroxysteroid dehydrogenase) [8]. This is a very impressive example of how gene duplication and divergence can lead to a shift in the function of the enzyme from metabolism to that of synthesis. It is this shift that we proposed was important in the 1990
BJ Letters
841
Table 1. ALIGN comparisons of Pseudomonas dihydrodiol dehydrogenases with Streptomyces coelicolor actilI protein, human 17f1-ydroxysteroid dehydrogenase, various dehydrogenases, Rhizobia proteins, ad adipocyte p27 protein The ALIGN analysis was used with the Dayhoff scoring matrix with a bias of 6 and a gap penalty of 8; 1000 comparisons of randomized sequences of these proteins were use for statistical analysis.
Dihydrodiol dehydrogenase
S. coelicolor actIII K. aerogenes ribitol dehydrogenase E. coli glucitol-6-phosphate dehydrogenase Human 17fl-hydroxysteroid
dehydrogenase Eubacterium bile acid 7-dehydroxylase R. meliloti nodG protein B. japonicumfixR protein Mouse adipocyte p27 protein B. megaterium glucose dehydrogenase
P. pseudoalcaligenes
P. putida
11.1 10.7 9.3
12.0 9.95 8.1
7.7
6.35
12.7 11.85 9.5 10.3 10.25
14.4 9.8 9.45 11.55 11.6
origins of stable molecules that were adapted for use in intercellular communications, as seen in Rhizobia-plant interactions and, of course, in steroid-mediated processes in mammals [8]. M. E. B. is a Visiting Scientist in the laboratory of Dr. Joseph J. Salvo in the Biological Sciences Laboratory, GE Research and Development, whose support is appreciated. I thank D. Abramowicz, J. Salvo, and J. Yates for comments on an earlier version of this manuscript.
Michael E. BAKER Department of Medicine, M-023, University of California, San Diego, La Jolla, CA 92093, U.S.A. Received 27 November 1989
Vol. 267
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