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JOuRNAL OF BACTERIOLOGY, Apr. 1981, p. 233-238
0021-9193/81/040233-06$02.00/0

Vol. 146, No. 1

Evolutionary Relationships Among y-Carboxymuconolactone
Decarboxylases
WU-KUANG YEH,* DON R. DURHAM,t PAUL FLETCHER,4 AND L. NICHOLAS ORNSTON
Department of Biology, Yale University, New Haven, Connecticut 06511
Received 17 October 1980/Accepted 19 January 1981

Acinetobacter calcoaceticus ADP 152 (17) were described recently. For immunodiffusion studies, the appropriate strains of A. vinelandii, P. putida, and A.
calcoaceticus were grown in 500-ml Erlenmeyer flasks
containing 150 ml of mineral medium (5, 14) supplemented with 10 mM p-hydroxybenzoate.
Purification of ycarboxymuconolactone decarboxylase from A. vinelandii All purification
procedures were performed between 0 to 40C. Buffer
A was 20 mM Tris-hydrochloride (pH 7.5) containing
25 pM dithiothreitol; buffer B was 10 mM Na2HPO4KH2PO4 (pH 7.0); and buffer C was 20 mM Trishydrochloride containing 0.1 M NaCl. Cell pastes with
a wet weight of 200 g were suspended in three volumes
of buffer A and disrupted by passage through an
American Instrument continuous-flow French pressure cell at 12,000 lb/in2. Unbroken cells and debris
were removed by centrifugation at 40,000 x g for 20
min. The resultant crude extract (Table 1 step 1) was
brought to 30% saturation by the addition of ammonium sulfate followed by centrifugation at 40,000 x g
for 20 min. Ammonium sulfate treatment was repeated
on the supernatant fraction until 75% saturation was
reached. After centrifugation, the protein pellet was
dissolved in buffer A (Table 1, step 2) and dialyzed
against three changes of this buffer over 48 h. The
dialysis was applied onto a DEAE-cellulose column
(5 by 30 cm) previously equilibrated with buffer A.
The column was washed with three volumes of the
tobacter vinelandii OP was obtained from Paul E. same buffer, after which a continuous linear gradient,
Bishop, North Carolina State University, Raleigh, constructed from 0 to 0.3 M NaCl in buffer A in a total
N.C. Large-scale growth of A. vinelandii was accom- volume of 6 liters, was applied. Fractions of 15 ml were
plished at 30°C in a New Brunswick Fermacell CF-130 collected at a flow rate of 60 ml/h, and those containfermentor containing 100 liters of modified Burk me- ing y-carboxymuconolactone decarboxylase activity,
dium (14). Pseudomonas putida PRS 2260 (4) and which eluted between 0.16 to 0.19 M NaCl, were pooled
t Present address: Department of Microbiology, Medical (Table 1, step 3). The DEAE-celluose eluate was
College of Virginia, Virginia Commonwealth University, Rich- treated with ammonium sulfate, and the protein pellet
that precipitated between 45 to 75% saturation was
mond, VA 23298.
$ Present address: Department of Microbiology, East Car- dissolved in buffer B (Table 1, step 4) and dialyzed
olina University, Greenville, NC 27834.
thoroughly against the same buffer. The dialysate was
233

Immunological comparisons conducted with
protocatechuate oxygenase (EC 1.13.11.3) (2)
and y-carboxymuconolactone decarboxylase
(EC 4.1.1.44) (1), enzymes of the protocatechuate branch of the ,-ketoadipate pathway, indicated that the Azotobacter species enzymes are
evolutionarily homologous to isofunctional enzymes formed by fluorescent Pseudomonar species. As described here and elsewhere (1, 10, 13),
isofunctional enzymes formed by members of
other bacterial genera appear to be immunologically distant, and these organism govern
expression of the enzymes with induction patterns unlike those shared by Azotobacter and
Pseudomonas species (12). The conclusions
drawn from immunological evidence are supported by chemical and physical data presented
here. The results show that the y-carboxymuconolactone decarboxylases of Azotobacter and
Pseudomonas species are closely related to each
other and more distantly related to the evolutionarily homologous y-carboxymuconolactone
decarboxylase of Acinetobacter species.
MATERIALS AND METHODS
Bacterial strains and growth conditions. Azo-

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-y-Carboxymuconolactone decarboxylase (EC 4.1.1.44) from Azotobacter vinehndii resembled the isofunctional enzymes from Acinetobacter calcoaceticus
and Pseudomonas putida. All three decarboxylases appeared to be hexamers
formed by association of identical subunits of about 13,300 daltons. The A.
vinelandii and P. putida decarboxylases cross-reacted immunologically with each
other, and the NHrterminal amino acid sequences of the enzymes differed in no
more than 7 of the first 36 residues. In contrast, the A. calcoaceticus decarboxylase
did not cross-react with the decarboxylase from A. vinelandii or P. putida; the
NH2-terminal amino acid sequences of these enzymes diverged about 50% from
the NH2-terminal amino acid sequence of the A. calcoaceticus decarboxylase.

234

YEH ET AL.

J. BACTERIOL.

TABLE 1. Purification of y-carboxymuconolacione decarboxylase from A. vinelandii
Step
1. Crude extract ....................
2. First 30 to 75% saturated ammonium
sulfate fraction .................
.......
3. First DEAE-cellulose eluate
4. Second 45 to 75% saturated ammonium sulfate fraction ............
5. Second DEAE-cellulose eluate .....
6. Third 40 to 65% saturated ammonium
sulfate fraction ...............
7. Sephadex G-200 elugte ....... .....
8. Sephadex G-100 eluate ....... .....
9. Quaternary aminoethyl-Sephadex
eluate .........................

Volume Total activity Total protein
(mg)
(U)
(ml)
17,670
34,000
610

8,290

Sp act
(U/mg)
1.92

100

Purification
(fold)
1.0

Yield (%)

32,000

1,575

3.63
20.3

89
94

1.9
10.6

33
120

31,350
26,400

726
356

43.2
74.2

92
78

22.5
38.6

7
32
35

23,920
19,840
12,600

248
30.4
10.0

96.5
653
1,260

70
58
37

50.3
340
656

12

6,040

4.8

1,258

18

655

30,130

applied onto a second DEAE-cellulose column (1.6 by
20 cm) previously equilibrated with buffer B. The
column was washed with 2 volumes each of 0.01, 0.05,
and 0.1 M sodium potassium phosphate buffer, and
then a continuous linear gradient, constructed from
0.1 M sodium potassium phosphate buffer to the same
buffer containing 0.3 M NaCl in a total volume of 300
ml, was applied. Fractions of 3 ml were collected at a
flow rate of 20 ml/h, and those containing the decarboxylase activity were pooled (Table 1, step 5) and
fractionated with ammonium sulfate. The protein pellet that precipitated between 40 to 65% saturation was
dissolved in buffer A (Table 1, step 6) and applied
onto a Sephadex G-200 column (2.5 by 100 cm) previously equilibrated with buffer A. Fractions of 3 ml
were collected at a flow rate of 14 ml/h, and those
containing a specific activity of the decarboxylas6
greater than 600 U/mg were combined (Table 1, step
7) and concentrated with ammonium sulfate (0 to 70%
saturation). The resultant pellet was dissolved in
buffer A and loaded onto a Sephadex G-100 column
(2.5 by 40 cm) previously equilibrated with buffer A.
The eluate containing the decarboxylase activity (Table 1, step 8) was concentrated with ammonium sulfate
(O to 70% saturation), dialyzed thoroughly against
buffer C, and applied onto a quaternary aminoethylSephadex column (0.9 by 8 cm) previously equilibrated
with buffer C. The column was washed with 10 volumes of the same buffer, and then a continuous linear
gradient, constructed from 0.1 to 0.4 M NaCl in 20
mM Tris-hydrochloride (pH 7.2) in a total volume of
50 ml, was applied. Fractions of 2 ml were colletted at
a flow rate of 10 ml/h, and those containing the
decarboxylase activity were pooled (Table 1, step 9).
The quaternary aminoethyl-Sephadex eluate was
stored at 40C in the presence of ammonium sulfate at
70% saturation.
Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis was performed with 10% disc
gels (9). The subunit size of A. vinelandii decarboxylase was estimated by sodium dodecyl sufate-gel electrophoresis (15) with previously described standard
proteins (17).
Amino acid analysis. The amino acid composition
of A. vinelandii decarboxylase was determined by a
previously described procedure (4) and analyzed with
a computer program (8) for the minimum molecular
weight, which is the subunit size, of the enzyme.

Njlrterminal amino acid sequence determination. Previously published procedures (16) were
used for determination of the NHr-terminal amino
acid sequence of A. vinelandii decarboxylase.
Serological studies. Antisera against A. vinelandii, P. putida, and A. cakoaceticus decarboxylases

were prepared (2), and the method of Stanier et al.
(13) was used to examine serological cross-reaction on
Ouchterlony double-diffusion plates (7).
Chemicals. Chemicals were described previously
(2, 17). Quaternary aminoethyl-Sephadex was obtained from Pharmacia Fine Chemicals.

RESULTS

Purity of A. vinelandiU decarboxylase.

The most purified preparation of A. vinelandii
decarboxylase (Table 1, step 9) possessed a specific activity of 1,260 U/mg. When subjected to
electrophoresis on 10% polyacrylamide gels, the
decarboxylase preparation migrated as a major
band and a slight minor band (Fig. 1). The minor
band may have been an electrophoretically different form of the decarboxylase because, as
described below, the preparation was immunologically homogeneous, and the NH2-terminal
amino acid sequence of the enzyme was determined without detectable contamination.
Molecular weight and subunit size determinations. The molecular weight of the A.
vinelandii decarboxylase, close to the molecular
weights of the A. calcoaceticus and P. putida
decarboxylases, was estimated to be 85,500 by
gel filtration (Fig. 1). The size of the A. vinelandii decarboxylase subunit was determined as
13,300 by sodium dodecyl sulfate-gel electrophoresis and as 13,460 by computer-aided analysis
of the amino acid composition of the enzyme (8).
Serological properties. Antisera prepared
against the purified A. vinelandii decarboxylase
formed a single precipitin band when diffused
against a crude extract of A. vinelandii cells in
which the enzyme had been induced (Fig. 2).
This band formed a spur with a precipitin band
formed by a P. putida extract containing the

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310
500

.

RELATIONSHIPS AMONG BACTERIAL ENZYMES

VOL. 146, 1981

>seLi"ova.;

3

c

2
zo

leY_waehd _Y&Gem_ 15t0001
*p_nuut-l,$-d_phwhW 130,000

0

C

31

3

ex trac,

-j

t

7-

c cxr
ruse ex
ra

Al 17,0_

Xv4iSoVsati kimsu

A4e I n e to ba
;w1;'. t er
*; cr;
e e - tr ;acr

60,000

X _~~~~~~~4600
-- -4S/
otwto
65

20
'A

\t

o.
d

tsba5 cter

n

7S

t:

"""~mm Ke,OOO

aW
4

Ac itie C kI

n a S'

rulee

235

2~eus dwjmI

._

U

pr*g
$). re-n Z.vr

kxtr<,

12

aneota;<.

e

030

0GM

045

p,ritire

20

LO

.

eanzym

Oc

A putre ena72c

Kav

FIG. 1. Insert depicts

a

stained 10% polyacryl-

50 pg of A. vinelandii decarbox9) had migrated electrophoretically. The graph shows data indicating the molecular
weights ofdecarboxylases determined by filtration on
a standardized Bio-Gel agarose A 1.5m column (2.6

amide gel on which
ylase (Table 1, step

by 100 cm).

FIG. 2. Double-diffusion plates showing the unmunological cross-reactions of enzymes with A.
vinelandii decarboxylase. The center wells received
0.2 ml (containing approximately 3 mg ofprotein) of
antiserum against the A. vinelandii decarboxylase.
The outer weUs of the top plate received crude extracts of cells in which the decarboxylase had been
induced bygrowth withp-hydroxybenzoate. The outer
wells of the bottom plate received the indicated
amounts ofpurified decarboxylases.

decarboxylase (Fig. 2). No precipitin band was
formed with extracts of uninduced A. vinelandii
and P. putida cells (not shown) or with A. calcoaceticus extract containing the enzyme (Fig.
2). Similar precipitin patterns were formed when
purified decarboxylase was substituted for crude
seudr£ .)n i
extract in the outer welaLLFig. 2). Thus, the A.
----?.];l pt
Stre enz ,: e
Psaeudomonas
vinelandii decarboxylase appears to be an img pure enzyfie
munologically homogeneous preparation that
cross-reacts strongly with the P. putida decarboxylase and not at all with the A. calcoaceticus
decarboxylase.
The conclusion that the A. vinelandii and P.
putida decarboxylases resemble each other more
closely than they resemble the A. calcoaceticus
decarboxylase is fortified by the immunodiffusion patterns shown in Fig. 3. Antisera prepared
against the P. putida decarboxylase form a precipitin band with the P.putida andA. vinelandii
decarboxylases, but not with the A. calcoacetiAzo
P-seuidomoolas
tno bac t ear
p1 re a- .z* e
cus decarboxylase (Fig. 3). Antisera prepared
p.g pure. nznaave
against the A. calcoaceticus decarboxylase
FIG. 3. Double-diffusion plates on which purified
formed a precipitin band with this enzyme, but
from different bacterial genera were
not with the corresponding decarboxylase from decarboxylases
diffused against antiserum prepared against P. puA. vinelandii or P. putida (Fig. 3).
tida decarboxylase (center well, top) and against
Amino acid composition. The amino acid
antiserum prepared against A. calcoaceticus decarcomposition of the A. vinelandii decarboxylase boxylase (center well, bottom).
is shown in Table 2. Quantitative comparison of
amino acid compositions may be achieved by hydrolysate. In over 5,000 pairwise comparisons,
measurement of SAQ, the sugm of the square of Marchalonis and Weltman (3) found that an
the difference in mole fraction of each amino SAQ of less than 50 invariably reflected a strucacid that can be readily quantitated in a protein tural similarity that was revealed by comparison
P

20

20

Downloaded from http://jb.asm.org/ on March 4, 2018 by guest

z.o tobac ter

20 :

025

. . .-.

]
r-~
us

0

;

020

nas
>1e"0
te
axac
eX
Ac in^^

EL.^

236

YEH ET AL.

J. BACTIOL.

TABLE 2. Amino acid composition of A. vinelandii

y-carboxymuconolactone decarboxylase
Amino acid

Asx
Thr
Ser
Glx
Pro
Gly
Ala

Amino acid residues per
13,460 daltonse

position (8).
b Estimated as cysteic acid after performic acid oxidation (4).
c
Determined after hydrolysis of a protein sample
with 3 N mercaptoethanesulfonic acid (4).

of amino acid sequences. With the A. vinelandii
decarboxylase as reference we found that SAQs
for the amino acid compositions of the A. calcoaceticus and P. putida decarboxylases were 9
and 24, respectively.
NH2-terminal amino acid sequence. Quantitative measurements for the first 49 cycles in
the NH2-terminal amino acid sequence determination of the A. vinelandii decarboxylase are
shown in Table 3.

DISCUSSION
Relationships among y-carboxymuconolactone decarboxylases from A. cacoaceticus, A. vinelandii, and P. putikL y-Carboxymuconolactone decarboxylases from the three
bacterial genera shared some properties: the enzymes appeared to be hexamers formed by association of identical subunits of about 13,300
daltons (11), and the amino acid compositions of
the proteins were similar (17). On the other
hand, the specific activity of the A. calcoaceticus
decarboxylase (140 U/mg) was substantially
lower than the specific activities of the A. vinelandii and P. putida decarboxylases (1,260 and
1,310 U/mg, respectively), and the latter two
decarboxylases cross-reacted immunologically
with each other, but not with the A. calcoaceticus decarboxylase. Thus, it appears that all
three decarboxylases were derived from a com-

46
47
48
49
a

LI (138)

G (16)
D (10)
LI (67)

G (12)
D (11)
I (8)

(Cys or
Ser)'
Gly
Asp
Ile

PTH, Phenylthiohydantoin.

bBH-ORG, Back hydrolysis-extracted organic phase.
BH-AQU, Back hydrolysis-remaining aqueous pham.
d ResultS
indicate the singe-letter amino acid designation
c

and (within parentheses) the number of nanomoles recovered.
"The only two amino acid residues that could not be
identified by the procedures used in this sequence determination are cysteine and serine.
fPTH-leucine and PTH-isoleucine were coeluted by the
gas chromatographic technique.
' PTH-asparagine was distinguished from PTH-asparatic
acid by high-pressure liquid chromatography.

Downloaded from http://jb.asm.org/ on March 4, 2018 by guest

11.26
5.95
6.10
..................
15.14
..................
4.95
..................
9.86
..................
13.17
..................
1.14
Cys
9.04
Val ..................
3.05
Met ..................
6.21
Ile .
....................
11.70
Leu ..................
2.63
Tyr ..................
3.66
Phe ..................
4.12
His ..................
4.76
Lys ..................
7.91
Arg ..................
1.18
TrpC ..................
a From computer-aided analysis of amino acid com..................
..................

TABLE 3. Automated sequence analysis of A.
vinelandii 'y-carboxymuconolactone decarboxylase:
identification of amino acid residues
Cycle PTH- Me3Si BH_ORGb BH-AQUe Residue
Met
1 M (193)d M (181)
D (125)
D (73)
2
Asp
E (166)
E (68)
Glu
3
K (37)
4
Lys
E (125)
E (68)
Glu
5
6
R(17) Arg
R(4)
Y (101)
Y (46)
7 Y (18)
Tyr
D (65)
D (47)
8
Asp
A (115)
A (53)
Ala
9 A (131)
G (106)
G (66)
10 G (129)
Gly
M (82)
Met
11 M (66)
Q (20)
E (49)
12
Gln
E (36)
V (33)
Val
V (153)
13 V (208)
R (8)
R (3)
14
Arg
R (3)
R (14) Arg
15
A (104)
A (47)
Ala
16 A (114)
V (75)
V (33)
Val
17 V (97)
L (35)
Leu
18
G (33)
G (26)
19 G (38)
Gly
D (31)
D (25)
20
Asp
A (85)
A (43)
Ala
21 A (72)
H (7)
His
22
V (26)
Val
V (145)
23 V (230)
D (48)
D (25)
24
Asp
R (2)
R (7)
25
Arg
26
(Cys or
Ser)'
L (26)
Leu
27 LIf (181) LI (119)
K (10)
28
Lys
D (11)
D (22)
Asnw
29
L (25)
Leu
30 LI (163) LI (117)
Thr
T (14)
31
Pro
P (11)
32 P (39)
F (14)
Phe
F (34)
33 F (32)
D (20)
Asne
D (7)
34
E (23)
Glu
E (46)
35
E (25)
Glu
E (42)
36
F (11)
Phe
F (31)
37 F (32)
E (22)
Gln
38
Q (5)
E (26)
Glu
E (24)
E (30)
39
Met
M (14)
40 M (9)
Be
LI (59)
41 LI (97)
I(11)
Thr
T (12)
42
Arg
R(3)
43
R(1)
H (3)
His
44
A (19)
A (45)
45
(Ala)

VOL. 146, 1981

4), whereas comparison of the A. vinelandii

the structural genes for the A.
vinelandii and P. putida decarboxylases seem
to have diverged recently relative to their divergence from the A. calcoaceticus decarboxylase.
These conclusions are strengthened by comparison of the NH2-terminal amino acid sequences
of the decarboxylases. The data allow a threeway comparison of 34 of the first 36 residues.
The A. vinelandii and P. putida sequences were
identical in 83% of the compared positions (Fig.
mon ancestor;

t
DecarbaKyffisa
Dwcarblzy1a"

1
tMi

2

ASP

sequence with the A. calcoaceticus sequence
reveals an identity of 47% (Fig. 5).
Early events in the evolution of y-carboxymuconolactone decarboxylases and muconolactone isomerases. y-Carboxymuconolactone decarboxylases and muconolactone isomerases, enzymes that mediate analogous biochemical reactions, appear to share a common
ancestral gene. Alignment of the NH2-terminal

8 9 10 11 12 13 14 15 16 17 18
3
6
7
TYR ASP ALA CLY MET GLN VAL ARC ARC AIA VAL L0
GW
LT4 GLU ARG

1 2 3
MT ASP OLU

5
LYS

8

7

6

CLE ARG

9

TYR ASP ALA

10 11 12 13 14 15 16 1718
GLY MET GLE VAL ARG ARG ALA VAL LWU

25

Asotct r

Docarbcay

237

CYS LIU

GLY

ASP ALA HIS VAL ASP ARC

19
GLV

ASP ALA HIS VAL ASP ARC CYS LW

20 21

22 23

PM

LVU PM

LYS ASK

AS

OW

OLU

25 26 27 28 29 30 31 32 33 34 35 36
CLY LYS L ASK ASP
CLY

24

FIG. 4. Comparison the NH2-terminal amino acid sequences of A. vinelandii and P. putida y-carboxymuconolactone decarboxylases. Identical residues are enclosed in boxes.

of

1

D

t

ecarb

2

6

3

4 5
[7 ASPGLU LYS GLV AG

Dcarzy
.
1Mr

""e e

1

2
ASK

3

4

ASP

a.y l

Aotoctr
D carboxyl

19

GLT

20

21 22

ASP ALA

8

7

TY7

9

10 11 12

fLYMCT GLK

13 14 15 16
ARCIAR AIG

VAL

17 18
VAL

LZU

6 7 8 9 10 11 12 13 14 15 16 17 18
CL
LW
OW
LW
GLCLV
C
LCU OLUVAL ARC
5

NIV

30 31

23 242526 272829
ASP

ARGCTCS

LW

LYS

AS

LW

TER

32 3334
ASK

35

LU

36
OW

20 21 22 23 24 25 S26 U27 28 29 30 31 32 33 34 35 36
AM
OW L
AS
ARC UASP
FIG. 5. Comparison of the NH2-terminal amino acid sequences of A. vinelandii and A. calcoaceticus
carboxymuconolactone decarboxylases. Identical residues are enclosed in boxes.

AC"tobsCtw
-,Las

19

Acinetobacter

D*car-=-Y"-e

1

Azotobacter

1

MET

Peudomonee

1

Decarboxylase

MET

lemerans

Peudomo

24

LYS

ASP

12

-A&-

ASP

A6

12
jASP

25
SEX

P

PU

4

2

NET ASH

DecarboxyFlase

Acinetobacter

GL

SS

26
IVAL

8

GLU

-A-

3

4

5

7

61

GLU LYSGUARTY
3

4

GOU

LYS

6
5
GLNIARGI

27 28 29

GLU

LYS

ALA

-A-

24 25 26 27 28 29
LYS GW

-A-

LYS ALA ASP GLU

7
TYR

8
ASP

8

y-

10 11 12

LYS GLN GLY

ARC

MOE

ALAGLY
9

ASP AIA

LEU GLU
11 12
MET GLN

10 11 12
GLY

MET GLN

30 31 32 33 34 35
SER GLN GLU LEU GLN

m

130 131 32 33343
MG
IMA

IL

FIG. 6. A portion of the NH2-terninal amino acid sequence of -y-carboxymuconolactone decarboxylase
be conserved within the primary structure of muconolactone isomerase. Numbers indicate the
positions of residues in the primary sequences of the proteins. Boxes enclose residues at positions where
identical residues are found in the decarboxylase-isomerase comparison.
appears to

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DsAGl

RELATIONSHIPS AMONG BACTERIAL ENZYMES

238

YEH ET AL.

ACKNOWLEBGMNEN1
We thank Gary Davis for his expert technical asitance.
This work was supported by grant PCM7724884 from the
National Science Foundation and Public Health Service
grants GM 21714 and GM 25487 from the National Institutes
of Health.

LITERATURE CTED
1. Durham, D. R., and L N. Ornton. 1980. Homologous
structural genes and similar induction pattern in Azotobacter spp. and Pseudomonas spp. J. Bacteriol. 143:

834-840.
2. Durham, D. R., L A. Stirling, L N. Oruton, and J.
J. Perry. 1980. Intergeneric evolutionary homology
revealed by the study of protocatechuate 3,4 dioxygenase fromAzotobacter vinelandul Biochemistry 19:149-

156.
3. Marchalonis, J. J., and J. K. WeltmanL 1971. Relatedness among proteins: a new method of estimation and
its application to immunoglobulins. Comp. Biochem.
Physiol. B 38:609-626.
4. McCorkle, G. M., W. K. Yeh, P. Fletcher, and L N.
Ornton. 1980. Repetitions in the NH2-terminal amino
acid a&quence of fi-ketoadipate enol-lactone hydrolase
from Pseudomonas putida. J. Biol. Chem. 255:63356341.
5. Ornston, L N., and R. Y. Stanier. 1966. The conversion

6.
7.
8.
9.

10.

11.

of catechol and protocatechuate to jS-ketoadipate by
Pseudomonas putida. L. Biochemisty. J. Biol. Chem.
241: 3776-3786.
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Downloaded from http://jb.asm.org/ on March 4, 2018 by guest

amino acids of the enzymes reveals sequence
imilarity suggesting a low overall homology
(17). In addition, the NH2-terminal amino acid
sequence of the decarboxylase appears to be
conserved within the primary structure of the
muconolactone isomerase: the tetrapeptide extending from residues 2 through 5 in the A.
vineandii decarboxylase is represented at residues 26 through 29 in the amino acid sequence
of the P. putida muconolactone isomerase (Fig.
6). This is consistent with the proposal that, as
genes for the ,B-ketoadipate pathway became
established, oligonucleotide substitution mutations placed sequences coding for peptides in
novel structural contexts (6, 18).

J. BACTERIOL.



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