Mechanistic Studies on Members of the Tautomerase Superfamily: Insights to Secondary
Catalytic promiscuity is a common theme in the tautomerase superfamily, varying
considerably in magnitude (ref: Wang, S.C., Person, M.D., Johnson, W.J., Jr., and
Whitman, C.P. (2003) Reactions of trans-3-Chloroacrylic acid dehalogenase with
acetylene substrate: Consequences of and evidence for a hydration reaction, Biochemistry
42, 8762-8773). Members of the tautomerase superfamily are identified by their ß-α-ß
structural fold motif along with a catalytic N-terminal proline. Three members of the
superfamily that have been identified are enzymes involved in the biodegradation of the
nematicide 1,3-dichloropopene (Scheme 1). Trans-3-Chloroacrylic Acid Dehalogenase
(CaaD) and Cis-3-Chloroacrylic Acid Dehalogenase(cis-CaaD) are involved in the
hydration of their respective isomers of Chloroacrylic Acid (2) to generate malonate
semialdehyde (3). Malonate semialdehyde decarboxylase (MSAD) then catalyzes the
product to generate acetaldehyde (4).
CaaD is one of the most highly studied enzymes within the tautomerase
superfamily. The gene encoding the protein was cloned from Pseudomonas pavonaceae
170, a bacteria that can utilize 1,3-dichloropopene as its sole source of carbon (Ref:
Poelarends, G. J., Saunier, R., and Janssen, D. B. (2001) J. Bacteriol. 183, 4269-4277).
The enzyme is a heterohexemer consisting of 3 alpha subunits and 3 beta subunits of 75
and 70 residues, respectively. The catalytic N-terminal proline has been found to reside
on the ß-subunit. While mutations of the N-terminal proline on the ß subunit shows a
complete loss of activity, an alanine mutation at the α subunit proline displays no change
in activity. The activity of CaaD was proposed to go thru a hydration mechanism. This
was verified in two key experiments. In the first experiment the N-terminal ß proline for
CaaD was found to have a pKa of 9.2, thereby allowing for the proline to function as a
hydrogen donor at physiological conditions (Ref: Azurmendi, H. F.; Wang, S. C.;
Massiah, M. A.; Whitman, C. P.; Mildvan, A. S. Biochemistry 2003, 42, 8619).
The second piece of evidence lies in a secondary substrate for CaaD. The
compound 2-oxo-3-pentynoate (5) was found to be a potent inhibitor for 4-oxalocrotonate
tautomerase (4-OT), another member of the tautomerase superfamly. This is due in part
to its catalytic proline having a depressed pKa, about 6.4. At physiological conditions,
the proline functions as a base, attacking the triple bond of 2-OP rendering the enzyme
covalently modified (Scheme 2). Because the pKa of the proline in CaaD is 9.2, it
cannot function as a base. Instead, it is proposed to donate a proton to the substrate
giving the product acetopyruvate (Scheme 3, 6).
(Scheme 2, inactivation of 4-OT by 2-OP)
make image of this...
O H2O O O
A crystal structure of CaaD was solved, thus expanding the knowledge of the
protein. Two arginines, at position 8 and 11 both on the ß subunit, are proposed to help
bring the negative portion of the substrate while a glutamine at postion 52 on the α
subunit is proposed to activate a water molecule. The N-terminal Proline on the ß subunit
is thought to provide a proton to the final product. A mutation in any 4 of these residues
erases any dehalogenase activity for CaaD.
Based on crystallographic and mutational studies two mechanisms for the
dehalogenation of trans-3-chloroacrylic acid has been proposed (Scheme 2.. will rename
to Scheme 4 and redraw).
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Evidence as to which mechanism is the correct one is currently being investigated
(Robertson B.A. and Whitman C.P. unpublished). One insight to the correct mechanism
was recently uncovered. The compound phenylenolpyruvate (7) was found to be a
substrate for CaaD, with the product identified as phenylpyruvate (8 FIX THE
SCHEME AND ID).
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This compound is
Based on a secondary activity of CaaD, Scheme 2 route B was found to be the more
favorable of the two proposed mechanisms (Ref: Poelarends, G. J., Johnson, W.H.,
Serrano, H., and Whitman C.P. (2007) Biochemistry 46, 9596-9604).
MSAD, also cloned from Pseudomonas pavonaceae 170 is a decarboxylase and
member of the tautomerase superfamily. This XXX amino acid
cis-CaaD was cloned from coryneform bacterium strain FG41. It is involved in
the dehalogenation of the cis-isomer of CAA.
Two new members of the tautomerase superfamily have recently been identified.
The first, a gene encoding a protein identified as FG41 MSAD was cloned from
coryneform bacterium strain FG41. The gene lies downstream of the gene encoding the
cis-CaaD protein. Sequence alignment of this protein along with MSAD and a few select
homologues shows high sequence homology (Figure XX). Based on the high sequence
similarity of this protein with MSAD it was proposed that this protein was also a
decarboxylase, which was verified (Table XX). The secondary hydratase activity of
MSAD is highly depressed in FG41 MSAD. One residue identified as being catalytically
important in MSAD, arginine XX is a glutamine in FG41 MSAD. A mutation of the
glutamine in FG41 MSAD increases the hydratase activity, without substantially altering
the wild-type decarboxylase activity. An unpublished crystal structure of FG41 MSAD
has recently been solved which will now allow for alignment of the active sites of the 2
decarboxylases (Guo, Y., Serrano, H., Whitman, C.P., Hackert, M.L. unplublished). A
residue, threonine 55 in MSAD, is an alainine in FG41 MSAD.
The second new member of the tautomerase superfamily was a protein from the
gene cg10061(check name! 62?) isolated from the bacteria Corynebacterium glutamicum.
The protein, named Cg10062 or CgX, was identified as a possible cis-CaaD homolog
based off a BlastP index search. Sequence alignment shows XX sequence homology
between CgX and cis-CaaD. The expressed protein was shown to have both the cis-
CaaD dehalogenase activity along with the secondary hydratase activity using 2-OP.
Interestingly, CgX also exhibits CaaD dehalogenase activity. There is low-level
homology between CgX and the X subunit of CaaD. Mutations of the proposed catalytic
residues that align with cis-CaaD eliminate the XX activities. A crystal structure of this
enzyme has been solved which allows for alignment of this protein with both cis-CaaD
and CaaD. Although CgX exhibits dehalogenase activities for both isomers of 3-
chloroacrylic acid, it is not very efficient.
The major objective of this proposal will be to first determine the residues
involved in the decarboxylation and hydration mechanism for MSAD and FG41 MSAD.
Secondly, the residues involved in separating the two isomer-specific dehalogenase
activity will be determined for CgX. Finally, if possible, CgX will be turned into a more
efficient dehalogenase. In order to do this, the following specific aims will be carried
out: 1.) Establish pKa value for Pro-1 for FG41 MSAD and CgX 2.) Site directed
mutagenesis on position 55 and 73(?) for MSAD and FG41 MSAD, 3.) site directed
mutations will be carried out on CgX at positions (write the ones down here) to determine
which residue is involved with the various activities, and 4) establish a working assay to
screen for enhanced activity for one isomer of 3-chloroacrylic acid.
This investigation will first examine the role that Pro-1 plays in both enzymes. A
pKa around 9 will establish the role for proline to function as a general acid in their
respective activities. In the proposed MSAD mechanism, the decarboxylation step
produces ethenol, which rapidly tautomerizes to acetaldehyde. Pro-1 is proposed to
function as the proton donor in this mechanism. A similar role for FG41 MSAD would
be proposed if the pKa of the active site proline is ~9. In the proposed CaaD and cis-
CaaD mechanisms, Pro-1 is proposed to act as the proton donor at C-2. A pKa of ~9 for
CgX for the active site proline will also allow CgX to follow a similar mechanism.
The use of (NMR stuff here, reference the msad paper? Or the l8r paper) to
determine the active site proline of members of the tautomerase superfamily has been
used in the past (specific aim #1). Also use Scott’s info here…
A pKa of 6 will show that proline functions as a general base, while also showing
that either enzyme uses a different mechanism for catalysis. In the case of FG41 MSAD,
an active site proline with a pKa of 6.4, which is seen in other members of the
tautomerase superfamily along with a mutant of MSAD, would allow FG41 MSAD to
function as a Schiff Base (Reference Joe’s work here for D37N). In the case of CgX, an
active site proline having a depressed pKa would show XXXXXXXXXXX.
Insight to the active site proline’s pKa is found using the compound 2-OP. It has
been shown that members of the tautomerase superfamily that contain prolines with
depressed pKas(6.5 and below) are covalently modified by 2-OP, due to the fact the the
proline functions as a general base and directly attacks the triple bond of 2-OP (Reference
sue’s paper for this). Active site prolines with a pKa ~9 however have been shown to
activate water and utilize 2-OP as a substrate, or in the case of FG41 MSAD, not become
covalently modified by 2-OP. Because CgX and FG41 MSAD have both been
demonstrated to hydrate the compound, neither would be expected to have a depressed
Sequence alignment of MSAD and FG41 MSAD shows high similarity between
the 2 enzymes. A mutation of the glutamine at position 73 from FG41 MSAD to an
arginine showed increased hydratase activity(specific aim #2). This mutant, along with
wild-type will be used when constructing the Ala55Thr mutation. <add info why it
should work>. Along these lines, a mutation of MSAD a position 73 from an arginine to
a glutamne shows no activity for decarboxylation or hydration. A mutation of wild-type
and Arg73Gln of position 55’s threonine to alanine will thus be constructed to probe the
necessity for this residue in either activity. <results>
Low level dehalogenase activity of CgX will next be probed(specific aim #3).
For the cis-CaaD activity, binding of the substrate to the active site, or release of the
product appears to be the rate limiting state, based on kcat and Km results. Because of
Finally, if the above experiments work and the two isomer specific dehalogenases
can be isolated, CgX will then be converted into a more efficient CaaD or cis-CaaD
without retaining the other activity(specific aim #4). This will be accomplished by
constructing a dual expression system