Galactaro δ-lactone isomerase: Lactone isomerization by a member of the amidohydrolase superfamily

Article abstract of DOI:10.1021/bi5000492

Agrobacterium tumefaciens strain C58 can utilize d-galacturonate as a sole source of carbon via a pathway in which the first step is oxidation of D-galacturonate to D-galactaro-1,5-lactone. We have identified a novel enzyme, D-galactarolactone isomerase (GLI), that catalyzes the isomerizaton of D-galactaro-1,5-lactone to D-galactaro-1,4-lactone. GLI, a member of the functionally diverse amidohydrolase superfamily, is a homologue of LigI that catalyzes the hydrolysis of 2-pyrone-4,6-dicarboxylate in lignin degradation. The ability of GLI to catalyze lactone isomerization instead of hydrolysis can be explained by the absence of the general basic catalysis used by 2-pyrone-4,6-dicarboxylate lactonase.

Full text of DOI:10.1021/bi5000492

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Galactaro δ‑Lactone Isomerase: Lactone Isomerization by a Member
of the Amidohydrolase Superfamily
Jason T. Bouvier,†,§,†F,§i, o‡ na P. Groninger-Poe, Matthew Vetting, Steven C. Almo,∥
†,§
∥
and John A. Gerlt*
†
‡
§
Department of Biochemistry, Department of Chemistry, and Institute for Genomic Biology, University of Illinois at
Urbana-Champaign, Urbana, Illinois 61801, United States
∥
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, United States
*
S Supporting Information
drawn to this pathway in Agrobacterium tumefaciens strain C58
ABSTRACT: Agrobacterium tumefaciens strain C58 can
utilize D-galacturonate as a sole source of carbon via a
pathway in which the first step is oxidation of D-
galacturonate to D-galactaro-1,5-lactone. We have identi-
fied a novel enzyme, D-galactarolactone isomerase (GLI),
that catalyzes the isomerizaton of D-galactaro-1,5-lactone
to D-galactaro-1,4-lactone. GLI, a member of the function-
ally diverse amidohydrolase superfamily, is a homologue of
LigI that catalyzes the hydrolysis of 2-pyrone-4,6-
dicarboxylate in lignin degradation. The ability of GLI to
catalyze lactone isomerization instead of hydrolysis can be
explained by the absence of the general basic catalysis used
by 2-pyrone-4,6-dicarboxylate lactonase.
because it includes a reaction catalyzed by a member of the
2
functionally diverse enolase superfamily. This enzyme,
designated D-galactarolactone cycloisomerase (Gci, Atu3139),
catalyzes the ring opening of D-galactaro-1,4-lactone to yield 2-
keto-3-deoxy-L-threo-hexarate [hereafter designated 5-keto-4-
deoxy-D-galactarate or 5-keto-4-deoxy-D-glucarate (KDG)] via a
β-elimination reaction that is initiated by abstraction of the
2
proton adjacent to the carboxylate group.
+
In this pathway, an NAD -dependent uronate dehydrogenase
(
Udh, Atu3143) oxidizes the β-pyranose form of D-
galacturonate to D-galactaro-1,5-lactone (δ-lactone). The
assumption has been that the δ-lactone nonenzymatically
isomerizes to D-galactaro-1,4-lactone (γ-lactone). Following this
isomerization, Gci catalyzes the ring opening reaction of the γ-
lactone to produce KDG that undergoes dehydration and
decarboxylation to form α-ketoglutarate semialdehyde, which is
oxidized to α-ketoglutarate, an intermediate in the citric acid
D-Galacturonate, the primary constituent of pectin, is an
abundant polymer found in plant cell walls. Following
hydrolysis by pectinases, D-galacturonate can be utilized as a
sole carbon source by many soil bacteria. Several pathways are
known for catabolism of D-galacturonate, including the
1−4
cycle.
Atu3139 is located in a gene cluster that includes Atu3138
and Atu3140 (Figure 1B); the transcription of the genes in this
cluster is upregulated when D-galacturonate is the carbon
source (unpublished results). This gene cluster also encodes
orthologues of KduI and KduD that are involved in an alternate
D-galacturonate utilization pathway that involves isomerization
1
oxidative pathway shown in Figure 1A. Our attention was
5
and reduction to produce 2-keto-3-deoxy-D-gluconate. On the
basis of the sequence similarity, the closest functionally
characterized homologue of the protein encoded by Atu3140
is a KDG dehydratase/decarboxylase (KdgD). Because KDG is
the product of the reaction catalyzed by Gci, the proximity of
these genes is not surprising. However, the function of the
protein encoded by Atu3138 was unknown.
The protein encoded by Atu3138 (UniProt accession
number A9CEQ7) is a member of the amidohydrolase
superfamily (AHS), a very large functionally diverse enzyme
6
superfamily. A9CEQ7 is a member of Pfam PF04909 (6287
sequences in release 27) and InterPro IPR006992 (11876
sequences in release 45.0). 2-Pyrone-4,6-dicarboxylate lacto-
nase (PDC lactonase or LigI) is a structurally and
7
mechanistically characterized member of these families.
Figure 1. (A) D-Galacturonate oxidative catabolic pathway. (B) Gene
cluster.
Received: January 12, 2014
Published: January 23, 2014
©
2014 American Chemical Society
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dx.doi.org/10.1021/bi5000492 | Biochemistry 2014, 53, 614−616

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PDC, the substrate for LigI, is structurally similar to D-
galactaro-δ-lactone: both are carboxy-substituted δ-lactones. In
the pathway shown in Figure 1A, D-galactaro-δ-lactone has been
proposed to isomerize to D-galactaro-γ-lactone nonenzymati-
cally. However, on the basis of the proximity of the genes
encoding A9CEQ7 and Gci and the structural similarity
between PDC and D-galactaro-δ-lactone, we hypothesized
that A9CEQ7 catalyzes the δ- to γ-lactone isomerization and
therefore is a “missing” enzyme in this pathway. At neutral pH,
the nonenzymatic isomerization is fast, so previous investigators
may have assumed there would be no need for a catalyst. Also,
because Gci catalyzes the ring opening reaction of D-galactaro-
γ-lactone to KDG, no need for a lactonase is apparent, i.e., a
reaction similar to that catalyzed by LigI. Here we report that
A9CEQ7 is a galactaro δ-lactone isomerase (GLI), a novel
activity for the AHS.
The reaction progress was monitored with a polarimeter. In
Figure 3, the black line shows the change in the optical rotation
D-Galactaro-δ-lactone was prepared in D O via bromine
2
8
oxidation of D-galacturonate. This material could not be
distinguished by H nuclear magnetic resonance (NMR)
spectroscopy from the product obtained by the NAD -
1
+
Figure 3. Polarimetric profiles of 5.5 mM D-galactaro-δ-lactone
without enzyme (black diamonds), with A9CEQ7 (red squares), and
with Gci (blue circles). Profile of 5.5 mM D-galactaro-γ-lactone with
Gci (green triangles).
dependent Udh (Atu3143). The synthetic δ-lactone is stable
indefinitely at pD 4.8 and −80 °C.
The progress of the A9CEQ7-catalyzed reaction was
1
1
monitored by H NMR spectroscopy (Figure 2). The H
during the nonenzymatic isomerization at pD 6.4; the red line
shows the change in the optical rotation during the reaction in
the presence of A9CEQ7. The kinetic parameters were
−
1
measured for the A9CEQ7-catalyzed reaction: k = 440 s ,
cat
4
−1 −1
and k /K = 8.3 × 10 M s . The rate enhancement is 6.8
10 . The rate of the nonenzymatic reaction is dependent on
cat
m
5
×
pH, with a pD of 6.4 providing sufficient kinetic stability of the
δ-lactone to allow confident rate measurements.
We also investigated the specificity of Gci for the γ- and δ-
lactones. In Figure 3, the green line shows the progress of the
reaction when Gci is added to γ-lactone produced in the
absence of A9CEQ7; the blue line shows the progress of the
reaction when Gci is added to a reaction mixture containing the
δ-lactone. These reactions reach the same final optical rotation.
The rate constants for the nonenzymatic isomerization of the δ-
lactone to the γ-lactone (black line) and the Gci-catalyzed
production of KDG in the absence of A9CEQ7 (blue line) are
the same, establishing that Gci does not catalyze the ring
opening of the δ-lactone.
1
On the basis of these results, we assign the D-galactarolactone
isomerase (GLI) function to A9CEQ7. Although several
lactonases have been identified in the AHS, this is the first
lactone isomerase reaction.
The structure of GLI was determined in the absence of a
ligand to 1.6 Å by molecular replacement using an ensemble
model of structural homologues from IPR006992. GLI has a
Figure 2. H NMR spectra of the isomerase substrate and product.
(
A) Synthetic δ-lactone immediately after the pD had been adjusted
from 4.8 to 6.4. (B) Time course of the reaction. (C) Reaction after 1
h. Resonances are color-coded to match the peaks associated with the
structures in Figure 1A.
NMR spectrum of the synthetic δ-lactone at pD 6.4 is shown in
Figure 2A; the red bars indicate the δ-lactone, the green bars
the γ-lactone, and the blue bars the residual D-galacturonate
from the synthesis. The spectra in Figure 2B (recorded at 2 min
intervals) show the progress of the reaction. As the reaction
proceeds, the intensities of the resonances associated with the
δ-lactone decrease as the intensities of the resonances
associated with the γ-lactone increase. After 1 h (Figure 2C),
the only resonances (in addition to those associated with the
residual D-galacturonate) are those associated with the γ-
lactone. No meso-galactarate is detected by hydrolysis of either
lactone.
typical amidohydrolase fold with a distorted (β/α) -TIM barrel.
8
The closest structural homologue as determined with
9
PDBeFold is LigI from Sphingomonas paucimobilius (Protein
Data Bank entry 4DI8) with a root-mean-square deviation
(rmsd) of 2.10 Å and a sequence identity of 27% over 253 Cα
atoms. Despite the low level of sequence identity and the high
rmsd values, LigI and GLI have very similar core TIM-barrel
structures (Figure 4A). As expected by the differences in their
substrates, LigI is a flat planar dicarboxylate lactone and GLI is
a monocarboxylate sugar lactone; most of their differences are
localized to the loops at the N-terminal end of the barrel that
play a role in substrate recognition. For example, Arg 130 in
6
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Biochemistry
Rapid Report
the boat conformer of the δ-lactone to enforce the proximity of
the 4-OH group and the lactone carbonyl group (Figure 5).
Figure 5. Proposed mechanism for A9CEQ7.
ASSOCIATED CONTENT
Supporting Information
A description of the experimental procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
*
S
Accession Codes
The atomic coordinates and structure factors for GLI have been
deposited as Protein Data Bank entry 4MUP.
AUTHOR INFORMATION
Corresponding Author
E-mail: j-gerlt@illinois.edu. Phone: (217) 244-7414. Fax:
217) 244-0508.
■
*
(
Funding
Use of the Advanced Photon Source, an Office of Science User
Facility operated for the U.S. Department of Energy (DOE)
Office of Science by Argonne National Laboratory, was
supported by the DOE under Contract DE-AC02-
06CH11357. Use of the Lilly Research Laboratories Collabo-
rative Access Team (LRL-CAT) beamline at Sector 31 of the
Advanced Photon Source was provided by Eli Lilly Co., which
operates the facility. This research was supported by National
Institutes of Health Grants P01GM071790 and U54GM093342
to J.A.G.
Figure 4. Structure of A9CEQ7. (A) Distorted (β/α) -TIM barrel.
8
A9CEQ7 (cyan) with supposed catalytic resides (yellow). LigI (4d8L)
is colored magenta. (B) Superimposition of catalytic residues with
those of LigI and the δ-lactone with PDC. Note an asparagine (N240)
has replaced an aspartate involved in activating a water at the end of β-
strand 8.
Notes
The authors declare no competing financial interests.
LigI, which coordinates the distal carboxylate of its substrate, is
a proline in GLI (Pro 123).
REFERENCES
■
Unlike many members of the amidohydrolase superfamily,
LigI does not require a divalent metal for its lactonase activity.
(
8
1) Richard, P., and Hilditch, S. (2009) Appl. Microbiol. Biotechnol.
2, 597−604.
2
+
2+
2+
2+
2+
2+
The presence of Ca , Co , Mg , Mn , Ni , or Zn does not
increase the rate of the GLI reaction. These results suggest GLI
also does not require a metal for catalytic activity.
̈
(2) Andberg, M., Maaheimo, H., Boer, H., Penttila, M., Koivula, A.,
and Richard, P. (2012) J. Biol. Chem. 287, 17662−17671.
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̈
(
In LigI, Asp 248 is the general base that activates a water
molecule for nucleophilic attack on the lactone carbonyl
(
̈
̈
M.,
7
group. A superposition of the active sites of GLI and the PDC-
(
2
(
6
liganded D248A mutant of LigI at pH 8.5 is shown in Figure
4
B. The active sites are essentially identical, with the notable
exception that in GLI Asn 240 is the structural homologue of
Asp 248 in LigI. The N240D substitution produced a 28-fold
(
−1
3
−1
decrease in activity (k = 16 s , and k /K = 3.6 × 10 M
M., Burley, S. K., Almo, S. C., and Raushel, F. M. (2012) Biochemistry
cat
cat
m
−
1
s ); no hydrolysis of the δ- or γ-lactone was observed in the
presence of the D240N mutant.
51, 3497−3507.
(8) Isbell, H. S., and Frush, H. L. (1943) J. Res. NBS 31, 33−44.
(9) Krissinel, E., and Henrick, K. (2004) Acta Crystallogr. D60,
Thus, the lactone isomerization reaction catalyzed by GLI
can be explained by the lack of a general base for lactone
hydrolysis. Inspection of the active site of GLI does not reveal
either a general base that would activate the 4-OH group of the
δ-lactone for intramolecular attack on the lactone carbonyl
group or a nucleophile that would allow the formation of an
acyl−enzyme intermediate that would partition between the δ-
and γ-lactones. Therefore, in analogy to the proposed
mechanism for the nonenzymatic reaction, we assume the
modest rate enhancement results from preferential binding of
2
256−2268.
6
16
dx.doi.org/10.1021/bi5000492 | Biochemistry 2014, 53, 614−616