Mechanism and Structure of γ-Resorcylate Decarboxylase

Article abstract of DOI:10.1021/acs.biochem.7b01213

γ-Resorcylate decarboxylase (γ-RSD) has evolved to catalyze the reversible decarboxylation of 2,6-dihydroxybenzoate to resorcinol in a nonoxidative fashion. This enzyme is of significant interest because of its potential for the production of γ-resorcylate and other benzoic acid derivatives under environmentally sustainable conditions. Kinetic constants for the decarboxylation of 2,6-dihydroxybenzoate catalyzed by γ-RSD from Polaromonas sp. JS666 are reported, and the enzyme is shown to be active with 2,3-dihydroxybenzoate, 2,4,6-trihydroxybenzoate, and 2,6-dihydroxy-4-methylbenzoate. The three-dimensional structure of γ-RSD with the inhibitor 2-nitroresorcinol (2-NR) bound in the active site is reported. 2-NR is directly ligated to a Mn²⁺ bound in the active site, and the nitro substituent of the inhibitor is tilted significantly from the plane of the phenyl ring. The inhibitor exhibits a binding mode different from that of the substrate bound in the previously determined structure of γ-RSD from Rhizobium sp. MTP-10005. On the basis of the crystal structure of the enzyme from Polaromonas sp. JS666, complementary density functional calculations were performed to investigate the reaction mechanism. In the proposed reaction mechanism, γ-RSD binds 2,6-dihydroxybenzoate by direct coordination of the active site manganese ion to the carboxylate anion of the substrate and one of the adjacent phenolic oxygens. The enzyme subsequently catalyzes the transfer of a proton to C1 of γ-resorcylate prior to the actual decarboxylation step. The reaction mechanism proposed previously, based on the structure of γ-RSD from Rhizobium sp. MTP-10005, is shown to be associated with high energies and thus less likely to be correct.

Full text of DOI:10.1021/acs.biochem.7b01213

Subscriber access provided by NORTH CAROLINA STATE UNIV
Article
Mechanism and Structure of #-Resorcylate Decarboxylase
Xiang Sheng, Yury patskovsky, Anna Vladimirova, Jeffrey B.
Bonanno, Steven C. Almo, Fahmi Himo, and Frank M. Raushel
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01213 • Publication Date (Web): 28 Dec 2017
Downloaded from http://pubs.acs.org on December 31, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street
N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 32
Biochemistry
1
2
3
4
5
6
7
8
9
Mechanism and Structure of γꢀResorcylate Decarboxylase
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ϕ
Ψ
ꢀ
Ψ
Xiang Sheng , Yury Patskovsky , Anna Vladimirova , Jeffrey B. Bonanno ,
Ψ*
ϕ*
ꢀ*
Steven C. Almo , Fahmi Himo , and Frank M. Raushel
ϕ
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SEꢁ106 91
ꢀ
Stockholm, Sweden; Department of Chemistry, Texas A&M University, College Station, Texas,
Ψ
7
7842, United States; Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx,
New York 10461, United States.
1
ACS Paragon Plus Environment

Biochemistry
Page 2 of 32
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT
γꢁResorcylate decarboxylase (γꢁRSD) has evolved to catalyze the reversible
decarboxylation of 2,6ꢁdihydroxybenzoate to resorcinol in a nonoxidative fashion. This enzyme
is of significant interest because of its potential for the production of γꢁresorcylate and other
benzoic acid derivatives under environmentally sustainable conditions. Kinetic constants for the
decarboxylation of 2,6ꢁdihydroxybenzoate catalyzed by γꢁRSD from Polaromonas sp. JS666 are
reported and the enzyme is shown to be active with 2,3ꢁdihydroxybenzoate, 2,4,6ꢁ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
trihydroxybenzoate and 2,6ꢁdihydroxyꢁ4 methylbenzoate. The threeꢁdimensional structure of γꢁ
RSD with the inhibitor 2ꢁnitroresorcinol (2ꢁNR) bound in the active site is reported. 2ꢁNR is
2
+
directly ligated to a Mn bound in the active site and the nitroꢁsubstituent of the inhibitor is
tilted significantly from the plane of the phenyl ring. The inhibitor exhibits a different binding
mode compared to the substrate bound in the previously determined structure of γꢁRSD from
Rhizobium sp. MTPꢁ10005. On the basis of the crystal structure of the enzyme from
Polaromonas sp. JS666, complementary density functional calculations were performed to
investigate the reaction mechanism. In the proposed reaction mechanism, γꢁRSD binds 2,6ꢁ
dihydroxybenzoate by direct coordination of the active site manganese ion to the carboxylate
anion of the substrate and one of the adjacent phenolic oxygens. The enzyme subsequently
catalyzes the transfer of a proton to C1 of γꢁresorcylate prior to the actual decarboxylation step.
The reaction mechanism proposed previously, based on the structure of γꢁRSD from Rhizobium
sp. JS666, is shown to be associated with high energies and thus less likely to be correct.
2
ACS Paragon Plus Environment

Page 3 of 32
Biochemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
γꢁResorcylate decarboxylase (γꢁRSD), also called 2,6ꢁdihydroxybenzoate decarboxylase
2,6ꢁDHBD), catalyzes the nonoxidative reversible decarboxylation of γꢁresorcylate (γꢁRS; 2,6ꢁ
(
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
dihydroxybenzoate) to resorcinol (1,3ꢁdihydroxybenzene) and CO as shown in Scheme 1a (1,
2
2). The resorcinol product of the decarboxylation activity is an important intermediate in the
fields of organic, pharmaceutical, agricultural and polymer chemistries, and γꢁRSD can be used
as a biocatalyst to produce this valuable compound (3). Recently, the carboxylation activity of γꢁ
RSD has attracted increasing attention as a potential biological alternative to the Kolbe–Schmitt
process (4), which requires harsh conditions in terms of pressure and temperature, for the
production of γꢁresorcylate and other benzoic acid derivatives (5-10).
Scheme 1. Reactions catalyzed by γꢁRSD (a) and LigW (b).
γꢁRSD has been characterized from several bacterial species, including Rhizobium sp.
MTPꢁ10005 (11), Rhizobium radiobacter WUꢁ0108 (12), Agrobacterium tumefaciens IAM12048
(13), Pandoraea sp. 12Bꢁ2 (14) and Rhodococcus jostii RHA1 (15). In the γꢁresorcylate
catabolic pathway identified in Rhizobium sp. MTPꢁ10005, the gene for γꢁRSD is downstream
from genes encoding a flavin reductase and a resorcinol hydroxylase and is upstream from genes
encoding a maleylacetate reductase and hydroxyquinol 1,2ꢁdioxygenase (16). The enzyme from
3
ACS Paragon Plus Environment

Biochemistry
Page 4 of 32
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Rhizobium sp. MTPꢁ10005 has been shown to be a reversible decarboxylase with reported
ꢁ1
4
ꢁ1 ꢁ1
kinetic parameters for k , K and k /K of 0.95 s , 71 µM and 1.3 x 10 M s , respectively,
cat
m
cat
m
for the decarboxylation of γꢁresorcylate (11). Crystal structures were later determined for the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
apoꢁenzyme and in the presence of substrate or inhibitor, demonstrating that the enzyme exists as
a homotetramer, of which each subunit consists of a (β/α) ꢁbarrel, three linkers and a Cꢁterminal
8
tail (17).
The enzyme 5ꢁcarboxyvanillate decarboxylase (LigW) from Novosphingobium
aromaticivorans DSM 12444, catalyzing the formation of vanillate via the nonoxidative
decarboxylation of 5ꢁcarboxyvanillate (Scheme 1b), is the closest structurally characterized
homologue of γꢁRSD from Rhizobium sp. JS666 with a 33% sequence identity. Both enzymes
are members of cog2159 of the amidohydrolase superfamily (AHS), and these enzymes require a
single divalent metal for catalysis (18). The structure of γꢁRSD from Rhizobium sp. JS666 has
previously been determined in complex with substrate (PDB id: 2DVU). A zinc ion was
observed in the active site and was coordinated by two histidines, one glutamate, one aspartate
and one oxygen atom of the substrate carboxylate group (17). In contrast, a manganese ion has
been identified in LigW, and the substrate is suggested to bind such that both the hydroxyl group
and the carboxylate group are coordinated to the metal ion (19). Due to the high similarity in the
reactions catalyzed by γꢁRSD and LigW (Scheme 1), it is of interest to compare their structures
and catalytic reaction mechanisms. Furthermore, since both enzymes belong to the
amidohydrolase superfamily, the members of which predominantly catalyze the hydrolysis of
ester and amide bonds, it is of substantial importance to understand how these enzymes have
evolved to catalyze the reversible decarboxylation of benzoic acid derivatives (18).
4
ACS Paragon Plus Environment

Page 5 of 32
Biochemistry
1
2
3
4
5
6
7
8
9
In the present study, we report the catalytic activity, substrate specificity and crystal
structure of γꢁRSD from Polaromonas sp. JS666 (locus tag: Bpro_2061) in complex with the
substrate analog inhibitor, 2ꢁnitroresorcinol. On the basis of the crystal structure reported here,
complementary theoretical calculations were performed with density functional theory to
investigate the reaction mechanism. The calculations show that γꢁRSD follows a very similar
mechanism as LigW. The mechanism proposed previously on the basis of the Znꢁstructure of the
enzyme from Rhizobium sp. MTPꢁ10005 is shown to be associated with high energies and thus
less likely to be correct.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MATERIALS and METHODS
Materials. All chemicals, buffers, and purification reagents were purchased from Sigmaꢁ
Aldrich unless otherwise specified. Pfu Turbo DNA polymerase and the E. coli strains BL21
(DE3) and XL1ꢁblue cells were obtained from Stratagene. The restriction enzyme DpnI was
purchased from New England BioLabs and oligonucleotides were obtained from Integrated Data
Technology through the Gene Technology Laboratory at Texas A&M University. Kanamycin,
isopropyl βꢁDꢁthiogalactopyranoside (IPTG), and LB broth were acquired from Research
Products International Corp. Protamine sulfate, Wizard Plus SV Miniprep DNA purification
system, and Vivaspin centrifugal concentrator (10 000 MWCO) were obtained from MP
Biomedicals LLC., Promega, and Fisher Scientific, respectively. Chromatographic gel filtration
columns were purchased from GE Healthcare. 2ꢁNitroresorcinol was purchased from Sigma
Aldrich.
Expression and Purification of γꢀResorcylate Decarboxylase (γꢀRSD) from
Polaromonas sp. JS666. The gene for γꢁRSD (Bpro_2061; gi: 91787937) from Polaromonas
5
ACS Paragon Plus Environment

Biochemistry
Page 6 of 32
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
sp. JS666 was chemically synthesized. The clone contained the codonꢁoptimized gene for γꢁ
RSD with an Nꢁterminal MetꢁVal extension and a Cꢁterminal AlaꢁGluꢁAsnꢁLeuꢁTyrꢁPheꢁGlnꢁ
SerꢁHis extension. The His ꢁtagged protein was used for crystallographic studies while the Cꢁ
6
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
terminal Hisꢁtag was removed for preparation of the enzyme used to measure the kinetic
parameters via standard siteꢁdirected mutagenesis protocols using the primers 5'ꢁ
GAGAACCTCTACTTCTAATAGCACCATCATCACCACCATꢁ3’ (forward) and 5’ꢁ
ATGGTGGTGATGATGGTGCTATTAGAAGTAGAGGTTCTCꢁ3’ (reverse). The
recombinant plasmid was transformed by electroporation into BL21 (DE3) cells, which were
plated on agar containing 50 µg/mL kanamycin. Six colonies were inoculated into 7 mL of LB
broth containing 7 µg/mL kanamycin and grown overnight at 37 °C. The starting cultures were
used to inoculate a total of 6.0 L of LB broth containing 50 µg/mL kanamycin and left to
incubate at 37 °C by shaking. Isopropyl βꢁthiogalactoside (IPTG) was added to a final
concentration of 0.5 mM to each of the flasks once the absorbance reached a range between 0.4ꢁ
0
.6 OD. Upon induction, 2.0 L of cells were supplemented with 1.0 mM MnCl . Expression of
2
o
the protein was achieved by shaking at 22 C overnight. The cells were collected by
o
centrifugation (8,000 rpm for 10 min) and stored at ꢁ80 C.
Hisꢁtagged protein for crystallographic studies was purified to homogeneity using general
methods, which have been described (20). Purification of the enzyme used to measure the
kinetic parameters followed the methods described below. The frozen cells were thawed at room
temperature and resuspended in 20 mM HEPES (pH 7.5) containing 20 mg
phenylmethanesulfonyl fluoride (PMSF) for every 5 g of cells. Lysis was achieved by
sonication, followed by centrifugation at 8,000 rpm for 10 min, and addition of 0.1 g of
protamine sulfate dissolved in 10 mL of 20 mM HEPES (pH 7.5) over a period of 45 min. The
6
ACS Paragon Plus Environment

Page 7 of 32
Biochemistry
1
2
3
4
5
6
7
8
9
solution was centrifuged at 8,000 rpm for 10 min and ammonium sulfate was added to the
supernatant solution to achieve 45% saturation. After stirring for 45 min, the suspension was
centrifuged at 8,000 rpm for 10 min. The pellet was reꢁsuspended in 10 mL of 20 mM HEPES,
pH 7.5, and loaded onto a 26/60 Superdex 200 gelꢁfiltration column. The protein was eluted
using 20 mM HEPES, pH 7.5, and further loaded on a ResourceꢁQ (GE Healthcare) anionꢁ
exchange column (6.0 mL). γꢁRSD was eluted using a linear gradient of 1.0 M NaCl in 20 mM
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
HEPES, pH 7.5. The purified protein was concentrated and subsequently stored at ꢁ80 C.
Determination of Kinetic Parameters. The decarboxylation of γꢁresorcylate by γꢁRSD
was monitored by following the decrease in absorbance at 305 nm using a differential molar
ꢁ1
ꢁ1
extinction coefficient (∆ε ) of 3,208 M cm . The decarboxylation of 2,3ꢁdihydroxybenzoate,
3
05
2
,4,6ꢁtrihydroxybenzoate, and 2,6ꢁdihydroxyꢁ4ꢁmethylbenzoate was monitored by following the
decrease in absorbance at 306, 256, and 304 nm using differential extinction coefficients (∆ε )
3
06
ꢁ1
ꢁ1
ꢁ1
ꢁ1
ꢁ1
ꢁ1
of 3680 M cm , (∆ε ) of 5953 M cm , and (∆ε ) of 2977 M cm , respectively. All
2
56
304
assays were conducted using a 96ꢁwell SpectraMax Plus384 UVꢁvis spectrophotometer and the
steadyꢁstate kinetic parameters were obtained using SoftMax Pro 5.0. Standard assay conditions
for the decarboxylation of 2,6ꢁdihydroxybenzoate, 2,3ꢁdihydroxybenzoate, 2,4,6ꢁ
trihydroxybenzoate, and 2,6ꢁdihydroxyꢁ4ꢁmethylbenzoate contained 50 mM HEPES (pH 7.0),
substrate (0 ꢁ 500 ꢂM, and 0.1 µM γꢁRSD in a final volume of 250 µL at 30 °C. All of the
kinetic constants were obtained using eqn. 1, where v is the initial velocity, E is the total enzyme
t
concentration, [A] is the substrate concentration, k is the turnover number, and K is the
cat
m
Michaelis constant.
v/E = k [A] / (K + [A])
(1)
t
cat
m
7
ACS Paragon Plus Environment

Biochemistry
Page 8 of 32
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Inhibition Studies. 2ꢁNitroresorcinol (2ꢁNR) was utilized as an inhibitor for the reaction
catalyzed by γꢁRSD. The inhibition experiments for 2ꢁNR contained 60 µM γꢁresorcylate, 50
mM HEPES, pH 7.0, and 1.0 µM γꢁRSD in a total volume of 250 µL at 30 °C. The apparent
app
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
inhibition constant (Ki ) for 2ꢁNR was obtained from a fit of the data to eqn. 2, where [I] is the
inhibitor concentration, v is the velocity in absence of inhibitor, and v is the velocity in presence
o
i
of inhibitor. The K value was obtained from eqn. 3, where [S] is the substrate concentration
d
used in the inhibition experiment and K is the Michaelis constant for the substrate.
m
app
app
vi/v˳ = Ki /([I] + Ki
)
(2)
(3)
app
Ki = K (1 + [S]/K )
d
m
Crystallization, Xꢀray Data Collection, and Structure Determination. γꢁRSD was
crystallized by the sittingꢁdrop vapor diffusion method. Concentrated (∼15 mg/mL) protein
solutions were mixed with an equal volume of precipitant (0.5 µL) and equilibrated at room
temperature against the same precipitant solution in clear tapeꢁsealed 96ꢁwell INTELLIꢁplates
(Art Robbins Instruments, Sunnyvale, CA). Crystallization screens were performed with a
PHENIX liquid handling robot (Art Robbins Instruments). The protein crystallized from the
mother liquor composed of 0.10 M sodium acetate, pH 4.6, 1.0 M dibasic ammonium citrate, and
5.0 mM MnCl . Crystals were soaked in this solution supplemented with 10 mM 2ꢁ
2
nitroresorcinol overnight, followed by flashedꢁcooling and storage in liquid nitrogen.
Xꢁray diffraction data were collected at 100K on the X29A beamline (National
Synchrotron Light Source, Brookhaven National Laboratory, Upton, NY) at a wavelength of
1.075Å. The diffraction data were processed and scaled with HKL3000 (21). The crystal
structure was determined by molecular replacement using the coordinates of Protein Data Bank
8
ACS Paragon Plus Environment

Page 9 of 32
Biochemistry
1
2
3
4
5
6
7
8
9
(
PDB) entry 3S4T as a starting model and PHASER software as implemented in CCP4 (22).
The initial model was built and refined using the automated ARP/WARP protocol (23). The
model was further refined using REFMAC5 (24) and manually adjusted using COOT
visualization and refinement software (25). The structure was deposited to the PDB as entry
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
QRO. All figures were produced using PYMOL (26). The data collection and refinement
statistics for the crystal structure are listed in Table 1.
Computational Details. All the calculations in the present study were preformed
employing the Gaussian 09 program (27) with the B3LYPꢁD3(BJ) hybrid density functional
method (28-31). Geometry optimizations were conducted with the 6ꢁ31G(d,p) basis set for C, N,
O and H and LANL2DZ pseudopotential for Mn and Zn (32). At the same level, frequency
calculations were preformed to obtain zeroꢁpoint energies (ZPE). To consider the effects of the
rest of the enzyme that is not included in the model, singleꢁpoint energy calculations were carried
out at the same level of theory as the geometry optimization using SMD solvation model with the
value of dielectric constant ε = 4 (33). To obtain more accurate energies, singleꢁpoint
calculations on the optimized structures were performed with LANL2DZ for Mn and Zn and the
larger basis set 6ꢁ311+G(2d,2p) for the other atoms. The ZPE and solvation effects are then
added to the large basis set energies (that include dispersion) and the final values are presented in
this paper. Following previous studies on other decarboxylation reactions, the entropy gain from
the release of CO is estimated to be equal to the translational entropy for the free molecule (34-
2
36). The value is calculated to be 11.1 kcal/mol at room temperature and this value is added to
the energy of the corresponding step.
Active Site Model. To investigate the detailed mechanism of the decarboxylation
reaction catalyzed by γꢁRSD, quantum chemical calculations employing the cluster approach
9
ACS Paragon Plus Environment

Biochemistry
Page 10 of 32
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
37-41) were performed on the basis of the crystal structure in complex with 2ꢁnitroresorcinol
reported here. A large active site model is designed by modifying the inhibitor to the natural
substrate, 2,6ꢁdihydroxybenzoate. The model consists of the metal cation along with its ligands
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Glu8, His10, His164, Asp287 and 2,6ꢁdihydroxybenzoate), six crystallographic water
molecules, residues that potentially form hydrogen bonds with the substrate directly or via water
molecules (Ser20, Asn128, His218, Glu221, Arg229, Asn234 and Glu291), residues that are πꢁ
stacked to the aromatic ring of the substrate (Phe23, Phe189 and Phe290), as well as other
residues that also contribute to the active site (Leu32, Leu36, Ala61, Gly184, Pro185, Thr186
and Ser262). All amino acids were truncated at the αꢁcarbons, and hydrogen atoms were added
manually. To preserve the overall structure of the active site during geometry optimizations, the
truncated αꢁcarbons and also a number of connected hydrogen atoms were kept fixed at their
crystallographic positions, as indicated in the figures. The Mnꢁcoordinated hydroxyl group of
the substrate is assumed to lose its proton upon binding to the metal, and the potential general
acid group Asp287 is modeled as its neutral state. The overall model consists of 317 atoms and
is neutral in charge.
RESULTS and DISCUSSION
Purification and Properties of γꢀRSD from Polaromonas sp. JS666. γꢁRSD was
purified to homogeneity using a simple protocol that involved ammonium sulfate fractionation,
gel filtration, and ion exchange chromatography. The purified enzyme was shown to catalyze
the decarboxylation of γꢁresorcylate to resorcinol, 2,3ꢁdihydroxybenzoate to catechol, 2,4,6ꢁ
trihydroxybenzoate to benzeneꢁ1,3,5ꢁtriol, and 2,6ꢁdihydroxyꢁ4ꢁmethylbenzoate to 5ꢁ
methylbenzeneꢁ1,3ꢁdiol. The catalytic constants for the decarboxylation reactions are presented
10
ACS Paragon Plus Environment

Page 11 of 32
Biochemistry
1
2
3
4
5
6
7
8
9
in Table 2 from a fit of the data to eqn. 1. The values of k and k /K for the decarboxylation
cat
cat
m
–1
4
–1 –1
of γꢁresorcylate are 0.44 ± 0.01 s and (1.3 ± 0.1) x 10 M s , respectively. The kinetic
constants for the other compounds found to be substrates for this enzyme are quite similar,
demonstrating that the enzyme is quite tolerant of the addition of other hydroxyl groups at C3,
C4, and C6.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
ꢁNitroresorcinol (2ꢁNR) was identified as an inhibitor of the reaction catalyzed by γꢁ
app
RSD. An apparent inhibition constant (Ki ) of 96 ± 8 ꢂM was obtained at a fixed substrate
concentration of 60 ꢂM (Figure 1) by a fit of the data to eqn. 2. The dissociation constant for
the binding of 2ꢁNR was determined to be 40 ± 4 ꢂM after correcting for the concentration of the
substrate used in the experiments with eqn. 3.
0.16
0.14
0.12
0.10
0.08
0
.06
.04
0
0.02
.00
0
0
100
200
300
400
500
600
[2-nitrobenzene-1,3-diol] uM
Figure 1. Inhibition of the reaction catalyzed by γꢁRSD in the presence of 2ꢁnitroresorcinol (2ꢁ
app
NR) with data fitted to eqn. 2, where Ki is the apparent inhibition constant.
Crystal Structure of γꢀRSD. The crystal structure of γꢁRSD from Polaromonas sp.
2
+
JS666 complexed with 2ꢁnitroresorcinol and Mn was determined to 1.65 Å resolution (PDB
entry 4QRO). The quaternary structure of γꢁRSD is a homotetramer, but the minimal catalytic
state is a homodimer (Figure 2). An essential arginine residue that is conserved in all of the
1
1
ACS Paragon Plus Environment

Biochemistry
Page 12 of 32
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
decarboxylases from cog2159 extends from the adjacent subunit into the active site and
presumably stabilizes the carboxylate substituent of the substrate.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Quaternary structure of γꢁRSD from Polaromonas sp. JS666 (PDB entry 4QRO). The
2
+
residues that coordinate the active site Mn are highlighted.
Each of the four subunits consists of a distorted (β/α) ꢁbarrel protein fold with the active
8
2
+
site and Mn embedded at the Cꢁterminal end of the barrel. A helical insertion domain is
observed (amino acids 12ꢁ39) with the side chain of Phe23 in a “herringꢁbone like” interaction
with the phenyl ring of the inhibitor at a van der Waals distance of 3.8 Å. The inhibitor is further
stabilized in the active site through πꢁπ stacking with Phe290 at a distance of 3.5 Å. His218 and
Glu221 form a hydrogenꢁbonded triad with Asp287 from βꢁstrand 8 as shown in Figure 3a. As
stated above, Arg229 from the adjacent monomer is within 3.2 Å of one of the oxygen atoms of
the C1 nitro group; Asn234 from the same subunit as Arg229 hydrogen bonds to the hydroxyl
group at C6 (Figure 3a).
1
2
ACS Paragon Plus Environment

Page 13 of 32
Biochemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Active site structures of (a) γꢁRSD from Polaromonas sp. JS6666 (PDB entry 4QRO),
(b) LigW from Novosphingobium aromaticivorans (PDB entry 4QRN), and (c) γꢁRSD from
Rhizobium sp. MTPꢁ10005 (PDB entry 2DVU).
1
3
ACS Paragon Plus Environment

Biochemistry
Page 14 of 32
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The single divalent cation is bound in the active site in an octahedral geometry (Figure
2
+
3
a). The residues important for binding Mn include a glutamate and a histidine from the ExH
motif found at the end of βꢁstrand 1 (Glu8 and His10), a histidine from βꢁstrand 5 (His164), and
an aspartate from βꢁstrand 8 (Asp287). The remaining two ligands include the nitroꢁsubstituent
of the inhibitor and the adjacent phenolic oxygen. Most notably, the geometry of the inhibitor
deviates significantly from planarity. In particular, the nitro group at C1 is tilted from the plane
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
+
of the phenyl ring plane with a torsion angle of 51°. Interestingly, both the Mn coordination
geometry and the mode of inhibitor binding are very similar to those observed in the very high
resolution Xꢁray structure of LigW in complex with 2ꢁnitrovanillate (Figure 3b) (19).
The expected high degree of similarity between γꢁRSD from Polaromonas sp. JS666 and
Rhizobium sp. MTPꢁ10005 was borne out by three dimensional structural alignments, which
revealed an agreement of 77% sequence identity and 0.36 Å RMSD over 317 aligned Cα atom
(PDB entry 4QRO compared to 2DVX). Likwise, γꢁRSD from Polaromonas aligned fairly well
with LigW from Sphingomonas paucimobilis, despite modest sequence identity (4QRO versus
4
INF, 37% sequence identity, 1.38 Å RMDS over 282 aligned Cα atoms).
Reaction Mechanism. The optimized structure of the enzymeꢁsubstrate complex (called
E:S) is shown in Figure 4. The substrate retains a very similar binding mode as the inhibitor in
the crystal structure. The carboxylate group forms hydrogen bonds with Asp287, Arg229, Wat1
and also the C6 hydroxyl group, which hydrogen bonds to Asn234. The deprotonated C2
hydroxyl group interacts with Ser20 and Asn128 through three water molecules. It is noteworthy
that this binding mode bears a very high similarity to that of LigW, suggesting a structural basis
for a similar reaction mechanism (19, 36). For γꢁRSD the reaction would likely start with the
1
4
ACS Paragon Plus Environment

Page 15 of 32
Biochemistry
1
2
3
4
5
6
7
8
9
protonation of the C1 atom of the substrate by Asp287, followed by CꢁC bond cleavage to
generate the CO and resorcinol products.
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Optimized structure of the active site model for γꢁRSD designed on the basis of the
crystal structure from Polaromonas sp. (PDB entry 4QRO). For clarity, only the polar
hydrogens and hydrogens on the substrate and truncated bonds are shown. Atoms fixed during
geometry optimization are shown in green. Selected distances are given in angstrom.
Starting from the enzymeꢁsubstrate complex E:S, the protonation of the substrate C1
carbon by Asp287 first takes place. This step is calculated to be rateꢁlimiting with a barrier of
1
4.8 kcal/mol, and the resulting intermediate (Int) is 8.1 kcal/mol higher in energy than E:S
(Figure 5). At the optimized transition state for this protonation (TS1), the distances of the
breaking OꢁH bond and the forming CꢁH bond are 1.29 Å and 1.40 Å, respectively (Figure 6).
Next, the CꢁC bond between the carboxylate group and the C1 atom breaks to form the products.
Interestingly, this decarboxylation process is easy with a calculated barrier of only 3.3 kcal/mol
1
5
ACS Paragon Plus Environment

Biochemistry
Page 16 of 32
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
relative to Int, that is 11.4 kcal/mol higher than E:S. At the corresponding transition state (TS2),
the length of the breaking CꢁC bond is 2.17 Å (Figure 6). The enzymeꢁproduct complex (E:P)
is 3.3 kcal/mol higher than E:S (Figure 5), including the contribution of the entropy gain from
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the release of CO as discussed in the Computational Details section. In the E:P structure, the
2
2+
Mn cation becomes fiveꢁcoordinated.
Figure 5. Calculated energy profile for the mechanism proposed on the basis of the DFT
calculations.
Very similar coordination was observed in the Xꢁray structure of LigW from
Sphingomonas paucimobilis complexed with the vanillate product (PDB entry 4L6D) (19). The
obtained energy profile here for γꢁRSD is also very similar to that calculated for LigW (36). For
both enzymes, the proton transfer step corresponds to a higher barrier than the decarboxylation
step. Also, the structures of the intermediates and transition states bear high similarities.
Alternative Mechanisms. As discussed above, several crystal structures of γꢁRSD from
another organism, Rhizobium sp. MTPꢁ10005, have been reported previously, one of which is an
enzymeꢁsubstrate complex (PDB entry 2DVU) (17). This enzyme was found to contain zinc in
the active site and the substrate binds to the active site such that one oxygen atom of the
carboxylate group, but none of the hydroxyl groups, coordinates to the metal ion (17). On the
1
6
ACS Paragon Plus Environment

Page 17 of 32
Biochemistry
1
2
3
4
5
6
7
8
9
basis of this structure, Asp287 was suggested to be in the deprotonated state, acting as a general
base to abstract a proton from the hydroxyl groups of the substrate to initiate the reaction.
Protonation of the C1 carbon can then take place, followed by a CꢁC bond cleavage to generate
the products (17).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Optimized structures of the stationary points along the proposed reaction pathway.
For clarity, only a small part of the model is shown here. Distances are given in angstrom.
1
7
ACS Paragon Plus Environment

Biochemistry
Page 18 of 32
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
It is interesting to compare the binding mode of the substrate in 2DVU with that in 4QRO
and also the feasibility of the reaction mechanisms using the quantum chemical approach
employed here. To this end, we have on the basis of the 2DVU structure constructed an active
site model (called E:SꢀZn, see Figure 7 for optimized structure) for γꢁRSD from Rhizobium sp.
This model consists of the same residues as that in E:S, but with a Zn instead of Mn and a
different binding mode for the substrate. In E:SꢀZn, both hydroxyl groups of the substrate are in
the neutral form, and the Asp287 residue is modeled to be in the deprotonated state since it is
suggested to act as a general base for the catalysis (17).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Optimized structure of the active site model for γꢁRSD designed on the basis of the
2
+
crystal structure from Rhizobium sp. (PDB entry 2DVU) containing Zn with the substrate
bound in a monodentate fashion by the carboxylate group. For clarity, only polar hydrogen
atoms and hydrogens on the substrate and truncated bonds are shown. Atoms fixed during
geometry optimization are shown in green.
1
8
ACS Paragon Plus Environment

Page 19 of 32
Biochemistry
1
2
3
4
5
6
7
8
9
Starting from E:SꢀZn, the structures of the intermediates involved in the corresponding
pathway were optimized (Figure 8). The first intermediate, formed by the proton transfer from
the substrate hydroxyl group to Asp287, is calculated to be 19.2 kcal/mol higher than E:SꢀZn.
The following intermediate, resulting from the proton transfer to C1 of the substrate, has an even
higher energy, >23 kcal/mol relative to E:SꢀZn, regardless of the identity of the proton source
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(either Asp287 or His218 via a water shuttle, see Scheme 2). These energies are thus
prohibitively high and the barriers leading to these intermediates should be even higher (not
calculated). Therefore, this mechanistic scenario should be ruled out, and it can be concluded
that the binding mode as reported in 2DVU is not a productive mode for catalysis.
Alternatively, we sought to establish whether or not it is possible that the substrate in the
Znꢁcontaining enzyme binds to the metal ion similarly to the Mnꢁdependent enzyme (4QRO), i.e.
in a bidentate binding mode as in Figures 3 and 4. In order to examine this possibility, another
active site model was constructed by manually repositioning the substrate in E:SꢀZn (see SI for
the optimized structure). The new enzymeꢁsubstrate complex is, however, calculated to have a
much higher energy when compared to E:SꢀZn, 14.9 kcal/mol, showing that for the Znꢁ
dependent enzyme the binding mode observed in 2DVU is highly preferred. Combined with the
prohibitively high barriers for the reaction pathway starting from E:SꢀZn as discussed above,
one might therefore speculate that the Znꢁbound form is not active for γꢁRSD.
1
9
ACS Paragon Plus Environment

Biochemistry
Page 20 of 32
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8. Optimized structures of the intermediates along the previously proposed reaction
pathway for the γꢁRSD from Rhizobium sp. shown in Scheme 2. In the case of Int2'ꢀZn (c), the
proton of Asp287 transfers to His218 spontaneously during the geometry optimization. For
clarity, only a small part of the model is shown here. The calculated energies relative to E:SꢀZn
are also given (in kcal/mol). Distances are given in angstrom.
20
ACS Paragon Plus Environment

Page 21 of 32
Biochemistry
1
2
3
4
5
6
7
8
9
Another alternative is whether for the Mnꢁdependent enzyme the substrate can bind as
that in 2DVU, i.e. only by the carboxylate group. By modifying the binding mode of the
substrate in E:S to resemble 2DVU, we optimized the geometry and calculated the energy of the
of this binding mode (see SI for optimized structure). The calculated energy difference between
the new structure and E:S is very small, only 0.4 kcal/mol in favor of the former one. This
shows that both binding modes are accessible for the Mnꢁdependent enzyme. However,
similarly to the case with E:SꢀZn, the intermediates along the reaction pathway were calculated
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(see SI) and the corresponding energies turned out to be prohibitively high. These calculations
thus show that the 2DVU binding mode is likely to be inactive for the Mnꢁdependent enzyme.
Conclusions. The catalytic activity, substrate specificity and crystal structure of γꢁRSD
have been reported. The values of k and k /K for the decarboxylation of γꢁresorcylate are
cat
cat
m
–
1
4
–1 –1
0
.44 ± 0.01 s and (1.3 ± 0.1) x 10 M s , respectively. In addition to the natural substrate, γꢁ
RSD is also shown to facilitate the decarboxylation of 2,3ꢁdihydroxybenzoate, 2,4,6ꢁ
trihydroxybenzoate and 2,6ꢁdihydroxyꢁ4 methylbenzoate. We described the crystal structure of
2+
the enzyme from Polaromonas sp. JS666 (locus tag: Bpro_2061) in complex with Mn and the
substrate analog inhibitor, 2ꢁnitroresorcinol. The binding mode of the inhibitor is different
compared to the substrate in the previously reported structure of the enzyme from Rhizobium sp.
2+
in complex with Zn , and instead shows high similarity to that seen in LigW complexed with 2ꢁ
nitrovanillate. Theoretical calculations employing density functional theory show that γꢁRSD
follows a very similar mechanism as LigW. The reaction starts with the protonation of the
carbon atom of the substrate, which is calculated to be the rateꢁlimiting step of the overall
reaction. A CꢁC bond cleavage then takes place to form the products. Additionally, the
mechanism proposed previously on the basis of the Znꢁstructure, involving binding of the
2
1
ACS Paragon Plus Environment

Biochemistry
Page 22 of 32
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
substrate to the metal ion only by its carboxylate group, is shown to be associated with high
energies both for the Znꢁ and Mnꢁbound enzymes.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Possible reaction mechanisms proposed on the basis of the structure of the Znꢁ
containing γꢁRSD from Rhizobium sp.
2
2
ACS Paragon Plus Environment

Page 23 of 32
Biochemistry
1
2
3
4
5
6
7
8
9
ꢀ
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
Additional results concerning the alternative mechanisms and Cartesian coordinates of
optimized structures.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
AUTHOR INFORMATION
Corresponding Authors
*
*
Eꢁmail for S. C. A.: steve.almo@einstein.yu.edu
Eꢁmail for F. H.: fahmi.himo@su.se
* Eꢁmail for F. M. R.: raushel@tamu.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported in part by the Knut and Alice Wallenberg Foundations (to F.H.), the
Robert A. Welch Foundation (Aꢁ840 to F.M.R.) and the National Institutes of Health (U54
GM093342 to S.C.A).
2
3
ACS Paragon Plus Environment

Biochemistry
Page 24 of 32
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
REFERENCES
1. Kluge, C., Tschech, A. and Fuchs, G. (1999) Anaerobic metabolism of resorcylic acids (mꢁ
dihydroxybenzoic acids) and resorcinol (1, 3ꢁbenzenediol) in fermenting and in a
denitrifying bacterium. Arch. Microbiol. 155, 68ꢁ74.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2. Iwasaki, Y., Kino, K., Nishide, H. and Kirimura, K. (2007) Regioselective and enzymatic
production of γꢁresorcylic acid from resorcinol using recombinant Escherichia coli cells
expressing a novel decarboxylase gene. Biotechnol Lett. 29, 819ꢁ822.
3
. Durairaj, R. B. (2005) Resorcinol: Chemistry, Technology, and Applications; SpringerꢁVerlag:
Berlin.
4
. Lindsey, A. S. and Jeskey, H. (1957) The Kolbeꢁschmitt reaction. Chem. Rev. 57, 583ꢁ620.
. Wuensch, C., Gross, J., Steinkellner, G., Lyskowski, A., Gruber, K., Glueck, S. M. and Faber,
K. (2014) Regioselective orthoꢁcarboxylation of phenols catalyzed by benzoic acid
decarboxylases: a biocatalytic equivalent to the Kolbe–Schmitt reaction. RSC Adv. 4, 9673ꢁ
5
9
679.
6. Ren, J., Yao, P., Yu, S., Dong, W., Chen, Q., Feng, J., Wu, Q. and Zhu, D. (2016) An
Unprecedented Effective Enzymatic Carboxylation of Phenols. ACS Catal. 6, 564ꢁ567.
7
. Pesci, L., Glueck, S. M., Gurikov, P., Smirnova, I., Faber, K. and Liese, A. (2015) Biocatalytic
carboxylation of phenol derivatives: kinetics and thermodynamics of the biological Kolbe–
Schmitt synthesis. FEBS J. 282, 1334ꢁ1345.
8
. Pesci, L., Kara, S. and Liese, A. (2016) Evaluation of the Substrate Scope of Benzoic Acid
(De) carboxylases According to Chemical and Biochemical Parameters. ChemBioChem 17,
1845ꢁ1850.
2
4
ACS Paragon Plus Environment

Page 25 of 32
Biochemistry
1
2
3
4
5
6
7
8
9
9
1
1
. Wuensch, C., Glueck, S. M., Gross, J., Koszelewski, D., Schober, M. and Faber, K. (2012)
Regioselective Enzymatic Carboxylation of Phenols and Hydroxystyrene Derivatives. Org.
Lett. 14, 1974ꢁ1977.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0. Plasch, K., Resch, V., Hitce, J., Popłoński, J., Faber, K. and Glueck, S.M. (2017)
Regioselective Enzymatic Carboxylation of Bioactive (Poly) phenols. Adv. Synth. Catal.
3
59, 959–965.
1. Yoshida, M., Fukuhara, N. and Oikawa, T. (2004) Thermophilic, reversible gammaꢁ
resorcylate decarboxylase from Rhizobium sp. strain MTPꢁ10005: purification, molecular
characterization, and expression. J. Bacteriol. 186, 6855ꢁ6863.
12. Ishii, Y., Narimatsu, Y., Iwasaki, Y., Arai, N., Kino, K. and Kirimura, K. (2004) Reversible
and nonoxidative gammaꢁresorcylic acid decarboxylase: characterization and gene cloning
of a novel enzyme catalyzing carboxylation of resorcinol, 1,3ꢁdihydroxybenzene, from
Rhizobium radiobacter. Biochem. Biophys. Res. Commun. 324, 611ꢁ620.
1
3. Yoshida, T., Hayakawa, Y., Matsui, T. and Nagasawa, T. (2004) Purification and
characterization of 2,6ꢁdihydroxybenzoate decarboxylase reversibly catalyzing nonoxidative
decarboxylation. Arch. Microbiol. 181, 391ꢁ397.
1
4. Matsui, T., Yoshida, T., Yoshimura, T. and Nagasawa, T. (2006) Regioselective
carboxylation of 1,3ꢁdihydroxybenzene by 2,6ꢁdihydroxybenzoate decarboxylase of
Pandoraea sp. 12Bꢁ2. Appl. Microbiol. Biotechnol. 73, 95ꢁ102.
15. Kasai, D., Araki, N., Motoi, K., Yoshikawa, S., Iino T., Imai S., Masai E. and Fukuda M.
2015) γꢁResorcylate catabolicꢁpathway genes in the soil actinomycete Rhodococcus jostii
RHA1. Appl. Environ. Microbiol. 81, 7656ꢁ7665.
(
2
5
ACS Paragon Plus Environment

Biochemistry
Page 26 of 32
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
6. Yoshida, M., Oikawa, T., Obata, H., Abe, K., Mihara, H. and Esaki, N. (2007) Biochemical
and genetic analysis of the γꢁresorcylate (2,6ꢁdihydroxybenzoate) catabolic pathway in
Rhizobium sp. strain MTPꢁ10005: identification and functional analysis of its gene cluster. J.
Bacteriol. 189, 1573ꢁ1581.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
7. Goto, M., Hayashi, H., Miyahara, I., Hirotsu, K., Yoshida, M. and Oikawa, T. (2006) Crystal
structures of nonoxidative zincꢁdependent 2,6ꢁdihydroxybenzoate (γꢁresorcylate)
decarboxylase from Rhizobium sp. strain MTPꢁ10005. J. Biol. Chem. 281, 34365ꢁ34373.
8. Seibert, C. M. and Raushel, F. M. (2005) Structural and Catalytic Diversity within the
Amidohydrolase Superfamily. Biochemistry 44, 6383−6391.
1
19. Vladimirova, A., Patskovsky, Y., Fedorov, A. A., Fedorov, E. V., Bonanno, J. B., Toro, R.,
Hillerich, B., Seidel, R., Richards, N. G. J., Almo, S. C. and Raushel, F. M. (2016) Substrate
Distortion and the Catalytic Reaction Mechanism of 5ꢁCarboxyvanillate Decarboxylase. J.
Am. Chem. Soc. 138, 826ꢁ836.
2
0. Sauder, J. M., Rutter, M. E., Bain, K., Rooney, I., Gheyi, T., Atwell, S., Thompson, D. A.,
Emtage, S., and Burley, S. K. (2008) High Throughput Protein Production and
Crystallization at NYSGXRC. Methods in Molecular Biology 426, 561ꢁ575.
1. Minor, W., Cymborowski, M., Otwinowski, Z. and Chruszcz, M. (2006) HKLꢁ3000: the
integration of data reduction and structure solution–from diffraction images to an initial
model in minutes. Acta Crystallogr., Sect. D: Biol. Crystallogr. 62, 859ꢁ866.
2
22. Collaborative Computational Project Number 4 The CCP4 Suit: Programs for Protein
Crystallography. (1994) Acta Crystallogr., Sect. D: Biol. Crystallogr. 50, 760−763.
2
3. Lamzin, V. S. and Wilson, K. S. (1993) Automated refinement of protein models. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 49, 129ꢁ147.
2
6
ACS Paragon Plus Environment

Page 27 of 32
Biochemistry
1
2
3
4
5
6
7
8
9
2
4. Murshudov, G. N., Vagin, A. A. and Dodson, E. J. (1997) Refinement of macromolecular
structures by the maximumꢁlikelihood method. Acta Crystallogr., Sect. D: Biol. Crystallogr.
53, 240ꢁ255.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
5. Emsley, P. and Cowtan, K. (2004) Coot: modelꢁbuilding tools for molecular graphics. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 60, 2126–2132.
6. Delano, W. L. (2002) The PyMOL Molecular Graphics System; Delano Scientific: San
Carlos, CA.
7. Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R.,
Scalmani, G., Barone, V., Mennucci, B., Petersson, G. A., Nakatsuji, H., Caricato, M., Li,
X., Hratchian, H. P., Izmaylov, A. F., Bloino, J., Zheng, G., Sonnenberg, J. L., Hada, M.,
Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y.,
Kitao, O., Nakai, H., Vreven, T., Montgomery, J. A., Jr., Peralta, J. E., Ogliaro, F.,
Bearpark, M., Heyd, J. J., Brothers, E., Kudin, K. N., Staroverov, Keith T., V. N.,
Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A., Burant, J. C., Iyengar, S. S.,
Tomasi, J., Cossi, M., Rega, N., Millam, J. M., Klene, M., Knox, J. E., Cross, J. B., Bakken,
V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R. E., Yazyev, O., Austin, A. J.,
Cammi, R., Pomelli, C., Ochterski, J. W., Martin, R. L., Morokuma, K., Zakrzewski, V. G.,
Voth, G. A., Salvador, P., Dannenberg, J. J., Dapprich, S., Daniels, A. D., Farkas, O.,
Foresman, J. B., Ortiz, J. V., Cioslowski, J. and Fox, D. J. (2013) Gaussian 09, revision
D.01; Gaussian, Inc.: Wallingford, CT.
2
8. Becke, A. D. (1993) Density functional Thermochemistry. III. The Role of Exact Exchange.
J. Chem. Phys. 98, 5648ꢁ5652.
2
7
ACS Paragon Plus Environment

Biochemistry
Page 28 of 32
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
9. Lee, C., Yang, W. and Parr, R. G. (1988) Development of the ColleꢁSalvetti Correlationꢁ
energy Formula into a Functional of the Electron Density. Phys. Rev. B 37, 785ꢁ789.
30. Grimme, S., Antony, J., Ehrlich, S. and Krieg, H. (2010) A Consistent and Accurate Ab
Initio Parametrization of Density Functional Dispersion Correction (DFTꢁD) for the 94
Elements H−Pu. J. Chem. Phys. 132, 154104.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
1. Grimme, S., Ehrlich, S. and Goerigk, L. (2011) Effect of the Damping Function in
Dispersion Corrected Density Functional Theory. J. Comput. Chem. 32, 1456ꢁ1465.
2. Hay, P. J. and Wadt, W. R. (1985) Ab Initio Effective Core Potentials for Molecular
Calculations. Potentials for the Transition Metal Atoms Sc to Hg. J. Chem. Phys. 82, 270ꢁ
283.
3
3
3. Marenich, A. V., Cramer, C. J. and Truhlar, D. G. (2009) Universal Solvation Model Based
on Solute Electron Density and on A Continuum Model of the Solvent Defined by the Bulk
Dielectric Constant and Atomic Surface Tensions. J. Phys. Chem. B 113, 6378ꢁ6396.
4. Lind, M. E. S. and Himo, F. (2014) Theoretical Study of Reaction Mechanism and
Stereoselectivity of Arylmalonate Decarboxylase. ACS Catal. 4, 4153ꢁ4160.
5. Sheng, X., Lind, M. E. S. and Himo F. (2015) Theoretical Study of the Reaction Mechanism
of Phenolic Acid Decarboxylase. FEBS J. 282, 4703ꢁ4713.
3
3
3
6. Sheng, X., Zhu, W., Huddleston, J. P., Xiang, D. F., Raushel, F. M., Richards, N. G., and
Himo, F. (2017) A Combined ExperimentalꢁTheoretical Study of the LigWꢁCatalyzed
Decarboxylation of 5ꢁCarboxyvanillate in the Metabolic Pathway for Lignin Degradation.
ACS Catal. 7, 4968ꢁ4974.
3
7. Siegbahn, P. E. M. and Himo, F. (2009) Recent developments of the quantum chemical
cluster approach for modeling enzyme reactions. J. Biol. Inorg. Chem. 14, 643ꢁ651.
2
8
ACS Paragon Plus Environment

Page 29 of 32
Biochemistry
1
2
3
4
5
6
7
8
9
3
8. Blomberg, M. R. A. and Siegbahn, P. E. M. (2010) Quantum chemical studies of protonꢁ
coupled electron transfer in metalloenzymes. Chem. Rev. 110, 7040ꢁ7061.
39. Siegbahn, P. E. M. and Himo, F. (2011) The quantum chemical cluster approach for
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
modeling enzyme reactions. Comput. Mol. Sci. 1, 323ꢁ336.
4
0. Blomberg, M. R. A., Borowski, T., Himo, F., Liao, R.ꢁZ. and Siegbahn, P. E. M. (2014)
Quantum chemical studies of mechanisms for metalloenzymes. Chem. Rev. 114, 3601ꢁ3658.
1. Himo, F. (2017) Recent Trends in Quantum Chemical Modeling of Enzymatic Reactions. J.
Am. Chem. Soc. 139, 6780ꢁ6786.
4
2
9
ACS Paragon Plus Environment