Angewandte
Chemie
one end of the b-sheet and includes b3 and b4 and a single
turn of helix, a3.
His56 and His89 residues donate hydrogen bonds from ND1
to carbonyl groups of Gly115 and Pro87, respectively (data
not shown). Nicotinic acid is tethered to the cation and
positioned between five hydrophobic residues; Phe21, Leu27,
Trp86, Tyr123, and Cys159 (Supporting Information, Fig-
ure S6). Trp86 NE1 and Tyr123 OH form a hydrogen bond to
hold these residues in place over the ligand. A further six
residues (Val29, Ile154, Ala155, Phe158, Ile184, and Leu186)
stabilize the hydrophobic environment around the nicotinic
acid and occlude the active site (Supporting Information,
Figure S6). In the absence of a ligand, the Zn2+ coordination
sphere is completed with a water as indicated in structures of
PhPncA and ScPncA.[6,7] Nicotinic acid O8 and O9 are 2.5
and 2.6 ꢀ, respectively, from Cys159 SG suggesting the
presence of a bifurcated hydrogen bond. The next nearest
functional group to Cys159 SG is Asp16 OD2 at a distance of
3.4 ꢀ. O9 accepts hydrogen bonds donated from main-chain
amides of cis-Ala155 and Cys159. Interaction with the cis-
Ala155 carbonyl group suggests that O8 is a hydroxy group
and that protonation of Asp16 OD2 may facilitate a second
hydrogen bond (Supporting Information, Figure S7). Alter-
natively, O8 as hydroxy group or if protonated may partic-
ipate in a bifurcated hydrogen bond with Asp16 and Ala155.
Asp16, together with Asp54 and Lys114 form a cluster of
interacting hydrophilic residues on one side of the ligand-
binding site. Lys114 NZ donates hydrogen bonds to
Asp16 OD1, Asp54 OD2, (Figure 2) and the main-chain
carbonyl group of Tyr123 (not shown). The close proximity
of Lys114 to Asp16 is likely to influence the pKa value. The
Asp16 carboxylate and main-chain amide groups form hydro-
gen bonds with Thr52 OG1 (Supporting Information, Fig-
ure S7), a pairing strictly conserved in PncA (Supporting
Information, Figure S8).
Gel filtration and analytical ultracentrifugation indicate
that AbPncA is a stable dimer in solution, approximate mass
47 kDa. The asymmetric unit of form I is a dimer (Supporting
Information, Figure S5), with an interface of 900 ꢀ2, which is
about 10% of the surface area of a subunit. In form II the
crystallographic twofold axis generates the same dimer. The
interactions that stabilize the dimer mainly involve residues
on a5 and b5. The AbPncA subunit resembles orthologues
from Pyrococcus horikoshii (PhPncA) and Saccharomyces
cerevisae (ScPncA).[6,7] Superpositions give r.m.s.d. values of
1.2 ꢀ for the overlay of an AbPncA subunit on either PhPncA
(44% sequence identity, 167 Ca atoms) or ScPncA (33%
identity, 191 Ca atoms).
The AbPncA active site is between the core and the
subdomain (Figure 1), and is formed by residues on b1, b2, the
b4–a4 loop, b5, and the b6–a6 turn. It is buried and
completely occluded from solvent by four polypeptide seg-
ments; the loops linking a3–b3, b5–a5, and b8–a9 together
with strand b4. Numerous hydrophobic residues (discussed
below) surround the active site and a gross conformational
change is probably required to permit substrate binding or
release of products.
The active site Cys159 is located at the N-terminus of a6 at
one side of the active site with the Zn2+ ion positioned on the
other side. The metal ion is held tightly in the AbPncA active
site as shown by activity being retained in the presence of
10 mm EDTA (data not shown). Octahedral coordination of
Zn2+ ion involves Asp54 OD2, His56 NE2, and His89 NE2,
two water molecules and nicotinic acid N5 (Figure 2). Cation–
ligand distances range from 2.11 to 2.28 ꢀ, consistent with
data on Zn2+ ligand geometry.[10] A network of hydrogen
bonds position the coordinating groups. One water molecule
forms hydrogen bonds with Ser62 OG and Asp121 OD2, the
other with Asp54 OD1 and Asp121 OD1. The coordinating
Amidation involves either acidic or basic hydrolysis and
the structures of the enzyme–product complex suggest that
basic hydrolysis applies in PncA. Acid hydrolysis would
involve nucleophilic water attacking the carbon of a proton-
ated amide. The hydrophobic environment on one side of the
amide and close interactions with functional groups on the
other renders it difficult to envisage how water could be
placed to attack C7. Nicotinic acid O9 accepts hydrogen
bonds from two amides so protonation at O9 would destabi-
lize the complex due to the proximity of the amides.
A more likely mechanism is indicated by a strictly
conserved and essential Cys159,[6,8] on the polar side of the
active site, ideally placed to attack the carbonyl carbon atom
in a manner similar to that proposed for other enzymes, for
example trypanothione synthetase amidase[11] and the nitri-
lase enzyme superfamily.[12] Nitrilases exploit a catalytic triad
consisting of a reactive cysteine, a lysine, and a glutamate. The
triad of PncA has a conservative difference with aspartate
replacing glutamate.
We propose a four-stage mechanism (Figure 3). In stage I,
substrate binds in the axial position displacing a water from
the Zn2+ coordination sphere. The waters coordinating to
equatorial sites of the metal ion are also held in position by
hydrogen-bonding interactions to the enzyme (Figure 2),
whereas the axial water lacks such a restraint. It is this
water that vacates the coordination sphere as substrate binds.
Figure 2. Active site of the nicotinic acid complex. The Zn2+ ion is a
blue-gray sphere, thin lines mark coordination to amino acid side
chains and two water molecules (marine spheres). Amino acids are in
stick representation: C gray, N blue, O red, S yellow. Nicotinic acid is
shown as a ball-and-stick model: C black, N blue, O red. Dashed lines
represent potential hydrogen bonds.
Angew. Chem. Int. Ed. 2009, 48, 9176 –9179
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
9177
Fyfe, Paul K.
