C O M M U N I C A T I O N S
Figure 3. Structure of SgTAM bound to the substrate analogue 11. (A) Electron density map (2Fo - Fc at 1.5σ) showing the active-site region. The
inhibitor is shown in black and the MIO cofactor red. (B) Representation of the major interactions and conformation of the enzyme with the bound analogue.
(C) View of the active site showing the electron density map (Fo - Fc at 2.0σ) calculated from cocrystals of analogue 12 showing the position of the methyl
group.
acid are His93 and Tyr415. These two residues form a bifurcated
hydrogen bond with the substrate phenol (Figure 3AB). The residue
corresponding to His93 has been implicated as a key specificity
determinant in tyrosine and phenylalanine ammonia lyases.7,8 The
carboxylate of the amino acid forms hydrogen bonds with Arg311
and Asn205. Overall, the active site contains a high percentage of
aromatic residues. The MIO is stacked with Phe356 (not illustrated)
and Tyr308 is adjacent to the methylene of the MIO. Tyr63 is
positioned in close proximity to the R and â-carbons of the inhibitor
and is well-positioned to assist in the elimination chemistry. Gly70
forms a hydrogen bond with the phenol of Tyr63 and both residues
are conserved in all MIO-based lyases and mutases. Tyr63 and
Gly70 reside on a loop that is often disordered in unliganded
ammonia lyase crystal structures. The phenolic oxygen of Tyr63
is positioned 3.2 Å away from the R-carbon and 3.4 Å from the
â-carbon of the bound inhibitor. Tyr63, in the phenolate state, can
act as a general base (Figure 4), shuttling the proton between the
R- and â- positions. The pKa of Tyr63 needs to be lowered in the
active site of SgTAM to favor the anionic form. Two aspects are
evident from the structure that contribute to a lower pKa: (i)
stabilization of the negative charge by the backbone hydrogen bond
of Gly70 and (ii) the significant number of R-helices with positive
dipoles pointed at the active site both will favor the phenolate. Also
consistent with this mechanism, the measured optimal pH for
SgTAM is ∼9.2b To confirm the importance of Tyr63 in the reaction
mechanism, site-directed mutagenesis was used to remove the
phenol by substituting phenylalanine. The resulting mutant, Tyr63Phe,
had no measurable aminomutase activity up to 1.9 mM substrate
concentration (see Supporting Information).
MIO adduct. Modeling of this orientation shows the active site can
accommodate this change while still maintaining binding interac-
tions between Arg311, His93, and Tyr415.
The presented structures of the aminomutase SgTAM provide
strong evidence that MIO-based enzymes use covalent catalysis
through the R-amine to direct the chemistry. For ammonia lyases,
a covalent adduct orients both an enzymatic base and the leaving
group for an E2-type elimination. For enzymes with aminomutase
activity, the conjugate 1,4-addition into the relatively nonelectro-
philic 4-hydroxycinnamate is facilitated by orienting the amino-
bound intermediate and maintaining the nucleophile in the neutral
form. Differentiation of the two catalytic pathways can be ac-
complished by retention of ammonia in the closed active site of
aminomutases.4
Acknowledgment. This work is supported in part by funds from
Boston College and the Damon Runyon Cancer Research Founda-
tion DRS-41-01 (S.D.B.) and NIH Grants CA78747 and CA113297
(B.S.). We thank Dr. Y. Li, Institute of Medicinal Biotechnology,
Chinese Academy of Medical Sciences, Beijing, China, for the
C-1027-producing S. globisporus strain and A. Orville and the staff
at the Brookhaven NSLS PXRR for assistance with X-ray data
collection.
Supporting Information Available: Full experimental details for
synthesis and characterization of inhibitors, co-complex crystallization,
structure determination and biochemical characterization of inhibitors/
mutant enzymes. This material is available free of charge via the Internet
References
The reaction of the related phenylalanine aminomutase has been
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the ammonium leaving group. The stereochemistry of the reverse
reaction of aminomutases (â-Tyr to R-Tyr) is variable. SgTAM
catalyzes the racemization of (S)-â-Tyr and a highly homologous
tyrosine aminomutase from chondramide biosynthesis produces the
opposite stereochemistry at the â-position, (R)-â-Tyr.3c The ster-
eochemical differentation can occur from rotation of the trans-
cinnamate intermediate to present the opposite face to the amine-
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Figure 4. Proposed mechanism for SgTAM activity.
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