C O M M U N I C A T I O N S
Figure 4. Two possible mechanisms for NfMI-catalyzed isomerization of maleate. In each, Cys76 performs a Michael-type nucleophilic attack on C2 of maleate,
saturating the C2dC3 double bond to allow free rotation. (a) Concomitant with nucleophilic attack of Cys76, Cys194 protonates the C2dC3 double bond, forming
a stable succinylcysteine intermediate. After C2-C3 bond rotation, deprotonation of C3 by Cys194 initiates release of fumarate and Cys76. (b) Nucleophilic attack
by Cys76 initiates delocalization of electrons to the C3-C4 bond, giving an enediolate intermediate stabilized by the dioxyanion hole including Cys194. C2-C3
bond rotation is followed by the reverse electron relay from the enediolate to Cys76, initiated by deprotonation of the C4 carboxylate by Cys194.
to be stabilized by a conserved dioxyanion hole, which provides it
References
1
1
with six hydrogen-bonding partners. In NfMI, this dioxyanion
hole is only partially conserved and binds the C1 carboxylate of
the intermediate rather than the C4, as might be expected from the
proposed mechanisms. Only four of the six H-bonding partners
persist in NfMI, as Leu77 and Val78 are found in place of Asn
and Thr (racemases) or Thr and Ser (AMDs) (Figure 2 and Figure
S7) Of these four, mutation of Tyr133 to Phe (Table 1) resulted in
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active site in NfMI. The putative dienolate, or succinylcarboxylate
of C4 in the C194A complex, is in fact bound by a dioxyanion
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among the superfamily members described to date.
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by the C194A structure remains the focus of future work. The
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within the Asp-Glu racemase superfamily that is more extensive
than envisaged previously. Overall, the results reveal an unprec-
edented biochemical mechanism for CdC bond isomerization and
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Supporting Information Available: Phylogenetic tree of Asp/Glu
racemase superfamily; protocols for cloning, expression, purification,
and crystallization of NfMI and mutants; data collection and refinement,
with statistics on WT NfMI and the C194A mutant; and kinetics assays
and pH/activity plot. This material is available free of charge via the
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JA1053576
J. AM. CHEM. SOC. 9 VOL. 132, NO. 33, 2010 11457