Xylonolactonase from Caulobacter crescentus Is a Mononuclear Nonheme Iron Hydrolase

Abstract

Caulobacter crescentus xylonolactonase (Cc XylC, EC 3.1.1.68) catalyzes an intramolecular ester bond hydrolysis over a nonenzymatic acid/base catalysis. Cc XylC is a member of the SMP30 protein family, whose members have previously been reported to be active in the presence of bivalent metal ions, such as Ca²⁺, Zn²⁺, and Mg²⁺. By native mass spectrometry, we studied the binding of several bivalent metal ions to Cc XylC and observed that it binds only one of them, namely, the Fe²⁺ cation, specifically and with a high affinity (K_d = 0.5 μM), pointing out that Cc XylC is a mononuclear iron protein. We propose that bivalent metal cations also promote the reaction nonenzymatically by stabilizing a short-lived bicyclic intermediate on the lactone isomerization reaction. An analysis of the reaction kinetics showed that Cc XylC complexed with Fe²⁺ can speed up the hydrolysis of d-xylono-1,4-lactone by 100-fold and that of d-glucono-1,5-lactone by 10-fold as compared to the nonenzymatic reaction. To our knowledge, this is the first discovery of a nonheme mononuclear iron-binding enzyme that catalyzes an ester bond hydrolysis reaction.

Figure 1

(A) Deconvoluted native mass spectra of Cc XylC in apo-form (blue) and holo-form (red). (B) Calculated mass spectra of the corresponding forms of Cc XylC. The most abundant isotopic masses are indicated.

Figure 2

Titration of Cc XylC with Fe²⁺.

Figure 3

Suggested total reaction of d-xylonolactone hydrolysis.