Background: Isatin hydrolase is involved in bacterial indole-3-acetic acid degradation and belongs to a structurally uncharacterized family. Results: We determined the first crystal structure of a functionally characterized metal dependent hydrolase of this overall fold. Conclusion:The new hydrolytic fold reveals a transient water wire that allow proton release. Significance: Proton transfer is a fundamental process important in the understanding of enzymes and membrane transporters.
The catalytic mechanism of the cyclic amidohydrolase isatin hydrolase depends on a catalytically active manganese in the substrate-binding pocket. The Mn2+ ion is bound by a motif also present in other metal dependent hydrolases like the bacterial kynurenine formamidase. The crystal structures of the isatin hydrolases from Labrenzia aggregata and Ralstonia solanacearum combined with activity assays allow for the identification of key determinants specific for the reaction mechanism. Active site residues central to the hydrolytic mechanism include a novel catalytic triad Asp-His-His supported by structural comparison and hybrid quantum mechanics/classical mechanics simulations. A hydrolytic mechanism for a Mn2+ dependent amidohydrolases that disfavour Zn2+ as the primary catalytically active site metal proposed here is supported by these likely cases of convergent evolution. The work illustrates a fundamental difference in the substrate-binding mode between Mn2+ dependent isatin hydrolase like enzymes in comparison with the vast number of Zn2+ dependent enzymes.
Two high resolution crystal structures of isatin hydrolase (IH) from the Baltic seabed bacteria Labrenzia aggregata are presented. The crystals structure capture both the apo and the product state. This hydrolase present a new fold and the first metal-dependent hydrolase with this fold to be functionally characterized [1]. The Isatin hydrolase catalyze the reaction that convert isatin to isatinate and belongs to a novel family of metalloenzymes that include bacterial kynurenine formamidase (KynB) also recently published, however hoste a binclear zink site in the active site[2] as compared to a single manganese in IH. The product state, mimicked by thioisatinate, has captured an additional water molecule that bridges the thioisatinate to a water channel and that could act as a proton wire and thus allows the proton to be released during the hydrolysis reaction only when the product is formed. The functional proton wire is therefore locked by thioisatinate and represents a unique catalytic feature trapped and visualized. Biochemical evidence for the proton wire is also presented as single point mutation from S225C enhances the Vmax of the enzyme. Ser-225 is the only side chain residue that is included in the proton wire. The molecular basis for thioisatinate recognition allows stronger and more confident identification of orthologous genes encoding isatin hydrolases within the prokaryotic kingdom. The isatin hydrolase orthologues found in human gut bacteria raise the question as to whether the indole-3-acetic acid degradation pathway is present in human gut flora.
Isatin is an endogenous inhibitor of monoamine oxidase B and is found in human blood and tissue. Increased levels of isatin have been linked to stress and anxiety in rodents and humans; however, the metabolism of isatin in humans is largely unknown. We have developed a fluorescence-based enzymatic assay that can quantify isatin in blood samples. A phase extraction of isatin followed by a second phase extraction combined with an enzymatic reaction performed by an isatin hydrolase is used to extract and quantify isatin in whole blood samples. This results in a purity of more than 95% estimated from RP-HPLC. The hydrophobic molecule isatin is in equilibrium between an organic and aqueous phase; however, conversion by isatin hydrolase to the hydrophilic product isatinate traps it in the aqueous phase, making this step highly specific for isatin. The described protocol also offers a novel method for fast and efficient removal of isatin from any type of sample. The isolated isatinate is converted chemically to anthranilate that allows fluorescent detection and quantification. Pig plasma isatin levels are quantified to a mean of 458 nM ± 91 nM. Biophysical characterization of the isatin hydrolase shows enzymatic functionality between pH 6 and 9 and at temperatures up to 50 °C. Isatin hydrolase is highly selective for manganese ions with a dissociation constant determined to be 9.5 μM. We deliver proof-of-concept for the enzymatic quantification of isatin in blood and provide a straightforward method for further investigation of isatin as a biomarker in human health.
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