A fundamental catalytic difference between zinc and manganese dependent enzymes revealed in a bacterial isatin hydrolase.

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.

The crystal structure of the regulatory domain of the human sodium-driven chloride/bicarbonate exchanger.

The sodium-driven chloride/bicarbonate exchanger (NDCBE) is essential for maintaining homeostatic pH in neurons. The crystal structure at 2.8 Å resolution of the regulatory N-terminal domain of human NDCBE represents the first crystal structure of an electroneutral sodium-bicarbonate cotransporter. The crystal structure forms an equivalent dimeric interface as observed for the cytoplasmic domain of Band 3, and thus establishes that the consensus motif VTVLP is the key minimal dimerization motif. The VTVLP motif is highly conserved and likely to be the physiologically relevant interface for all other members of the SLC4 family. A novel conserved Zn2+-binding motif present in the N-terminal domain of NDCBE is identified and characterized in vitro. Cellular studies confirm the Zn2+ dependent transport of two electroneutral bicarbonate transporters, NCBE and NBCn1. The Zn2+ site is mapped to a cluster of histidines close to the conserved ETARWLKFEE motif and likely plays a role in the regulation of this important motif. The combined structural and bioinformatics analysis provides a model that predicts with additional confidence the physiologically relevant interface between the cytoplasmic domain and the transmembrane domain.

Enzymatic detection and quantification assay of isatin, a putative stress biomarker in blood.

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.

A proton wire and water channel revealed in the crystal structure of isatin hydrolase.

The high resolution crystal structures of isatin hydrolase from Labrenzia aggregata in the apo and the product state are described. These are the first structures of a functionally characterized metal-dependent hydrolase of this fold. Isatin hydrolase converts isatin to isatinate and belongs to a novel family of metalloenzymes that include the bacterial kynurenine formamidase. The product state, mimicked by bound thioisatinate, reveals a water molecule that bridges the thioisatinate to a proton wire in an adjacent water channel and thus allows the proton released by the reaction to escape only when the product is formed. The functional proton wire present in isatin hydrolase isoform b represents a unique catalytic feature common to all hydrolases is here trapped and visualized for the first time. The local molecular environment required to coordinate thioisatinate 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.