The Tail of KdsC: CONFORMATIONAL CHANGES CONTROL THE ACTIVITY OF A HALOACID DEHALOGENASE SUPERFAMILY PHOSPHATASE

Tapan Biswas,Li Yi,Parag Aggarwal,Jing Wu,John R Rubin,Jeanne A Stuckey,Ronald W Woodard,Oleg V Tsodikov

doi:10.1074/jbc.m109.012278

Tapan Biswas, Li Yi + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m109.012278

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Abstract

The phosphatase KdsC cleaves 3-deoxy-D-manno-octulosonate 8-phosphate to generate a molecule of inorganic phosphate and Kdo. Kdo is an essential component of the lipopolysaccharide envelope in Gram-negative bacteria. Because lipopolysaccharide is an important determinant of bacterial resistance and toxicity, KdsC is a potential target for novel antibacterial agents. KdsC belongs to the broad haloacid dehalogenase superfamily. In haloacid dehalogenase superfamily enzymes, substrate specificity and catalytic efficiency are generally dictated by a fold feature called the cap domain. It is therefore not clear why KdsC, which lacks a cap domain, is catalytically efficient and highly specific to 3-deoxy-D-manno-octulosonate 8-phosphate. Here, we present a set of seven structures of tetrameric Escherichia coli KdsC (ranging from 1.4 to 3.06 A in resolution) that model different intermediate states in its catalytic mechanism. A crystal structure of product-bound E. coli KdsC shows how the interface between adjacent monomers defines the active site pocket. Kdo is engaged in a network of polar and nonpolar interactions with residues at this interface, which explains substrate specificity. Furthermore, this structural and kinetic analysis strongly suggests that the binding of the flexible C-terminal region (tail) to the active site makes KdsC catalytically efficient by facilitating product release.

Highlights

30594 JOURNAL OF BIOLOGICAL CHEMISTRY eukaryotes and prokaryotes, are responsible for a wide range of important biological functions, such as the biosynthesis of important metabolic molecules [2], transport [3], signal transduction [4, 5], transcription [6], and DNA repair [7]
Based on additional structural elements that are inserted in the Rossmann fold, haloacid dehalogenase superfamily (HADSF) enzymes are divided into three subfamilies
During our initial efforts to obtain a crystal structure of substrate (Kdo8P)- or product (Kdo)-bound E. coli KdsC, we observed an unusual binding of the C-terminal eight-residue region of one monomer into the active site of a neighboring monomer

Summary

The Tail of KdsC

CONFORMATIONAL CHANGES CONTROL THE ACTIVITY OF A HALOACID DEHALOGENASE SUPERFAMILY PHOSPHATASE*. In haloacid dehalogenase superfamily enzymes, substrate specificity and catalytic efficiency are generally dictated by a fold feature called the cap domain. 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase (KdsC, previously annotated as YrbI; EC 3.1.3.45), an archetypical HADSF enzyme from the C0 subfamily, displays a narrow substrate specificity [12, 13] and high catalytic efficiency [12]. Putative homologs of Escherichia coli KdsC phosphatase (Fig. 2) are present in all Gram-negative bacteria with the known genomic sequence Examples of these include YrbI from Haemophilus influenzae [16] and a putative phosphatase from Aquifex aeolicus (Protein Data Bank code 2p9j), which exhibit a tetrameric structure.

KdsC Phosphatase Structure and Mechanism

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION