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Introduction

Isocitrate dehydrogenase kinase/phosphatase (IDHK/P or AceK) is an E. coli enzyme which phosphorylates or dephosphorylates isocitrate dehydrogenase (IDH) on a specific serine residue (Ser113) thus regulating its activities. This is a regulatory mechanism which enables bacteria to bypass the Krebs cycle via the glyoxylate shunt in response to nutrient availability. AceK is expressed from a single gene, aceK. Both kinase and phosphatase activities reside on the same polypeptide and share the same active sites.

Structure

The AceK structure contains two functional domains: a kinase domain (KD) where the kinase, phosphatase and ATPase reactions occur, and a regulatory domain (RD) that helps form allosteric binding pockets involved in regulating the catalytic domain’s function. The AMP molecule is situated in a pocket at the interface between the KD and RD and acts as a bridge.

The regulatory domain, which comprises the amino-terminal half of the AceK sequence, is mainly composed of a-helices. It begins with two long, parallel a-helices that form a large hairpin structure, followed by two short, parallel a-helices that form a small hairpin segment. The regulatory domain is linked to the kinase domain by a 27-residue-long a-helix. This domain represents a unique protein fold with no structural homologues.

The kinase domain, which makes up the carboxy-terminal half of AceK, has a classic bi-lobe protein kinase fold with the ATP-binding cleft located at the interface between the two lobes. The N-terminal lobe consists mainly of a twisted, five-stranded, antiparallel b-sheet and two a-helices. The larger C-terminal lobe is predominantly a-helical with some stretches of antiparallel b-strands. The ATP molecule is under the cover of the five-stranded b-sheet and is shielded by loop-b3aC. Loop-b3aC shifts upwards or downwards to controls access to the ATP binding site. The substrate recognition loop (SRL) stretches out of the C-terminal lobe. This loop, together with loop-b3aC, forms a large cleft that is the ICDH binding site.

Function

Active Site

Regulation

IDHK/P monitors general metabolism by responding to the levels of a wide variety of metabolites. This ability of IDHK/P allows the IDH phosphorylation cycle to compensate for substantial perturbations of the system. Many of the regulatory effectors are derived from the end products of the glyoxylate bypass, and represent negative feedback inhibition mechanisms. Furthermore, Depletion in AMP levels signals that the cell requires energy and isocitrate will continue through the Krebs cycle with IDH dephosphorylated. Thus, AMP, 3-phosphoglycerate, and pyruvate belong to class of effectors which bind directly to ICDHK/P. They activate IDH phosphatase and inhibit both IDH kinase and the intrinsic ATPase activities. NADPH and isocitrate regulate IDHK/P through the IDH. NADPH and isocitrate inhibit IDH kinase, and NADPH also inhibits IDH phosphatase. However, they do not activate IDH phosphatase nor inhibit the IDH-independent ATPase activity. Surprisingly, no regulators that activate AceK kinase activity or inhibit AceK phosphatase activity have been identified, illustrating the stringent control of the glyoxylate bypass, which only needs to be activated when the cell is nutrient deprived.

Relevance

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