IUCr 2008 (Osaka, Japan) student award winners of the Larry Calvert CNC/IUCr Trust Fund Award

Daniel Lee

(Department of Biochemistry, Queen's University)

While protein tyrosine kinases (PTKs) have been extensively characterized in eukaryotes, far less is known about their emerging counterparts in prokaryotes. The inner-membrane Wzc/Etk protein belongs to the bacterial PTK family, which plays a critical role in regulating the polymerization and transportation of virulence-determining capsular polysaccharide (CPS). The kinase utilizes a unique two-step activation mechanism centering on the intraphosphorylation of a tyrosine residue, although the specific detail remains unknown. Herein we report the first crystal structure of a bacterial PTK, the C-terminal kinase domain of E. coli tyrosine kinase (Etk) at 2.5 Å resolution. The folding of the Etk kinase domain in bacteria differs markedly from that in eukaryotic PTKs. Based on the structure and supporting mass spectrometric evidence of the PTK observed, a unique activation mechanism is consequently proposed that involves the regulation of the phosphorylation of a single tyrosine residue at position 574 and its specific interaction with a previously unidentified key arginine residue at position 614 (R614) to unblock the active site.

Nobohiku Watanabe

(Department of Biochemistry, University of Alberta)

The lysine biosynthetic pathway is an attractive target for the development of new antibiotics or herbicides because it is absent in humans. LL-diaminopimelate aminotransferase (LL-DAP-AT) is a newly discovered enzyme in the novel lysine biosynthetic pathway in Chlamydia and plants. Previously, three different lysine biosynthetic pathways have been characterized in bacteria. However, none of the previous bacterial lysine biosynthetic pathways were found in Chlamydia or in plants. Recently, LL-DAP-AT was discovered to be the missing piece in Chlamydial and plant lysine biosynthetic pathways, and this enzyme bypasses three enzymatic pathways in the previously described bacterial lysine biosynthetic pathway. In order to understand the mechanism of this enzyme and to assist in the design of inhibitors, we have determined the three-dimensional structures of LL-DAP-AT from A. thaliana in native and with two substrate-analogues (LL-DAP-PLP, Glu-PLP) bound. LL-DAP-AT is a pyridoxal-5'-phosphate (PLP) dependent enzyme and belongs to the type I fold family of PLP-dependent enzymes. Comparison of the active site residues of LL-DAP-AT and aspartate aminotransferases revealed that the PLP binding residues in LL-DAP-AT are well conserved in both enzymes. However, Tyr37, Tyr152, Glu97 and Asn309 are unique to LL-DAP-AT. Tyr37 and Tyr152 are positioned to recognize distal carboxylate groups of both LL-DAP and glutamate. Glu97, Asn309 and water molecules form an array of hydrogen-bonds to stereospecifically recognize LL-DAP in the active site. Our studies revealed the unique stereospecific recognition mechanism used by this newly discovered LL-DAP-AT.

Jimin Zheng

(Department of Biochemistry, Queen's University)

The study of bacterial phosphorylation systems was advanced by the discovery of a phosphorylating activity in E. coli which regulates isocitrate dehydrogenase (IDH). This was the first prokaryote phosphorylation system to be identified in bacteria, and is the only known serine/threonine (Ser/Thr) phosphorylation system/pathway in E. coli. AceK is a 66,500-Dalton protein which uniquely possesses both kinase and phosphatase activities. This phosphorylation-dephosphorylation system modifies the Ser-113 residue on IDH. It is this modification that regulates the amount of isocitrate going through the glyoxylate bypass. IDH competes with isocitrate lyase in directing isocitrate through the Krebs' cycle or glyoxylate bypass, respectively. When the organism is grown on acetate, IDH is in its inactive phosphorylated form, thus inhibiting Krebs' cycle. Alternatively, a change of carbon source to glucose or pyruvate results in the activation of IDH by dephosphorylation, and the initiation of Krebs' cycle. AceK also demonstrates ATPase activity independent of IDH. Sequence comparison shows no similarities between AceK and the eukaryote Ser/Thr protein kinases. Therefore, as a distinct bifunctional protein, AceK may possess a novel kinase/phosphatase structural fold and (de)phosphorylation mechanism.The function of AceK and its involvement in the regulation of Krebs' cycle and the glyoxylate bypass is well-characterized, but its structural and mechanistic qualities have remained relatively unknown. Structural studies would enable analysis of AceK at both a macro and micro scale. The determination of the crystal structure could provide confirmation of the function of AceK by identifying the various kinase, phosphatase and ATPase domains and insights into the coordination of the kinase, phosphatase and ATPase activity of AceK. Of note, it is currently unknown how, or if at all, the active site changes conformation as it switches between kinase and phosphatase activity. Determination of the structure "caught" in both kinase and phosphatase modes would provide information on the manner by which this bifunctionality is achieved. Three Acek crystal forms were obtained and SAD datasets were collected at CHESS and BNL synchrotron source. The AceK structure is determined at 2.6 Å. The overall AceK structure displays a typical eukaryotic kinase folding.