Structural and mechanistic investigations on Salmonella typhimurium acetate kinase (AckA): identification of a putative ligand binding pocket at the dimeric interface

Background: Bacteria such as Escherichia coli and Salmonella typhimurium can utilize acetate as the sole source of carbon and energy. Acetate kinase (AckA) and phosphotransacetylase (Pta), key enzymes of acetate utilization pathway, regulate flux of metabolites in glycolysis, gluconeogenesis, TCA cycle, glyoxylate bypass and fatty acid metabolism. Results: Here we report kinetic characterization of S. typhimurium AckA (StAckA) and structures of its unliganded (Form-I, 2.70 angstrom resolution) and citrate-bound (Form-II, 1.90 angstrom resolution) forms. The enzyme showed broad substrate specificity with k(cat)/K-m in the order of acetate > propionate > formate. Further, the K-m for acetyl-phosphate was significantly lower than for acetate and the enzyme could catalyze the reverse reaction (i.e. ATP synthesis) more efficiently. ATP and Mg2+ could be substituted by other nucleoside 5'-triphosphates (GTP, UTP and CTP) and divalent cations (Mn2+ and Co2+), respectively. Form-I StAckA represents the first structural report of an unliganded AckA. StAckA protomer consists of two domains with characteristic beta beta beta alpha beta alpha beta alpha topology of ASKHA superfamily of proteins. These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound Methanosarcina thermophila AckA (MtAckA). Spectroscopic and structural analyses of StAckA further suggested occurrence of inter-domain motion upon ligand-binding. Unexpectedly, Form-II StAckA structure showed a drastic change in the conformation of residues 230-300 compared to that of Form-I. Further investigation revealed electron density corresponding to a citrate molecule in a pocket located at the dimeric interface of Form-II StAckA. Interestingly, a similar dimeric interface pocket lined with largely conserved residues could be identified in Form-I StAckA as well as in other enzymes homologous to AckA suggesting that ligand binding at this pocket may influence the function of these enzymes. Conclusions: The biochemical and structural characterization of StAckA reported here provides insights into the biochemical specificity, overall fold, thermal stability, molecular basis of ligand binding and inter-domain motion in AckA family of enzymes. Dramatic conformational differences observed between unliganded and citrate-bound forms of StAckA led to identification of a putative ligand-binding pocket at the dimeric interface of StAckA with implications for enzymatic function.

New insight into the architecture of oxy-anion pocket in unliganded conformation of GAT domains: A MD-simulation study

Human Guanine Monophosphate Synthetase (hGMPS) converts XMP to GMP, and acts as a bifunctional enzyme with N-terminal ``glutaminase'' (GAT) and C-terminal ``synthetase'' domain. The enzyme is identified as a potential target for anticancer and immunosuppressive therapies. GAT domain of enzyme plays central role in metabolism, and contains conserved catalytic residues Cys104, His190, and Glu192. MD simulation studies on GAT domain suggest that position of oxyanion in unliganded conformation is occupied by one conserved water molecule (W1), which also stabilizes that pocket. This position is occupied by a negatively charged atom of the substrate or ligand in ligand bound crystal structures. In fact, MD simulation study of Ser75 to Val indicates that W1 conserved water molecule is stabilized by Ser75, while Thr152, and His190 also act as anchor residues to maintain appropriate architecture of oxyanion pocket through water mediated H-bond interactions. Possibly, four conserved water molecules stabilize oxyanion hole in unliganded state, but they vacate these positions when the enzyme (hGMPS)-substrate complex is formed. Thus this study not only reveals functionally important role of conserved water molecules in GAT domain, but also highlights essential role of other non-catalytic residues such as Ser75 and Thr152 in this enzymatic domain. The results from this computational study could be of interest to experimental community and provide a testable hypothesis for experimental validation. Conserved sites of water molecules near and at oxyanion hole highlight structural importance of water molecules and suggest a rethink of the conventional definition of chemical geometry of inhibitor binding site.

Understanding the ex-ante cost of liquidity in the limit order book: A note

This paper estimates a new measure of liquidity costs in a market driven by orders. It represents thecost of simultaneously buying and selling a given amount of shares, and it is given by a single measure of ex-ante liquidity that aggregates all available information in the limit order book for a given number of shares. The cost of liquidity is an increasing function relating bid-ask spreads with the amounts available for trading. This measure completely characterizes the cost of liquidity of any given asset. It does not suffer from the usual ambiguities related to either the bid-ask spread or depth when they are considered separately. On the contrary, with a single measure, we are able to capture all dimensions of liquidity costs on ex-ante basis.

ICSF guide book: understanding the Work in Fishing Convention, 2007

This guidebook attempts to provide a quick overview of the Work in Fishing Convention, 2007, which was adopted in Geneva, Switzerland, in June 2007 at the 96th International Labour Conference (ILC) of the International Labour Organization (ILO). It does not purport to provide interpretation of any provisions of the Convention and should not in any way be treated as a substitute for the actual provisions it contains. This guidebook is intended mainly to help those unfamiliar with the Convention and the working of the ILO and the ILC, gain some understanding of the relevant issues. In particular, it is hoped that the guidebook will aid fish workers and their organizations understand the possible benefits and implications of the Convention for artisanal and small-scale fisheries in developing countries.

Towards accessible e-book platforms

Outcomes of a piece of research into the accessibility of e-books. LTD

Towards accessible e-book platforms

A small scale project was funded to explore the accessibility of e-book platforms, offer specific confidential advice for participating partners and provide generic guidance for publishers and aggregators.

Good practice guidance for library and information professionals - moving towards accessible e-book platforms

Good practice guidance for publishers and procurers of e-book platforms based on the benefits and barriers identified by robust testing using real people with disabilities