Unraveling the B. pseudomallei Heptokinase WcbL: From Structure to Drug Discovery

Abstract

SummaryGram-negative bacteria utilize heptoses as part of their repertoire of extracellular polysaccharide virulence determinants. Disruption of heptose biosynthesis offers an attractive target for novel antimicrobials. A critical step in the synthesis of heptoses is their 1-O phosphorylation, mediated by kinases such as HldE or WcbL. Here, we present the structure of WcbL from Burkholderia pseudomallei. We report that WcbL operates through a sequential ordered Bi-Bi mechanism, loading the heptose first and then ATP. We show that dimeric WcbL binds ATP anti-cooperatively in the absence of heptose, and cooperatively in its presence. Modeling of WcbL suggests that heptose binding causes an elegant switch in the hydrogen-bonding network, facilitating the binding of a second ATP molecule. Finally, we screened a library of drug-like fragments, identifying hits that potently inhibit WcbL. Our results provide a novel mechanism for control of substrate binding and emphasize WcbL as an attractive anti-microbial target for Gram-negative bacteria.

Carbohydrate Kinases: A Conserved Mechanism Across Differing Folds

“…GHMP kinases belong to a wider structural fold that includes diverse proteins. These include several carbohydrate kinases: in addition to the galactokinase for which the family is named, these include N-acetylgalactosamine (GalNAc) kinase [84], galacturonic acid kinase [85], arabinose kinase [86], and one group of heptose-7-phosphate kinases [87]. Human galactokinase is important for galactose catabolism (via the Leloir pathway), and it has been suggested as a target for drug interventions to suppress galactosemia (caused by an accumulation of the product galactose-1-phosphate when the next Leloir pathway enzyme is absent) [10,[88][89][90]; or, for use in enzyme replacement therapy in the (rarer) galactosemias that is caused by a defect in galactokinase [91].…”

“…One case of a kinase that can also use GTP and ITP with lower activity has been reported [33]. As with other carbohydrate kinases, a magnesium ion is also required for activity [87,98].…”

“…The two domains are not strictly separated in the protein sequence, with the very C-terminus of the protein contributing to the domain that is largely made from the N-terminal parts of the protein [99,100]. Some members of the family are monomeric, whilst others are obligate dimers [36,87]. The protein active site is formed in a cleft between the two domains.…”

“…The protein active site is formed in a cleft between the two domains. The carbohydrate substrate is held in a deep pocket that is formed between the two domains, whilst the nucleotide binds along a shallow groove between the two domains with parts of the ribose ring exposed to solvent [87]. The nucleotide binding site is present before the carbohydrate binds to the enzyme, similarly to the RKs.…”

“…Eukaryotic galactokinases show an ordered mechanism, where ATP binds the enzyme before galactose [100][101][102], as do N-acetylgalactosamine kinases [92]. Plant galactokinases and bacterial heptose-7-phosphate kinases show the opposite mechanism, an ordered mechanism with the carbohydrate binding first [87,103]; whilst, E. coli galactokinase shows a random sequential mechanism [104].…”