The emerging multi-drug resistant bacteria, commonly referred to as superbugs, claim the lives of hundreds of thousands of individuals each year, highlighting the urgent need for new and improved antibiotics. Structural insights into the mechanism of action of essential enzymes unique to bacteria provide a rational basis for novel therapeutics design and discovery.
The membrane-integral glycerol 3-phosphate acyltransferase (GPAT), PlsY, catalyses the committed step in phospholipid biosynthesis by acylating glycerol 3-phosphate to form lysophosphatidic acid. It has no eukaryotic homologs and is essential in most Gram-positive bacteria such as multi-drug resistant Enterococcus faecium and Streptococcus pneumoniae, identified by the World Health Organization as the most threatening of human pathogens. What sets PlsY apart is that it uses the highly unusual acyl phosphate, as its acyl donor. This is in contrast to other GPATs which employ acyl-carrier protein and acyl-CoA as donors. Taking advantage of this unique feature, previous studies have identified several acyl phosphate analogous as potential PlsY-inhibiting antimicrobials. Such drug discovery efforts would benefit greatly from high resolution structures for rational drug design, as well as convenient enzymatic assays.
In a recent Nature communications issue (https://www.nature.com/articles/s41467-017-01821-9), a research team led by Dr. Dianfan Li, a principle investigator in Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences, report the X-ray structure of the PlsY from Aquifex aeolicus at an atomic (1.48 Å) resolution using crystals growing in a lipid bilayer environment termed the lipid cubic phase. The structure revealed an unreported fold with a surprising seven transmembrane helices, as opposed to a reported five transmembrane helices model. Despite that it is generally challenging to obtain Michaelis-Menten complex structures due to the transit nature of enzyme-substrate interactions, we have solved four additional structures of PlsY in complex with its two substrates and products. Together with mutagenesis data, this palette of five structures forms the basis of a completely different acylation mechanism that is of the ‘substrate assisted catalysis’ type, differentiating PlsY with other acyltransferases that employ a critical ‘aspartate-histidine’ dyad for catalysis. In addition to informing the catalytic mechanism of PlsY, the reported structures suggest routes by which the substrates enter and the products exit the active site. They also help explain why the lipid product, lysophosphatidic acid, is such a potent inhibitor of the enzyme.
The crystals used for structure determination were grown in the lipid cubic phase. With a view to demonstrating the functional relevance of these structures, it was important to show that PlsY was enzymatically active in the cubic phase, a somewhat unusual membrane mimetic. Accordingly, a coupled assay was developed wherein the phosphate-releasing activity of PlsY reconstituted in the cubic mesophase was quantified using a fluorescently labelled inorganic phosphate-biosensor. Compared to the existing PlsY activity assay that uses 14C-labelled glycerol 3-phosphate, this new assay is continuous, does not use radioisotopes, and can be performed in a 96-well format for high throughput screening. The latter is critically important for drug discovery applications. The method should prove generally applicable for a range of phosphate generating membrane enzymes.
The structures also showed that PlsY’s active site is relatively inflexible, a particularly attractive feature for in silico drug screening applications. In addition, sequence analysis showed that the active site is highly conserved between different species. Thus, the structure should provide a useful template to generate reliable homology model for virtual screening of small molecules as inhibitors; and the biochemical assay should provide guidance for further iterative structure-based inhibitor design, which may ultimately contribute to antibiotic discovery.
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