Combining Functional and Structural Genomics to Sample the Essential Burkholderia Structome

Abstract

BackgroundThe genus Burkholderia includes pathogenic gram-negative bacteria that cause melioidosis, glanders, and pulmonary infections of patients with cancer and cystic fibrosis. Drug resistance has made development of new antimicrobials critical. Many approaches to discovering new antimicrobials, such as structure-based drug design and whole cell phenotypic screens followed by lead refinement, require high-resolution structures of proteins essential to the parasite.Methodology/Principal FindingsWe experimentally identified 406 putative essential genes in B. thailandensis, a low-virulence species phylogenetically similar to B. pseudomallei, the causative agent of melioidosis, using saturation-level transposon mutagenesis and next-generation sequencing (Tn-seq). We selected 315 protein products of these genes based on structure-determination criteria, such as excluding very large and/or integral membrane proteins, and entered them into the Seattle Structural Genomics Center for Infection Disease (SSGCID) structure determination pipeline. To maximize structural coverage of these targets, we applied an “ortholog rescue” strategy for those producing insoluble or difficult to crystallize proteins, resulting in the addition of 387 orthologs (or paralogs) from seven other Burkholderia species into the SSGCID pipeline. This structural genomics approach yielded structures from 31 putative essential targets from B. thailandensis, and 25 orthologs from other Burkholderia species, yielding an overall structural coverage for 49 of the 406 essential gene families, with a total of 88 depositions into the Protein Data Bank. Of these, 25 proteins have properties of a potential antimicrobial drug target i.e., no close human homolog, part of an essential metabolic pathway, and a deep binding pocket. We describe the structures of several potential drug targets in detail.Conclusions/SignificanceThis collection of structures, solubility and experimental essentiality data provides a resource for development of drugs against infections and diseases caused by Burkholderia. All expression clones and proteins created in this study are freely available by request.

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

“…Among the class I enzymes, B. subtilis and E. coli PRPP synthases and human PRPP synthase isozyme 1 have been crystallized, and high-resolution structures have been determined (49)(50)(51)(52). A three-dimensional structure has been determined also for the PRPP synthase from the Gram-negative bacterium Burkholderia (Pseudomonas) pseudomallei strain 1710b (PDB code 3dah) (53). However, there are no biochemical data available for the latter enzyme.…”

Structure of the effector‐binding domain of deoxyribonucleoside regulator DeoR from Bacillus subtilis

“…Interestingly, ribose-5-phosphate isomerase A from Burkholderia thailandensis (PDB code 3UW1) binds the linear form of ribose-5-phosphate in a position very similar to that of dR5P in C-DeoR (Fig. 2B) [11]. The sugar, however, does not form a covalent adduct in this B. thailandensis enzyme because the amino acid residue corresponding to Lys141 of C-DeoR is serine.…”

Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining

“…Besides sequences with only putative SDR activity, a well-studied human SDR enzyme, the dehydrogenase/ reductase SDR family member 4 (DHRS4) (62)(63)(64), and experi-mentally verified 3-ketoacyl-acyl carrier protein reductases from Vibrio harveyi (FabG) (65) and Burkholderia pseudomallei (66) were included.…”