General Base Swap Preserves Activity and Expands Substrate Tolerance in Hedgehog Autoprocessing.

Hedgehog (Hh) autoprocessing converts Hh precursor protein to cholesterylated Hh ligand for downstream signaling. A conserved active-site aspartate residue, D46, plays a key catalytic role in Hh autoprocessing by serving as a general base to activate substrate cholesterol. Here we report that a charge-altering Asp-to-His mutant (D46H) expands native cholesterylation activity and retains active-site conformation. Native activity toward cholesterol was established for D46H in vitro using a continuous FRET-based autoprocessing assay and in cellulo with stable expression in human 293T cells. The catalytic efficiency of cholesterylation with D46H is similar to that with wild type (WT), with kmax/KM = 2.1 × 103 and 3.7 × 103 M-1 s-1, respectively, and an identical pKa = 5.8 is obtained for both residues by NMR. To our knowledge this is the first example where a general base substitution of an Asp for His preserves both the structure and activity as a general base. Surprisingly, D46H exhibits increased catalytic efficiency toward non-native substrates, especially coprostanol (>200-fold) and epicoprostanol (>300-fold). Expanded substrate tolerance is likely due to stabilization by H46 of the negatively charged tetrahedral intermediate using electrostatic interactions, which are less constrained by geometry than H-bond stabilization by D46. In addition to providing fundamental insights into Hh autoprocessing, our findings have important implications for protein engineering and enzyme design.

Stabilizing Leaf and Branch Compost Cutinase (LCC) with Glycosylation: Mechanism and Effect on PET Hydrolysis.

Cutinases are polyester hydrolases that show a remarkable capability to hydrolyze polyethylene terephthalate (PET) to its monomeric units. This revelation has stimulated research aimed at developing sustainable and green cutinase-catalyzed PET recycling methods. Leaf and branch compost cutinase (LCC) is particularly suited toward these ends given its relatively high PET hydrolysis activity and thermostability. Any practical enzymatic PET recycling application will require that the protein have kinetic stability at or above the PET glass transition temperature (Tg, i.e., 70 °C). This paper elucidates the thermodynamics and kinetics of LCC conformational and colloidal stability. Aggregation emerged as a major contributor that reduces LCC kinetic stability. In its native state, LCC is prone to aggregation owing to electrostatic interactions. Further, with increasing temperature, perturbation of LCC's tertiary structure and corresponding exposure of hydrophobic domains leads to rapid aggregation. Glycosylation was employed in an attempt to impede LCC aggregation. Owing to the presence of three putative N-glycosylation sites, expression of native LCC in Pichia pastoris resulted in the production of glycosylated LCC (LCC-G). LCC-G showed improved stability to native state aggregation while increasing the temperature for thermal induced aggregation by 10 °C. Furthermore, stabilization against thermal aggregation resulted in improved catalytic PET hydrolysis both at its optimum temperature and concentration.

V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein.

Inteins mediate protein splicing, which has found extensive applications in protein science and biotechnology. In the Mycobacterium tuberculosis RecA mini-mini intein (ΔΔIhh), a single valine to leucine substitution at position 67 (V67L) dramatically increases intein stability and activity. However, crystal structures show that the V67L mutation causes minimal structural rearrangements, with a root-mean-square deviation of 0.2 Å between ΔΔIhh-V67 and ΔΔIhh-L67. Thus, the structural mechanisms for V67L stabilization and activation remain poorly understood. In this study, we used intrinsic tryptophan fluorescence, high-pressure nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations to probe the structural basis of V67L stabilization of the intein fold. Guanidine hydrochloride denaturation monitored by fluorescence yielded free energy changes (ΔGf°) of -4.4 and -6.9 kcal mol-1 for ΔΔIhh-V67 and ΔΔIhh-L67, respectively. High-pressure NMR showed that ΔΔIhh-L67 is more resistant to pressure-induced unfolding than ΔΔIhh-V67 is. The change in the volume of folding (ΔVf) was significantly larger for V67 (71 ± 2 mL mol-1) than for L67 (58 ± 3 mL mol-1) inteins. The measured difference in ΔVf (13 ± 3 mL mol-1) roughly corresponds to the volume of the additional methylene group for Leu, supporting the notion that the V67L mutation fills a nearby cavity to enhance intein stability. In addition, we performed MD simulations to show that V67L decreases side chain dynamics and conformational entropy at the active site. It is plausible that changes in cavities in V67L can also mediate allosteric effects to change active site dynamics and enhance intein activity.

Myxobacterial Predation: A Standardised Lawn Predation Assay Highlights Strains with Unusually Efficient Predatory Activity.

Myxobacteria prey upon a broad range of microorganisms. Lawn assays are commonly used to quantify myxobacterial predation-myxobacterial suspensions are spotted onto prey lawns, and monitored via spot expansion. The diversity in motility behaviours of myxobacterial strains and differing assay protocols in myxobacteriology laboratories led us to develop a highly-specified assay, which was applied to 28 myxobacterial strains preying on seven phytopathogenic prey species. Generally, prey organisms showed no qualitative differences in their susceptibility/resistance to myxobacterial predation. For most myxobacteria, prey did not stimulate, and in ~50% of cases actively hindered colony expansion. Only ~25% of predator/prey strain combinations exhibited greater colony expansion than in the absence of nutrients. The activity of predatory strains against different prey correlated, implying effective predators may have relatively non-specific predation mechanisms (e.g., broad specificity proteases/lipases), but no correlation was observed between predatory activity and phylogeny. Predation on dead (but intact) or lysed prey cells gave greater colony expansion than on live prey. Occasional strains grew substantially faster on dead compared to lysed cells, or vice-versa. Such differences in accessing nutrients from live, dead and lysed cells indicates there are strain-specific differences in the efficiencies/machineries of prey killing and nutrient acquisition, which has important implications for the ecology of myxobacterial predators and their prey.

Predatory Strategies of Myxococcus xanthus: Prey Susceptibility to OMVs and Moonlighting Enzymes.

Predatory outer membrane vesicles (OMVs) secreted by myxobacteria fuse readily with the outer membranes of Gram-negative bacteria, introducing toxic cargo into their prey. Here we used a strain of the myxobacterium Myxococcus xanthus that produces fluorescent OMVs to assay the uptake of OMVs by a panel of Gram-negative bacteria. M. xanthus strains took up significantly less OMV material than the tested prey strains, suggesting that re-fusion of OMVs with producing organisms is somehow inhibited. The OMV killing activity against different prey correlated strongly with the predatory activity of myxobacterial cells, however, there was no correlation between OMV killing activity and their propensity to fuse with different prey. It has previously been proposed that M. xanthus GAPDH stimulates the predatory activity of OMVs by enhancing OMV fusion with prey cells. Therefore, we expressed and purified active fusion proteins of M. xanthus glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase (GAPDH and PGK; moonlighting enzymes with additional activities beyond their roles in glycolysis/gluconeogenesis) to investigate any involvement in OMV-mediated predation. Neither GAPDH nor PGK caused lysis of prey cells or enhanced OMV-mediated lysis of prey cells. However, both enzymes were found to inhibit the growth of Escherichia coli, even in the absence of OMVs. Our results suggest that fusion efficiency is not a determinant of prey killing, but instead resistance to the cargo of OMVs and co-secreted enzymes dictates whether organisms can be preyed upon by myxobacteria.

A Genomic Survey of Signalling in the Myxococcaceae.

As prokaryotes diverge by evolution, essential 'core' genes required for conserved phenotypes are preferentially retained, while inessential 'accessory' genes are lost or diversify. We used the recently expanded number of myxobacterial genome sequences to investigate the conservation of their signalling proteins, focusing on two sister genera (Myxococcus and Corallococcus), and on a species within each genus (Myxococcus xanthus and Corallococcus exiguus). Four new C. exiguus genome sequences are also described here. Despite accessory genes accounting for substantial proportions of each myxobacterial genome, signalling proteins were found to be enriched in the core genome, with two-component system genes almost exclusively so. We also investigated the conservation of signalling proteins in three myxobacterial behaviours. The linear carotenogenesis pathway was entirely conserved, with no gene gain/loss observed. However, the modular fruiting body formation network was found to be evolutionarily plastic, with dispensable components in all modules (including components required for fruiting in the model myxobacterium M. xanthus DK1622). Quorum signalling (QS) is thought to be absent from most myxobacteria, however, they generally appear to be able to produce CAI-I (cholerae autoinducer-1), to sense other QS molecules, and to disrupt the QS of other organisms, potentially important abilities during predation of other prokaryotes.

Within-species variation in OMV cargo proteins: the Myxococcus xanthus OMV pan-proteome.

Extracellular membrane vesicles are produced by all domains of life (bacteria, archaea and eukaryotes). Bacterial extracellular vesicles (outer membrane vesicles or OMVs) are produced by outer membrane blebbing, and contain proteins, nucleic acids, virulence factors, lipids and metabolites. OMV functions depend on their internal composition, therefore understanding the proteome of OMVs, and how it varies between organisms, is imperative. Here, we report a comparative proteomic profiling of OMVs from strains of Myxococcus xanthus, a predatory species of Gram-negative myxobacteria whose secretions include secondary metabolites and hydrolytic enzymes, thought to be involved in prey lysis. Ten strains were chosen for study, of which seven had genome sequences available. The remaining three strains were genome sequenced allowing definition of the core and accessory genes and genome-derived proteins found within the pan-genome and pan-proteome respectively. OMVs were isolated from each strain and proteins identified using mass spectrometry. The M. xanthus OMV pan-proteome was found to contain tens of 'core' and hundreds of 'accessory' proteins. Properties of the OMV pan-proteome were compared with those of the pan-proteome deduced from the M. xanthus pan-genome. On average, 80% of 'core' OMV proteins are encoded by genes of the core genome, yet the OMV proteomes of individual strains contain subsets of core genome-derived proteins which only partially overlap. In addition, the distribution of characteristics of vesicle proteins does not correlate with the genome-derived proteome characteristic distribution. We hypothesize that M. xanthus cells package a personalized subset of proteins whose availability is only partially dictated by the presence/absence of encoding genes within the genome.

Timing and Scope of Genomic Expansion within Annelida: Evidence from Homeoboxes in the Genome of the Earthworm Eisenia fetida.

Annelida represents a large and morphologically diverse group of bilaterian organisms. The recently published polychaete and leech genome sequences revealed an equally dynamic range of diversity at the genomic level. The availability of more annelid genomes will allow for the identification of evolutionary genomic events that helped shape the annelid lineage and better understand the diversity within the group. We sequenced and assembled the genome of the common earthworm, Eisenia fetida. As a first pass at understanding the diversity within the group, we classified 363 earthworm homeoboxes and compared them with those of the leech Helobdella robusta and the polychaete Capitella teleta. We inferred many gene expansions occurring in the lineage connecting the most recent common ancestor (MRCA) of Capitella and Eisenia to the Eisenia/Helobdella MRCA. Likewise, the lineage leading from the Eisenia/Helobdella MRCA to the leech H. robusta has experienced substantial gains and losses. However, the lineage leading from Eisenia/Helobdella MRCA to E. fetida is characterized by extraordinary levels of homeobox gain. The evolutionary dynamics observed in the homeoboxes of these lineages are very likely to be generalizable to all genes. These genome expansions and losses have likely contributed to the remarkable biology exhibited in this group. These results provide a new perspective from which to understand the diversity within these lineages, show the utility of sub-draft genome assemblies for understanding genomic evolution, and provide a critical resource from which the biology of these animals can be studied.