The \u3cem\u3edapE\u3c/em\u3e-encoded \u3cem\u3eN\u3c/em\u3e-succinyl-l,l-diaminopimelic Acid Desuccinylase from \u3cem\u3eHaemophilus influenzae\u3c/em\u3e Contains Two Active-site Histidine Residues

The catalytic and structural properties of the H67A and H349A dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. On the basis of sequence alignment with the carboxypeptidase from Pseudomonas sp. strain RS-16, both H67 and H349 were predicted to be Zn(II) ligands. The H67A DapE enzyme exhibited a decreased catalytic efficiency (180-fold) compared with wild-type (WT) DapE towards N-succinyldiaminopimelic acid. No catalytic activity was observed for H349A under the experimental conditions used. The electronic paramagnetic resonance (EPR) and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to that of WT DapE after the addition of a single Co(II) ion. The addition of 1 equiv of Co(II) to H67A DapE provides spectra that are very different from those of the first Co(II) binding site of the WT enzyme, but that are similar to those of the second binding site. The EPR and electronic absorption data, in conjunction with the kinetic data, are consistent with the assignment of H67 and H349 as active-site metal ligands for the DapE from H. influenzae. Furthermore, the data suggest that H67 is a ligand in the first metal binding site, while H349 resides in the second metal binding site. A three-dimensional homology structure of the DapE from H. influenzae was generated using the X-ray crystal structure of the DapE from Neisseria meningitidis as a template and superimposed on the structure of the aminopeptidase from Aeromonas proteolytica (AAP). This homology structure confirms the assignment of H67 and H349 as active-site ligands. The superimposition of the homology model of DapE with the dizinc(II) structure of AAP indicates that within 4.0 Å of the Zn(II) binding sites of AAP all of the amino acid residues of DapE are nearly identical

Structural Evidence of a Major Conformational Change Triggered by Substrate Binding in DapE Enzymes: Impact on the Catalytic Mechanism

The X-ray crystal structure of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae (HiDapE) bound by the products of hydrolysis, succinic acid and l,l-DAP, was determined at 1.95 Å. Surprisingly, the structure bound to the products revealed that HiDapE undergoes a significant conformational change in which the catalytic domain rotates ∼50° and shifts ∼10.1 Å (as measured at the position of the Zn atoms) relative to the dimerization domain. This heretofore unobserved closed conformation revealed significant movements within the catalytic domain compared to that of wild-type HiDapE, which results in effectively closing off access to the dinuclear Zn(II) active site with the succinate carboxylate moiety bridging the dinculear Zn(II) cluster in a μ-1,3 fashion forming a bis(μ-carboxylato)dizinc(II) core with a Zn–Zn distance of 3.8 Å. Surprisingly, His194.B, which is located on the dimerization domain of the opposing chain ∼10.1 Å from the dinuclear Zn(II) active site, forms a hydrogen bond (2.9 Å) with the oxygen atom of succinic acid bound to Zn2, forming an oxyanion hole. As the closed structure forms upon substrate binding, the movement of His194.B by more than ∼10 Å is critical, based on site-directed mutagenesis data, for activation of the scissile carbonyl carbon of the substrate for nucleophilic attack by a hydroxide nucleophile. Employing the HiDapE product-bound structure as the starting point, a reverse engineering approach called product-based transition-state modeling provided structural models for each major catalytic step. These data provide insight into the catalytic reaction mechanism and also the future design of new, potent inhibitors of DapE enzymes

Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches

Expression profiling of the schizont and trophozoite stages of Plasmodium falciparum with a long-oligonucleotide microarray

BACKGROUND: The worldwide persistence of drug-resistant Plasmodium falciparum, the most lethal variety of human malaria, is a global health concern. The P. falciparum sequencing project has brought new opportunities for identifying molecular targets for antimalarial drug and vaccine development. RESULTS: We developed a software package, ArrayOligoSelector, to design an open reading frame (ORF)-specific DNA microarray using the publicly available P. falciparum genome sequence. Each gene was represented by one or more long 70 mer oligonucleotides selected on the basis of uniqueness within the genome, exclusion of low-complexity sequence, balanced base composition and proximity to the 3' end. A first-generation microarray representing approximately 6,000 ORFs of the P. falciparum genome was constructed. Array performance was evaluated through the use of control oligonucleotide sets with increasing levels of introduced mutations, as well as traditional northern blotting. Using this array, we extensively characterized the gene-expression profile of the intraerythrocytic trophozoite and schizont stages of P. falciparum. The results revealed extensive transcriptional regulation of genes specialized for processes specific to these two stages. CONCLUSIONS: DNA microarrays based on long oligonucleotides are powerful tools for the functional annotation and exploration of the P. falciparum genome. Expression profiling of trophozoites and schizonts revealed genes associated with stage-specific processes and may serve as the basis for future drug targets and vaccine development

Inhibition of the \u3cem\u3edapE\u3c/em\u3e-Encoded \u3cem\u3eN\u3c/em\u3e-Succinyl- ʟ, ʟ-diaminopimelic Acid Desuccinylase from \u3cem\u3eNeisseria meningitidis\u3c/em\u3e by ʟ-Captopril

Binding of the competitive inhibitor ʟ-captopril to the dapE-encoded N-succinyl-ʟ, ʟ-diaminopimelic acid desuccinylase from Neisseria meningitidis (NmDapE) was examined by kinetic, spectroscopic, and crystallographic methods. ʟ-Captopril, an angiotensin-converting enzyme (ACE) inhibitor, was previously shown to be a potent inhibitor of the DapE from Haemophilus influenzae (HiDapE) with an IC50 of 3.3 μM and a measured Ki of 1.8 μM and displayed a dose-responsive antibiotic activity toward Escherichia coli. ʟ-Captopril is also a competitive inhibitor of NmDapE with a Ki of 2.8 μM. To examine the nature of the interaction of ʟ-captopril with the dinuclear active site of DapE, we have obtained electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) data for the enzymatically hyperactive Co(II)-substituted forms of both HiDapE and NmDapE. EPR and MCD data indicate that the two Co(II) ions in DapE are antiferromagnetically coupled, yielding an S = 0 ground state, and suggest a thiolate bridge between the two metal ions. Verification of a thiolate-bridged dinuclear complex was obtained by determining the three-dimensional X-ray crystal structure of NmDapE in complex with ʟ-captopril at 1.8 Å resolution. Combination of these data provides new insights into binding of ʟ-captopril to the active site of DapE enzymes as well as important inhibitor–active site residue interaction’s. Such information is critical for the design of new, potent inhibitors of DapE enzymes

Functional responses of methanogenic archaea to syntrophic growth.

Methanococcus maripaludis grown syntrophically with Desulfovibrio vulgaris was compared with M. maripaludis monocultures grown under hydrogen limitation using transcriptional, proteomic and metabolite analyses. These measurements indicate a decrease in transcript abundance for energy-consuming biosynthetic functions in syntrophically grown M. maripaludis, with an increase in transcript abundance for genes involved in the energy-generating central pathway for methanogenesis. Compared with growth in monoculture under hydrogen limitation, the response of paralogous genes, such as those coding for hydrogenases, often diverged, with transcripts of one variant increasing in relative abundance, whereas the other was little changed or significantly decreased in abundance. A common theme was an apparent increase in transcripts for functions using H(2) directly as reductant, versus those using the reduced deazaflavin (coenzyme F(420)). The greater importance of direct reduction by H(2) was supported by improved syntrophic growth of a deletion mutant in an F(420)-dependent dehydrogenase of M. maripaludis. These data suggest that paralogous genes enable the methanogen to adapt to changing substrate availability, sustaining it under environmental conditions that are often near the thermodynamic threshold for growth. Additionally, the discovery of interspecies alanine transfer adds another metabolic dimension to this environmentally relevant mutualism

Structure and Function of Bacillus subtilis YphP, a Prokaryotic Disulfide Isomerase with a CXC Catalytic Motif†,‡

ABSTRACT: The DUF1094 family contains over 100 bacterial proteins, all containing a conserved CXCmotif, with unknown function. We solved the crystal structure of the Bacillus subtilis representative, the product of the yphP gene. The protein shows remarkable structural similarity to thioredoxins, with a canonical RβRβRββR topology, despite low amino acid sequence identity to thioredoxin. The CXC motif is found in the loop immediately downstream of the first β-strand, in a location equivalent to the CXXC motif of thioredoxins, with the first Cys occupying a position equivalent to the first Cys in canonical thioredoxin. The experimentally determined reduction potential of YphP is E0 =-130 mV, significantly higher than that of thioredoxin and consistent with disulfide isomerase activity. Functional assays confirmed that the protein displays a level of isomerase activity that might be biologically significant.We propose a mechanism by which the members of this family catalyze isomerization using the CXC catalytic site. The Bacillus subtilis yphP gene codes for a member of a superfamily of over 100 prokaryotic, highly conserved proteins (DUF1094), found predominantly in Firmicutes such as Staphy

Fixed-target serial crystallography at the Structural Biology Center

Serial synchrotron crystallography enables the study of protein structures under physiological temperature and reduced radiation damage by collection of data from thousands of crystals. The Structural Biology Center at Sector 19 of the Advanced Photon Source has implemented a fixed-target approach with a new 3D-printed mesh-holder optimized for sample handling. The holder immobilizes a crystal suspension or droplet emulsion on a nylon mesh, trapping and sealing a near-monolayer of crystals in its mother liquor between two thin Mylar films. Data can be rapidly collected in scan mode and analyzed in near real-time using piezoelectric linear stages assembled in an XYZ arrangement, controlled with a graphical user interface and analyzed using a high-performance computing pipeline. Here, the system was applied to two β-lactamases: a class D serine β-lactamase from Chitinophaga pinensis DSM 2588 and L1 metallo-β-lactamase from Stenotrophomonas maltophilia K279a

Chromosome landmarks and autosome-sex chromosome translocations in Rumex hastatulus, a plant with XX/XY1Y2 sex chromosome system

Rumex hastatulus is the North American endemic dioecious plant with heteromorphic sex chromosomes. It is differentiated into two chromosomal races: Texas (T) race characterised by a simple XX/XY sex chromosome system and North Carolina (NC) race with a polymorphic XX/XY1Y2 sex chromosome system. The gross karyotype morphology in NC race resembles the derived type, but chromosomal changes that occurred during its evolution are poorly understood. Our C-banding/DAPI and fluorescence in situ hybridization (FISH) experiments demonstrated that Y chromosomes of both races are enriched in DAPI-positive sequences and that the emergence of polymorphic sex chromosome system was accompanied by the break of ancestral Y chromosome and switch in the localization of 5S rDNA, from autosomes to sex chromosomes (X and Y2). Two contrasting domains were detected within North Carolina Y chromosomes: the older, highly heterochromatinised, inherited from the original Y chromosome and the younger, euchromatic, representing translocated autosomal material. The flow-cytometric DNA estimation showed ∼3.5 % genome downsizing in the North Carolina race. Our results are in contradiction to earlier reports on the lack of heterochromatin within Y chromosomes of this species and enable unambiguous identification of autosomes involved in the autosome-heterosome translocation, providing useful chromosome landmarks for further studies on the karyotype and sex chromosome differentiation in this species

KG-COVID-19: A Framework to Produce Customized Knowledge Graphs for COVID-19 Response.

Integrated, up-to-date data about SARS-CoV-2 and COVID-19 is crucial for the ongoing response to the COVID-19 pandemic by the biomedical research community. While rich biological knowledge exists for SARS-CoV-2 and related viruses (SARS-CoV, MERS-CoV), integrating this knowledge is difficult and time-consuming, since much of it is in siloed databases or in textual format. Furthermore, the data required by the research community vary drastically for different tasks; the optimal data for a machine learning task, for example, is much different from the data used to populate a browsable user interface for clinicians. To address these challenges, we created KG-COVID-19, a flexible framework that ingests and integrates heterogeneous biomedical data to produce knowledge graphs (KGs), and applied it to create a KG for COVID-19 response. This KG framework also can be applied to other problems in which siloed biomedical data must be quickly integrated for different research applications, including future pandemics