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Maltseva, Natalia

Publications and source records attributed to Maltseva, Natalia.

10 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Revealing reaction intermediates in one-carbon elongation by thiamine diphosphate/CoA-dependent enzyme family

2-Hydroxyacyl-CoA lyase/synthase (HACL/S) is a thiamine diphosphate (ThDP)-dependent versatile enzyme originally discovered in the mammalian α-oxidation pathway. HACL/S natively cleaves 2-hydroxyacyl-CoAs and, in its reverse direction, condenses formyl-CoA with aldehydes or ketones. The one-carbon elongation biochemistry based on HACL/S has enabled the use of molecules derived from greenhouse gases as biomanufacturing feedstocks. We investigated several HACL/S family members with high activity in the condensation of formyl-CoA and aldehydes, and distinct chain-length specificities and kinetic parameters. Our analysis revealed the structures of enzymes in complex with acyl-CoA substrates and products, several covalent intermediates, bound ThDP and ADP, as well as the C-terminal active site region. One of these observed states corresponds to the intermediary α–carbanion with hydroxymethyl-CoA covalently attached to ThDP. This research distinguishes HACL/S from related sub-families and identifies key residues involved in substrate binding and catalysis. These findings expand our knowledge of acyloin-condensation biochemistry and offer attractive prospects for biocatalysis using carbon elongation.

2-hydroxyacyl-CoA lyase↗
DOE OSTI2024

Epitopes recognition of SARS-CoV-2 nucleocapsid RNA binding domain by human monoclonal antibodies

Coronavirus nucleocapsid protein (NP) of SARS-CoV-2 plays a central role in many functions important for virus proliferation including packaging and protecting genomic RNA. The protein shares sequence, structure, and architecture with nucleocapsid proteins from betacoronaviruses. The N-terminal domain (NP RBD ) binds RNA and the C-terminal domain is responsible for dimerization. After infection, NP is highly expressed and triggers robust host immune response. The anti-NP antibodies are not protective and not neutralizing but can effectively detect viral proliferation soon after infection. Two structures of SARS-CoV-2 NP RBD were determined providing a continuous model from residue 48 to 173, including RNA binding region and key epitopes. Five structures of NP RBD complexes with human mAbs were isolated using an antigen-bait sorting. Complexes revealed a distinct complement-determining regions and unique sets of epitope recognition. This may assist in the early detection of pathogens and designing peptide-based vaccines. Mutations that significantly increase viral load were mapped on developed, full length NP model, likely impacting interactions with host proteins and viral RNA.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A high-throughput structural system biology approach to increase structure representation of proteins from Clostridioides difficile

ABSTRACT Clostridioides difficile causes life-threatening gastrointestinal infections. It is a high-risk pathogen due to a lack of effective treatments, antimicrobial resistance, and a poorly conserved genomic core. Herein, we report 30 X-ray structures from a structure genomics pipeline spanning 13 years, representing 10.2% of the X-ray structures for this important pathogen.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The Enzymatic Activity of Inosine 5'-Monophosphate Dehydrogenase May Not Be a Vulnerable Target for Staphylococcus aureus Infections

Many bacterial pathogens, including Staphylococcus aureus, require inosine 5'-monophosphate dehydrogenase (IMPDH) for infection, making this enzyme a promising new target for antibiotics. Although potent selective inhibitors of bacterial IMPDHs have been reported, relatively few have displayed antibacterial activity. Here we use structure-informed design to obtain inhibitors of S. aureus IMPDH (SaIMPDH) that have potent antibacterial activity (minimal inhibitory concentrations less than 2 μM) and low cytotoxicity in mammalian cells. The physicochemical properties of the most active compounds were within typical Lipinski/Veber space, suggesting that polarity is not a general requirement for achieving antibacterial activity. In this work, five compounds failed to display activity in mouse models of septicemia and abscess infection. Inhibitor-resistant S. aureus strains readily emerged in vitro. Resistance resulted from substitutions in the cofactor/inhibitor binding site of SaIMPDH, confirming on-target antibacterial activity. These mutations decreased the binding of all inhibitors tested, but also decreased catalytic activity. Nonetheless, the resistant strains had comparable virulence to wild-type bacteria. Surprisingly, strains expressing catalytically inactive SaIMPDH displayed only a mild virulence defect. Collectively these observations question the vulnerability of the enzymatic activity of SaIMPDH as a target for the treatment of S. aureus infections, suggesting other functions of this protein may be responsible for its role in infection.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

2'-O methylation of RNA cap in SARS-CoV-2 captured by serial crystallography

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog ( m7 GpppA m2'-O ). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2

SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme’s active site. Our findings provide new insights for the development of uracil scaffold-based drugs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Structural and biochemical analysis of the metallo-β-lactamase L1 from emerging pathogen Stenotrophomonas maltophilia revealed the subtle but distinct di-metal scaffold for catalytic activity

Emergence of Enterobacteriaceae harboring metallo-beta-lactamases (MBL) has raised global threats due to their broad antibiotic resistance profiles and the lack of effective inhibitors against them. We have been studied one of the emerging environmental MBL, the L1 from Stenotrophomonas maltophilia K279a . We determined several crystal structures of L1 complexes with three different classes of beta-lactam antibiotics (penicillin G, moxalactam, meropenem, and imipenem), with the inhibitor captopril and different metal ions (Zn +2 , Cd +2 , and Cu +2 ). All hydrolyzed antibiotics and the inhibitor were found binding to two Zn +2 ions mainly through the opened lactam ring and some hydrophobic interactions with the binding pocket atoms. Without a metal ion, the active site is very similarly maintained as that of the native form with two Zn +2 ions, however, the protein does not bind the substrate moxalactam. When two Zn +2 ions were replaced with other metal ions, the same di-metal scaffold was maintained and the added moxalactam was found hydrolyzed in the active site. Differential scanning fluorimetry and isothermal titration calorimetry were used to study thermodynamic properties of L1 MBL compared with New Deli Metallo-beta-lactamase-1 (NDM-1). Both enzymes are significantly stabilized by Zn +2 and other divalent metals but showed different dependency. These studies also suggest that moxalactam and its hydrolyzed form may bind and dissociate with different kinetic modes with or without Zn +2 for each of L1 and NDM-1. Our analysis implicates metal ions, in forming a distinct di-metal scaffold, which is central to the enzyme stability, promiscuous substrate binding and versatile catalytic activity. Statement: The L1 metallo-beta-lactamase from an environmental multidrug-resistant opportunistic pathogen Stenotrophomonas maltophilia K279a has been studied by determining 3D structures of L1 enzyme in the complexes with several beta-lactam antibiotics and different divalent metals and characterizing its biochemical and ligand binding properties. We found that the two-metal center in the active site is critical in the enzymatic process including antibiotics recognition and binding, which explains the enzyme's activity toward diverse antibiotic substrates. This study provides the critical information for understanding the ligand recognition and for advanced drug development.

59 BASIC BIOLOGICAL SCIENCES↗