Biology subjects

Evdokimova, E.

Publications and source records attributed to Evdokimova, E..

3 recordsLinked to original sources

bioRxiv2022

Ni(II) binding affinity and specificity of solute binding proteins: the importance of the double His motif and variable loop revealed by structural and mutational studies

Extracytoplasmic solute binding proteins (SBPs) are molecular shuttles involved in the cellular uptake of various small molecules and metal ions including Ni(II). Our previous study with the Ni(II) binding proteins (NiBPs) CjNikZ from Campylobacter jejuni and CcNikZ-II from Clostridium carboxidivorans demonstrated they were able to bind Ni(II) at low micromolar affinity without the need for additional chelators. Here, we determined the crystal structure of apo CcNikZ-II, which revealed a Ni(II) binding site comprised of the highly conserved double His (HH-)prong (His511, His512) and a short variable (v-)loop nearby (Thr59-Thr64, TEDKYT). Alanine scanning mutagenesis of the CcNikZ-II Ni(II) binding site identified Glu60 and His511 as essential for high affinity binding to Ni(II). Phylogenetic analysis of >4000 SBP sequences demonstrated the presence of two clusters of proteins containing the HH-prong with CcNikZ-II and CjNikZ. To provide insights into the role of the double His-prong and v-loop sequence in Ni(II) binding of NiBPs, nine purified CcNikZ-II homologues containing the HH-prong and v-loop were screened using an automated screening workflow. Metal binding assays with purified homologous NiBPs revealed high Ni(II) binding affinity without requirement for chelators indicating that the double His prong represents a signature motif for the presence of Ni(II) binding activity in SBPs. The engineered CcNikZ-II variants with the wild type v-loop (TEDKYT) replaced with v-loops from NiBPs with higher affinity showed up to an order of magnitude higher affinity for Ni(II). In addition, the v-loop appears to play a role in metal ion specificity as purified wild type and engineered NiBPs with different v-loop sequences showed distinct metal profiles. This work paves way for metalloprotein engineering of NiBPs towards biocatalytic and metal recovery applications.

biochemistry↗

bioRxiv2022

Thermophilic carboxylesterases from hydrothermal vents of the volcanic island of Ischia active on synthetic and biobased polymers and mycotoxins

Hydrothermal vents have a widespread geographical distribution and are of high interest for investigating microbial communities and robust enzymes for various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of Firmicutes (Geobacillus and Brevibacillus spp.), Proteobacteria and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate (PHB) and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus spp., and to some extent, Fontimonas and Schleiferia spp. The screening at 37-70{o}C of metagenomic fosmid library from above enrichment cultures resulted in identification and successful production in Escherichia coli of three hydrolases (IS10, IS11 and IS12), all derived from yet uncultured Chloroflexota and showing low sequence identity (33-56%) to characterized enzymes. Enzymes exhibited maximal esterase activity at temperatures 70-90{o}C, with IS11 showing the highest thermostability (90% activity after 20 min incubation at 80{o}C). IS10 and IS12 were highly substrate-promiscuous and hydrolysed all 51 monoester substrates tested. Enzymes were active with polyesters (PLA and polyethylene terephthalate model substrate, 3PET) and mycotoxin T-2 (IS12). IS10 and IS12 had a classical /{beta} hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in the hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 [A] revealed the presence of the N-terminal {beta}-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 includes catalytic residues Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain encloses the catalytic cleft like a lid contributing to substrate binding. Thus, this study has identified novel thermotolerant carboxylesterases with a broad substrate range including polyesters and mycotoxins for potential applications in biotechnology. IMPORTANCEHigh-temperature-active microbial enzymes are important biocatalysts for many industrial applications including recycling of synthetic and biobased polyesters increasingly used in textiles, fibres, coatings and adhesives. Here, we have discovered three novel thermotolerant carboxylesterases (IS10, IS11 and IS12) from high-temperature enrichment cultures from the Ischia hydrothermal vents incubated with biobased polymers. The identified metagenomic enzymes originated from uncultured Chloroflexota and showed low sequence similarity to known carboxylesterases. Active sites of IS10 and IS12 had the largest "effective volumes" among the characterized prokaryotic carboxylesterases and exhibited high substrate promiscuity, including hydrolysis of polyesters and mycotoxin T-2 (IS12). Though less promiscuous compared to IS10 and IS12, IS11 had a higher thermostability with high temperature optimum (80-90 {o}C) for activity, hydrolysed polyesters, and its crystal structure revealed an unusual lipocalin domain likely involved in substrate binding. The polyesterase activity in these enzymes makes them attractive candidates for further optimisation and potential application in plastics recycling.

microbiology↗

bioRxiv2021

Structural and molecular rationale for the diversification of resistance mediated by the Antibiotic_NAT family

The environmental microbiome harbors a vast repertoire of antibiotic resistance genes (ARGs) which can serve as evolutionary predecessors for ARGs found in pathogenic bacteria, or can be directly mobilized to pathogens in the presence of selection pressures. Thus, ARGs from benign environmental bacteria are an important resource for understanding clinically relevant resistance. Here, we conduct a comprehensive functional analysis of the Antibiotic_NAT family of aminoglycoside acetyltransferases. We determined a pan-family antibiogram of 21 Antibiotic_NAT enzymes, including 8 derived from clinical isolates and 13 from environmental metagenomic samples. We find that environment-derived representatives confer high-level, broad-spectrum resistance, including against the atypical aminoglycoside apramycin, and that a metagenome-derived gene likely is ancestral to an AAC(3) gene found in clinical isolates. Through crystallographic analysis, we rationalize the molecular basis for diversification of substrate specificity across the family. This work provides critical data on the molecular mechanism underpinning resistance to established and emergent aminoglycoside antibiotics and broadens our understanding of ARGs in the environment.

microbiology↗