Biology subjects

Paquete, C. M.

Publications and source records attributed to Paquete, C. M..

8 recordsLinked to original sources

bioRxiv2026

A new player in the biochemistry of Anammox bacteria: a multidomain HAO-like protein

Anaerobic ammonium-oxidizing (anammox) bacteria are essential players in the global nitrogen cycle, responsible for converting ammonium and nitrite directly to nitrogen gas. Anammox bacteria have unique features such as a specialized cellular compartment - the anammoxosome. Candidatus (Ca.) Brocadia pituitae genome, as other anammox bacteria, encodes for a diversity of hydroxylamine oxidoreductase (HAO) paralogs, often pointed out as the enzymes responsible for most of the reactions of the anammox cycle. One of this Ca. B. pituitae HAO paralogs is an 840-amino acids protein, named here as BpMHAO, that stands out for its unprecedented domain organization, which includes a multicopper oxidase-like (MCo-like) domain followed by the HAO-like one. Sequence and structural analyses classified this MCo-like domain as homologous to the small laccase family. Spectroscopic characterization revealed a distinct UV-visible spectrum, tentatively assigned to the T3 center, whereas the EPR spectra confirmed the presence of T1, T2 and T3 copper centers. Enzymatic studies demonstrated limited laccase and oxygen-dependent ferroxidase activities. On the other hand, enzymatic assays performed in cell extracts from Escherichia coli and Shewanella oneidensis, harbouring the recombinant HAO-like domain, exhibited a robust hydroxylamine reductase activity using methyl viologen as the electron donor. Our results showed that the BpMHAO potentially plays a role in the anammox process/reactions by converting hydroxylamine into hydrazine. This feature can be relevant to anammox bacteria either by i) mitigating unwanted hydroxylamine, obtained by incorrect formation of this compound, by converting it into hydrazine and enabling its use in the anammox reaction or ii) using hydroxylamine from the outside medium as a substitute for ammonium, delivering hydrazine directly to the last step of the cycle, short-circuiting its first steps.

biochemistry↗

bioRxiv2025

The modular evolution of multiheme cytochromes c bucks the general trend observed in proteins

Multiheme cytochromes c are versatile enzymes which contribute to key steps in diverse biogeochemical cycles of elements and are also key players in microbial electrochemical technologies. We previously showed that these enzymes evolve by grafting and pruning of cytochrome c modules. Here, we extended our analysis and found that grafting can also involve the incorporation of domains of other protein families and of small peptide sequences that affect the heme coordination environment. We show that the addition of hemes to multiheme cytochromes c occurs exclusively by integrating heme-binding peptides and that gain of hemes is equally probable along the protein sequence. By contrast, heme loss occurs by the loss of heme-binding peptides and the accumulation of point mutations. Notably, heme-binding motif loss is disproportionately more prevalent at the position nearer to the N-terminus. This observation contrasts with the general trend observed in proteins, which are usually more conserved at the N-terminus, and likely reflects the way in which multiheme cytochromes c are assembled. Overall, this work completes the picture of how multiheme cytochromes c evolved to achieve the diversity of structures and functions found in nature, and sets them apart from other proteins with respect to the drivers for their evolution. This has contributed to the diversity of roles that they play in various biogeochemical cycles and has implications for engineering artificial variants to enhance biotechnological applications.

biochemistry↗

bioRxiv2025

Polyphasic discrimination of Shewanella seohaensis from closely related species and a whole-genome multilocus (wgMLST) scheme for the evaluation of diversity within this Shewanella clade

Shewanella is an environmentally ubiquitous genus with significant roles in bioelectrochemical applications and human infections. However, identification problems involving Shewanella putrefaciens, Shewanella xiamenensis, and Shewanella seohaensis, have been reported, potentially hindering research progress in these areas. In this study, we explored how to discriminate between these species. By comparing the genomes of Shewanella spp. available in public databases with that of the newly sequenced strain DSM9451, we showed that this strain is a member of the species S. seohaensis. Of the eight public genomes associated with this species, only two were correctly identified in public databases. Phenotypic analysis revealed distinct features of S. seohaensis with respect to S. putrefaciens and Shewanella decolorationis. However, only differences in the intensity of biochemical reactions were observed between S. xiamenensis and S. seohaensis. To discriminate between these closely related species and explore their diversity, a whole-genome multilocus sequence typing scheme was developed. The scheme distinguished related species and revealed significant diversity among S. seohaensis and S. xiamenensis isolates, suggesting that both species may harbor isolates with significantly different metabolic properties. These differences are so wide within the clade that may explain why these two species are difficult to distinguish. The identification of exclusive genes of each species allowed the design of a simple molecular method to differentiate S. seohaensis from closely related species, which will help in clarifying its role in human infections and environmental processes. ImportanceMisidentification within the Shewanella genus, particularly between S. putrefaciens, S. seohaensis, and S. xiamenensis, has been reported and misperceives scientific research on Shewanella spp. in diverse fields, including both biotechnological applications and human infections. Near-complete 16sRNA gene sequencing fails to correctly classify many Shewanella species, and MALDI-TOF systems used in clinical microbiology laboratories are suboptimal species-level identification. In this study, we identified phenotypic characteristics that can guide differentiation and classification, and building upon the identification of species-specific genes, we suggest an accurate and cost-effective molecular test as an alternative to genome sequencing. The proposed wgMLST scheme allows the exploration of species and strain diversity, highlighting the limitations of generalizing results from studies of a single strain. As an emergent pathogen and biotechnological candidate, the proper identification by a single molecular test will enhance the insights about these species towards biotechnology development and public health safety.

microbiology↗

bioRxiv2024

A Survey of the Desulfuromonadia cytochromome provides a glimpse of the unexplored diversity of multiheme cytochromes in nature

Multiheme cytochromes (MHC) provide prokaryotes with a broad metabolic versatility that contributes to their role in the biogeochemical cycling of the elements. However, MHC were isolated and studied in detail only from a limited number of species. To obtain a broader view of the diversity of MHC, we employed bioinformatics tools to study the cytochromome encoded in the genomes of the Desulfuromonadia class. We found that MHC predicted to be extracellular are the least conserved and present higher diversity. Although the most prevalent MHC have homologues already characterized, nearly half of the MHC families in the Desulforomonadia class have no known homologues and AlphaFold2 was employed to predict their 3D structures. This work illuminates for the first time the universe of experimentally uncharacterized cytochromes that are likely to contribute to the metabolic versatility and to the fitness of Desulfuromonadia in diverse environmental conditions and to drive biotechnological applications.

biochemistry↗

bioRxiv2023

Role of the inner membrane cytochrome ImcH in Geobacter extracellular electron transfer and energy conservation

Electroactive bacteria combine the oxidation of carbon substrates with an extracellular electron transfer (EET) process that discharges electrons to an electron acceptor outside the cell. This process involves electron transfer through consecutive redox proteins that efficiently connect the inner membrane to the cell exterior. In this study, we isolated and characterized the quinone-interacting membrane cytochrome c ImcH from Geobacter sulfurreducens, which is involved in the EET process to high redox potential acceptors. Our work provides evidence that ImcH is electroneutral, as it transfers electrons and protons to the same side of the membrane, contributing to the maintenance of a proton motive force, and plays a central role in recycling the menaquinone pool. ImportanceGeobacter sulfurreducens is a model electroactive bacterium, widespread in the environment and of significant interest for biotechnological applications. Its ability to form thick and conductive biofilms on top of conducting surfaces makes this microbe very useful in bioelectrochemical systems for the production of energy or added value products. To explore Geobacter spp. as a biocatalyst it is essential to understand its metabolism, particularly the molecular mechanisms for extracellular electron transfer and energy conservation. Our results reveal the importance of ImcH in both processes, identifying this protein as a major player on Geobacter metabolism.

biochemistry↗

bioRxiv2022

A new paradigm of multiheme cytochrome evolution by grafting and pruning protein modules

Multiheme cytochromes play key roles in diverse biogeochemical cycles, but understanding the origin and evolution of these proteins is a challenge due to their ancient origin and complex structure. Up until now, the evolution of multiheme cytochromes composed by multiple redox modules in a single polypeptide chain was proposed to occur by gene fusion events. In this context, the pentaheme nitrite reductase NrfA and the tetraheme cytochrome c554 were previously proposed to be at the origin of the extant octa- and nonaheme cytochromes c involved in metabolic pathways that contribute to the nitrogen, sulfur and iron biogeochemical cycles by a gene fusion event. Here, we combine structural and character-based phylogenetic analysis with an unbiased root placement method to refine the evolutionary relationships between these multiheme cytochromes. The evidence show that NrfA and cytochrome c554 belong to different clades, which suggests that these two multiheme cytochromes are products of truncation of ancestral octaheme cytochromes related to extant ONR and MccA, respectively. From our phylogenetic analysis, the last common ancestor is predicted to be an octaheme cytochrome with nitrite reduction ability. Evolution from this octaheme framework led to the great diversity of extant multiheme cytochromes analised here by pruning and grafting of protein modules and hemes. By shedding light into the evolution of multiheme cytochromes that intervene in different biogeochemical cycles, this work contributes to our understanding about the interplay between biology and geochemistry across large time scales in the history of Earth.

biochemistry↗

bioRxiv2022

Evidence for quinol oxidase activity of ImoA, a novel NapC/NirT family protein from the neutrophilic Fe(II) oxidizing bacterium Sideroxydans lithotrophicus ES-1

The freshwater chemolithoautotrophic Gram-negative bacterium Sideroxydans lithotrophicus ES-1 oxidizes Fe(II) at the cell surface. In this organism, it is proposed that the monoheme cytochrome MtoD from the Mto pathway transfer electrons across the periplasm to an inner membrane NapC/NirT family tetraheme cytochrome encoded by Slit_2495, for which we propose the name ImoA (inner membrane oxidoreductase). ImoA has been proposed to function as the quinone reductase, receiving electrons from iron oxidizing extracellular electron uptake pathway to reduce the quinone pool. In this study, ImoA was cloned on a pBAD plasmid vector and overexpressed in Escherichia coli. Biochemical and spectroscopic characterization of the purified ImoA reveals that this 26.5 kDa cytochrome contains one high-spin and three low-spin hemes. Our data show that ImoA can function as a quinol oxidase and is able to functionally replace CymA, a related NapC/NirT family tetraheme cytochrome required for anaerobic respiration of a wide range of substrates by Shewanella oneidensis. We demonstrate that ImoA can transfer electrons to different periplasmic proteins from S. oneidensis including STC and FccA, but in a manner that is distinct from that of CymA. Phylogenetic analysis shows that ImoA is clustered closer to NirT sequences than to CymA. This study suggests that ImoA functions as a quinol oxidase in S. oneidensis and raises questions about the directionality and/or reversibility of electron flow through the Mto pathway in S. lithotrophicus ES-1. ImportanceFe(II)-oxidizing bacteria play an important role in the biogeochemical cycling of iron, representing a promising class of organisms for the development of novel biotechnological processes, including bioelectrosynthesis. These organisms perform extracellular electron transfer, taking up electrons from Fe(II) outside of the cell, possibly through a porin-cytochrome complex in the outer membrane. The electrons are then transferred to the quinone pool in the inner membrane via periplasmic and inner membrane electron transfer proteins. In this paper, we produced and characterized the NapC/NirT family tetraheme cytochrome ImoA, encoded by Slit_2495, an inner membrane protein from the Gram-negative Fe(II)-oxidizing bacterium Sideroxydans lithotrophicus ES-1, proposed to be involved in extracellular electron transfer to the quinone pool. We show that ImoA may function instead as a quinol oxidase. The obtained insights represent the first step in understanding mechanisms of electron flow in S. lithotrophicus ES-1 and may lead towards practical biotechnological applications of Fe(II)-oxidizing bacteria.

biochemistry↗

bioRxiv2019

How thermophilic Gram-positive organisms perform extracellular electron transfer: characterization of the cell surface terminal reductase OcwA

Extracellular electron transfer is the key process underpinning the development of bioelectrochemical systems for the production of energy or added-value compounds. Thermincola potens JR is a promising Gram-positive bacterium to be used in these systems because it is thermophilic. In this paper we describe the structural and functional properties of the nonaheme cytochrome OcwA, which is the terminal reductase of this organism. The structure of OcwA, determined at 2.2[A] resolution shows that the overall-fold and organization of the hemes are not related to other metal reductases and instead are similar to that of multiheme cytochromes involved in the biogeochemical cycles of nitrogen and sulfur. We show that, in addition to solid electron acceptors, OcwA can also reduce soluble electron shuttles and oxyanions. These data reveal that OcwA can take the role of a respiratory swiss-army knife allowing this organism to grow in environments with rapidly changing availability of terminal electron acceptors without the need for transcriptional regulation and protein synthesis.\n\nImportanceThermophilic Gram-positive organisms were recently shown to be a promising class of organisms to be used in bioelectrochemical systems for the production of electrical energy. These organisms present a thick peptidoglycan layer that was thought to preclude them to perform extracellular electron transfer (i.e. exchange catabolic electrons with solid electron acceptors outside of the cell). In this manuscript we describe the structure and functional mechanisms of the multiheme cytochrome OcwA, the terminal reductase of the Gram-positive bacterium Thermincola potens JR found at the cell surface of this organism. The results presented here show that this protein is unrelated with terminal reductases found at the cell surface of other electroactive organisms. Instead, OcwA is similar to terminal reductases of soluble electron acceptors. Our data reveals that terminal oxidoreductases of soluble and insoluble substrates are evolutionarily related, providing novel insights into the evolutionary pathway of multiheme cytochromes.

biochemistry↗