Biology subjects

Mendoza, J.

Publications and source records attributed to Mendoza, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Design of readers for pathological PTM proteoforms in Alzheimer's disease

Alzheimer's disease (AD) is characterized by heterogeneous Tau phosphorylation and aberrant amyloid-beta (Abeta) processing, yet molecular tools that distinguish closely related molecular states of tau and Abeta with predefined specificity remain difficult to develop. Here, we designed de novo protein readers targeting three disease-associated phospho-Tau regions and N-terminal pyroglutamate-modified Abeta (N3pG-Abeta). Screening 192 designs per target identified 51 modification-selective hits among 768 candidates (6.6%). Representative readers exhibited nanomolar to subnanomolar affinities for modified peptides, discriminated authentic modifications from unmodified or substitution-based controls, and preferentially recognized modified full-length targets. We further programmed local modification specificity by interface redesign, generating pT231 readers conditional on the phosphorylation state of neighboring S235 and S237. A 2.0-angstrom crystal structure of an N3pG-Abeta reader closely matched the computational design model (interface C-alpha RMSD = 0.52 angstrom), and redesign of pT231 readers tuned apparent binding affinities from greater than 1 uM to below 1 nM. In a human 3D neuronal culture with both pathological Abeta and tau, selective readers targeting N3pG-Abeta, Tau pT231, and Tau pT217 recognized disease-associated signals, exhibiting colocalization with related antibodies and much higher signal in the AD line than the control. Tau pT231 readers further quantitatively distinguish AD patients and control human brain lysates in dot-blot assays. Together, these results establish genetically encoded readers with programmable specificity for defined Tau and Abeta modification states, enabling molecular-state-resolved detection in human disease models and biological samples, and lay a foundation for controlled pathological molecular perturbation.

bioengineering↗

bioRxiv2026

Programming N-linked glycan composition through de novo protein design

N-linked glycans are ubiquitous post-translational modifications that mediate diverse biological processes, including cell signaling and adhesion, molecular recognition, and host-pathogen interactions. Despite their importance, N-linked glycans have largely remained beyond the reach of biomolecular design due to a lack of methods capable of jointly modeling glycans and protein structure. Here, we develop a general all-atom computational pipeline for creating de novo glycoproteins in which the amino acid sequence encodes both the location and composition of N-linked glycans. The method uses three-dimensional protein structure to tune glycan accessibility to processing enzymes in the eukaryotic secretory pathway. Systematic increases in steric restriction progressively reduced processing, shifting glycans from heterogeneous complex-type structures toward increasingly underprocessed oligomannose-type species resembling those found on many viral glycoproteins. Despite this programmed restriction, the resulting oligomannose glycans remained accessible for biological recognition, inhibiting uropathogenic Escherichia coli adhesion to human bladder cells and binding mannose-binding lectin. These results establish steric control of N-glycan processing as a genetically encodable design principle and extend computational protein design to functional glycoproteins with programmed composition.

biochemistry↗

bioRxiv2026

Complex enzyme active site scaffolding by iteratively detuned catalytic guidance

De novo enzyme design requires sequences whose structures reproduce prescribed catalytic geometries. This remains challenging for active sites that couple multiple catalytic residues, cofactors and substrates. Here we introduce the Catalytic Guidance Engine (CaGE), which optimizes designed sequences to satisfy complex sets of functional constraints by alternating constraint guided structure prediction trajectories with sequence design, gradually fading the strength of the constraint information over cycles to nudge the designed sequence towards satisfying all the constraints. We train RF3 to accept atom-pair distances and other design conditions, and use classifier-free guidance to fade their influence over multiple design cycles. We also develop training-free motif guidance protocols which operate at inference time by directly adjusting selected atoms towards the design target geometry during diffusion sampling. CaGE improves catalytic-motif geometry across a diverse enzyme benchmark and yields sequences that recover complex target active sites in independent AF3 predictions. We experimentally demonstrate the power of CaGE by designing active enzymes with complex catalytic sites: a nonheme-iron design positions four active-site residues around an iron centre and a transition-state ligand, and a PLP-dependent design positions four catalytic residues around a PLP cofactor with multiple functional groups. Active site substitutions and a 2.7[A] crystal structure support the designed mechanisms and structures. Thus, CaGE provides a route to functional de novo enzymes with complex active sites.

bioengineering↗

bioRxiv2026

Supramammillary projections to the lateral preoptic area drive dopamine release and active behavior

The supramammillary nucleus (SuM) is known to promote active, approach-oriented behaviors by engaging ventral tegmental area (VTA) dopamine neurons projecting to the nucleus accumbens (NAc), primarily via a glutamatergic SuM[->]medial septum (MS) pathway. However, the role of other SuM projections in this process remains unclear. Here we combined anatomical tracing, fiber photometry, chemogenetic, and optogenetic approaches to investigate the SuM[->]lateral preoptic area (LPO) pathway. We found that SuM[->]LPO neurons possess extensive collateral projections and are inhibited during consummatory behavior but activated by aversive and unexpected stimuli. Chemogenetic activation of these neurons reduced water intake and immobility during stress, while inhibition enhanced the appetitive phase for ingestive behavior and fear-related immobility. Optogenetic stimulation of SuM[->]LPO terminals reinforced behavior and elicited dopamine release in the NAc. These findings identify the SuM[->]LPO pathway as a distinct circuit that, alongside the SuM[->]MS pathway, engages mesolimbic dopamine signaling to facilitate active behavioral responses to environmental challenges.

neuroscience↗

bioRxiv2026

Orexinergic Input to the Supramammillary Region Links Threat Salience to Approach Behavior and Dopamine Release

Orexin (hypocretin) neurons (OXN) broadcast arousal-related signals across the forebrain, yet how their inputs shape motivated behavior remains poorly understood. The supramammillary region (SuM), a posterior hypothalamic structure implicated in arousal, active coping and reward-seeking, receives dense orexin innervation. Here, we define how orexinergic terminals in the SuM (OXNSuM) integrate threat salience with approach motivational processes. Using viral tracing and RNAscope, we show that SuM neurons express both orexin receptor subtypes, with type 2 receptors predominating. Fiber photometry recordings from OXNSuM terminals reveal strong activation by footshock and conditioned threat cues, modest responses to salient sensory stimuli, and pronounced suppression during water consumption. Optogenetic stimulation of OXNSuM terminals reinforces instrumental responding, biases real-time place preference, and elicits rapid, stimulation-locked dopamine release in the nucleus accumbens. These findings identify an orexin-to-SuM pathway that is engaged by uncertainty and threat yet promotes approach behavior through coupling to mesolimbic dopamine, revealing a mechanism linking hypothalamic arousal signals to adaptive motivational responses. SIGNIFICANCE STATEMENTThe supramammillary region (SuM) has emerged as a critical node linking arousal, threat processing, and motivated behavior, yet the sources and functions of its modulatory inputs remain unclear. Here, we identify orexinergic projections to the SuM as a circuit mechanism that couples threat-related salience with approach behavior and nucleus accumbens dopamine signaling. We show that orexin terminals in the SuM are activated by stressful and uncertain stimuli but suppressed during consummatory behavior, and that selective activation of these terminals is positively reinforcing and drives rapid dopamine release. These findings reveal a pathway through which orexin neurons promote active coping and reward-seeking, advancing our understanding of how hypothalamic neuromodulation shapes mesolimbic function and adaptive behavioral responses.

neuroscience↗

bioRxiv2026

Controlling metal-carbonate phase, form, and function through de novo protein design

Biomineralization enables living systems to construct hybrid materials by controlling the location, orientation, and polymorph of inorganic crystals with proteins and other biomolecules. Despite decades of study, the molecular principles underlying these processes remain difficult to harness in engineered materials, in part because native biomineralization proteins are often intrinsically disordered, heterogeneous, or insoluble. Here we show that de novo designed protein interfaces can be assembled into reconfigurable two-dimensional arrays which template calcite nanocrystals. By fine-tuning RFdiffusion2 on repeat protein scaffolds, we further enable the design of protein architectures which selectively form aragonite, a metastable polymorph of calcium carbonate, in nucleation conditions that otherwise result in a mixture of phases. Extending beyond inorganics found in biological systems, we show that lattice-matched protein designs template cobalt carbonate formation: a flat helical repeat protein interface promotes unconfined growth, whereas soluble D3 cage assemblies yield more homogenous cobalt carbonate nanocrystals confined to the interior of the cage. These protein-cage cobalt carbonate hybrid materials function as electrocatalysts for alkaline water splitting. Our results demonstrate the potential of deep learning-based methods to unlock the structural and functional activity of protein-mineral composites.

biochemistry↗

bioRxiv2026

Generative design of programmable asymmetric β-barrel nanopores

Protein nanopores are powerful tools for molecular sensing, sequencing, and separation, but designing pores with programmable function remains challenging. Native homo-oligomeric transmembrane {beta} barrels (TMBs) are used for these applications, but their uniform lumens limit spatial resolution and analyte discrimination. Although monomeric TMBs can be designed using energy-based methods, these approaches remain highly manual and limited to structural design rather than function. Here, we present a generative AI framework for TMB design, with backbone and sequence design models trained on a curated distillation set. We characterized 48 designs spanning 0.7-1.5 nm in diameter, diverse lumen chemistries, and hydrophobic thicknesses. Crystal structures closely match the design models. We demonstrate that our method produces customizable nanopores for ion sensing, DNA translocation, and transport across polymer membranes.

biochemistry↗

bioRxiv2026

Behavioral dynamics of different stages of sexual motivation in male and female rats

Sexual motivation is a complex concept involving both the initial drive to begin mating and the motivation to sustain copulation. Disruptions in sexual motivation are often observed in psychiatric disorders. This study proposes that sexual motivation can be divided into two distinct components: sexual incentive motivation and the drive to sustain copulation. To investigate this, we utilized the Motivation to Continue Copulation (MCC) test, which measures effort (nose pokes) to gain access to a sexual reward, and compared it with the Sexual Incentive Motivation (SIM) test and standard copulation tests. Male and female rats were trained on a fixed ratio (FR) 1 schedule using cheese rewards before transitioning to sexual rewards. After six FR1 sessions, the effort required increased to FR5 and progressive ratio (PR) schedules. Results revealed that sexual incentive motivation, measured by the SIM test, was higher in males after sexual experience, while females maintained consistent levels. In the MCC test, both males and females exhibited increased motivation to continue copulation with experience, but the motivation declined in the 2nd ejaculatory series. These findings demonstrate that sexual motivation comprises distinct components. The MCC test effectively measures the drive to sustain copulation, while the SIM test assesses incentive motivation. This distinction is crucial for advancing behavioral neuroscience and understanding sexual dysfunction in psychiatric conditions.

neuroscience↗

bioRxiv2025

Modelling the effect of V1a receptor antagonism and its potential therapeutic effect in circadian disorders

BackgroundThe suprachiasmatic nucleus (SCN) is the central circadian clock in mammals, regulating many daily physiological and behavioral rhythms. Dysregulation of the SCN is associated with various circadian disorders, highlighting the potential therapeutic benefits of targeting its neurons and output pathways. Vasopressin signaling is one of the main regulators of the synchronicity and the functional output of the SCN. MethodsWe investigated the effect of a single dose (30mg/kg) of the vasopressin V1a receptor (V1aR) antagonist, balovaptan, on resynchronization of locomotor activity rhythms in mice after a 6-hour phase advance of the light-dark cycle. To mechanistically model the effect of V1aR antagonism, we developed a mathematical framework simulating the SCN, its control of circadian biomarkers (melatonin, core body temperature), and the impact of V1aR antagonism. ResultsA single administration of the V1a antagonist balovaptan significantly accelerated resynchronization of locomotor activity rhythms to new light-dark cycles. To mechanistically understand this effect, we devised a mathematical model of the SCN that successfully captures this accelerated synchronization of circadian rhythms under V1aR antagonism. Additionally, the model replicates well-established SCN behaviors in both humans and rodents, including the phase response curve triggered by a light pulse at various circadian phases, and the desynchronization of the SCN observed in forced desynchronization experiments. Mechanistically, our model suggests that weakening vasopressin signaling via V1aR antagonism strengthens the SCNs resistance to internal desynchronization. Additionally, our model suggests a strong link between the endogenous period (tau) and the phase of circadian biomarkers, with longer tau values resulting in delayed biomarker rhythms. Importantly, the model predicts that V1aR antagonism induces a phase advance proportional to tau. The model predicts that individuals with longer endogenous periods, who consequently exhibit greater phase delays in their circadian rhythms, could experience more substantial phase advances in response to V1aR antagonism. DiscussionWe show that targeting V1aR is enough to cause a faster resynchronization to a new light-dark cycle in the jet lag paradigm and establish a computational framework for investigating its therapeutic potential in circadian rhythm disorders. This framework, adaptable to incorporate pharmacodynamic data, can be used to design clinical trials evaluating V1aR antagonism for treating circadian disorders.

neuroscience↗

bioRxiv2025

VadK, a Non-Canonical Kinase that Regulates the Methylcitrate Cycle and is Essential for Mycobacterium tuberculosis Virulence

The evolution of new enzymatic functions is constrained and guided by the architecture of an organisms metabolic and regulatory networks as well as by environmental constraints. Here, we identify a previously uncharacterized kinase that has evolved from pyruvate phosphate dikinase (PPDK). Through biochemical and systems-level analyses, we show that this enzyme, encoded by Rv1127c in Mycobacterium tuberculosis (Mtb), has diverged from its ancestral role in central carbon metabolism to function as a histidine kinase in pathogenic mycobacteria and related species. We designate this enzyme Virulence Associated DiKinase (VadK), reflecting its ability to autophosphorylate and role in coordinating metabolism and virulence. VadK is essential for the utilization of exogenous carbon sources critical for survival within the host and is required for Mtb pathogenicity in murine models of tuberculosis. Furthermore, VadK interacts with key enzymes of the methylcitrate cycle, and 13C-metabolic flux analysis indicates that it fine-tunes flux through this pathway, with elevated flux proving growth limiting. Together, these findings identify VadK as a previously unrecognized regulatory kinase that integrates metabolic control with virulence in Mtb, revealing a new facet of metabolic regulation in bacterial pathogenesis and a potential target for therapeutic intervention.

microbiology↗

bioRxiv2025

A periplasmic protein complex mediates cell envelope integrity and intrinsic multidrug resistance in Mycobacterium tuberculosis

The intrinsic drug resistance of Mycobacterium tuberculosis (Mtb) is a major barrier to effective tuberculosis (TB) treatment, largely due to its complex, impermeable cell envelope. We identified a periplasmic protein complex comprising FecB and Rv3035 that is essential for maintaining envelope integrity and mediating intrinsic multidrug resistance in Mtb. FecB interacts with Rv3035, forming a stable heterodimer that associates with the cell envelope biosynthesis protein AftB. We report the structures of Rv3035 alone and in complex with FecB and identify critical residues for complex formation and function. Co-essentiality and genetic interaction analyses support a functional link between FecB, Rv3035 and AftB, an arabinofuranosyltransferase which synthesizes arabinogalactan and lipoarabinomannan. Loss of FecB or Rv3035 disrupted AftB-mediated arabinan synthesis, suggesting that these proteins support AftBs enzymatic activity. Importantly, FecB is required for Mtb virulence in mice, underscoring its physiological relevance. These findings highlight FecB, Rv3035 and AftB as promising therapeutic targets.

microbiology↗

bioRxiv2025

Bacteria deliver a microtubule-binding protein into mammalian cells to promote colonization

Pathogenic Bordetella bacteria infect the ciliated respiratory epithelia of mammalian and avian hosts. Several bacterial proteins mediate host cell adhesion, but filamentous hemagglutinin (FhaB) is a principal adhesin because mutants lacking this protein exhibit profound colonization defects. Here, we show that FhaB carries a C-terminal microtubule-binding domain (FhaB-CT), which is translocated into the host-cell cytoplasm to promote bacterial colonization. Cryogenic electron microscopy of microtubule-bound FhaB-CT shows that the domain binds primarily to -tubulin through a network of polar interactions. Live-cell microscopy of infected tracheal explants reveals that FhaB-CT delivery is required for Bordetella to occupy a niche at the base of cilia on airway epithelia. Finally, we demonstrate that the microtubule-binding domain is required for long-term colonization of the mouse nasal cavity by B. pertussis. These observations suggest that the FhaB-CT domain is delivered into motile cilia, where it interacts with axonemal microtubules. We propose that Bordetella initially adhere to the tips of cilia, then deploy multiple FhaB adhesin molecules to migrate to the base of the cilial forest. This mechanism enables Bordetella to resist removal by the mucociliary escalator that clears the respiratory tract of microbes and debris.

microbiology↗

bioRxiv2024

Lineage-specific neutralising antibodies after SARS-CoV-2 mild disease. Immune boosting effect of vaccination

We followed a group of 105 non-vaccinated individuals after Alpha or Delta SARS-CoV-2 mild disease, measuring the viral shedding (qRT-PCR, dPCR and subgenomic RNA-E) and humoral response (commercial immunoassay and pseudovirus and live virus neutralisation) up to six months. Sixty nine patients received a vaccination boost during the follow-up period (n=95). Subgenomic RNA-E showed a shorter period until negativity (mean 2.2 weeks) compared to gRNA (mean 5.2 weeks). A high correlation between qRT-PCR and dPCR was found for viral load estimation, even when no nucleic acid extraction was used in dPCR (R2 = 0.87). Post-convalescent sera showed the strongest neutralisation against the variant of natural exposure, while the neutralisation capacity against Omicron variants was significantly lower compared to the other variants. Additionally, the results suggested that commercial immunoassays may not accurately predict protection against a different variant than the variant of exposure. An immune boosting effect of the SARS-CoV-2 vaccination was evident. Variant-specific neutralising antibodies were detected one month after natural infection. Although short lived, maximum igG response was observed after hybrid immunisation (natural infection + vaccination). This study also points to potential improvements in the clinical management of SARS-CoV-2 cases. Firstly, subgenomic RNA-E is a potentially more accurate biomarker of infectivity than current qRT-PCRs using genomic RNA as target. Secondly, accuracy of high-throughput immunoassays must be validated in order to estimate specific protection and organise vaccination campaigns. Our findings could play a role in the current implementation of SARS-CoV-2 vaccine programs. Author SummaryYears after SARS-CoV-2 related infections challenged the healthcare systems of the whole world, the optimal strategy to deal with diagnosis, quarantines or vaccination patterns is still a matter of debate. The interplay between infectivity and immunity in the different circulating variants is complex, and qPCR for diagnosis may extend quarantines, as detection of viral RNA does not necessarily mean that the virus remains infectious. We studied a group of patients infected with pre-Omicron variants to study how their variant-specific antibodies reacted to the past and present variants of SARS-CoV-2. In both Alpha and Delta, antibodies neutralise their own variants better than other variants that came before and after. Perhaps most importantly, neutralisation was lowest against Omicron variants. We followed the viral shedding dynamics of the patients, testing different PCR techniques and targets. Subgenomic RNA was detectable in nasopharyngeal samples for a shorter time than genomic RNA and it has been suggested as a good marker of infectivity. Using sgRNA instead of genomic RNA as a PCR target could reduce hospital bed occupation and quarantine time. Overall, we hope that these results could help guide pandemic and diagnostic control in the future.

microbiology↗

bioRxiv2024

Orchestrating Improbable Chemistries: Structural Snapshots of B12-Dependent Methionine Synthase's Catalytic Choreography

Cobalamin (vitamin B12) and its derivatives play an essential role in biological methylation, with cobalamin-dependent methionine synthase (MS) serving as a canonical example. MS catalyzes multiple methyl transfers within a single, dynamic multi-domain architecture that has proven challenging to study, hampering efforts to elucidate its catalytic mechanism(s). Utilizing a thermostable MS homolog and non-native cobalamin cofactors, we have captured crystal structures of transient conformational states of MS, including those directly involved in folate demethylation and homocysteine methylation. These snapshots reveal the mechanistic significance of five-coordinate, His-off methylcobalamin in homocysteine methylation and highlight the crucial role of the folate-binding domain and interdomain linkers in orchestrating the intricate structural rearrangements required for catalysis. This expanded conformational ensemble, including the unexpected capture of novel Cap-on conformations, underscores the remarkable plasticity of MS, exceeding previous estimations. Our findings provide crucial insights into the catalytic mechanism of MS, laying the foundation for harnessing cobalamins biocatalytic potential and elucidating how nature exploits protein dynamics to facilitate complex transformations.

biochemistry↗

bioRxiv2023

Development of a sensor for disulfide bond formation in diverse bacteria

In bacteria, disulfide bonds contribute to the folding and stability of proteins important for processes in the cellular envelope. In E. coli, disulfide bond formation is catalyzed by DsbA and DsbB enzymes. DsbA is a periplasmic protein that catalyzes disulfide bond formation in substrate proteins while DsbB is an inner membrane protein that transfers electrons from DsbA to quinones, thereby regenerating the DsbA active state. Actinobacteria including mycobacteria use an alternative enzyme named VKOR which performs the same function as DsbB. Disulfide bond formation enzymes, DsbA and DsbB/ VKOR represent novel drug targets because their inhibition could simultaneously affect the folding of several cell envelope proteins including virulence factors, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. We have previously developed a cell-based and target-based assay to identify molecules that inhibit the DsbB and VKOR in pathogenic bacteria, using Escherichia coli cells expressing a periplasmic {beta}-Galactosidase sensor ({beta}-Galdbs) which is only active when disulfide bond formation is inhibited. Here we report the construction of plasmids that allow fine-tuning of the expression of the {beta}-Galdbs sensor and can be mobilized into other gram-negative organisms. As an example, when harbored in P. aeruginosa UCBPP-PA14, {beta}-Galdbs behaves similarly as in E. coli and the biosensor responds to the inhibition of the two DsbB proteins. Thus, these {beta}-Galdbs reporter plasmids provide a basis for identifying novel inhibitors of DsbA and DsbB/VKOR against multi-drug resistant, gram-negative pathogens and to further study oxidative protein folding in diverse gram-negative bacteria. ImportanceDisulfide bonds contribute to the folding and stability of proteins in the bacterial cell envelope. Disulfide bond-forming enzymes represent new drug targets against multidrug-resistant bacteria since inactivation of this process would simultaneously affect several proteins in the cell envelope, including virulence factors, toxins, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. Identifying the enzymes involved in disulfide bond formation in gram-negative pathogens as well as their inhibitors can contribute to the much-needed antibacterial innovation. In this work, we developed sensors of disulfide bond formation for gram-negative bacteria. These tools will enable the study of disulfide bond formation and the identification of inhibitors for this crucial process in diverse gram-negative pathogens.

microbiology↗

bioRxiv2023

Structural basis of S-adenosylmethionine-dependent Allosteric Transition from Active to Inactive States in Methylenetetrahydrofolate Reductase

Methylenetetrahydrofolate reductase (MTHFR) is a pivotal flavoprotein connecting the folate and methionine methyl cycles, catalyzing the conversion of methylenetetrahydrofolate to methyltetrahydrofolate. Human MTHFR (hMTHFR) undergoes elaborate allosteric regulation involving protein phosphorylation and AdoMet-dependent inhibition, though other factors such as subunit orientation and FAD status remain understudied due to the lack of a functional structural model. Here, we report crystal structures of Chaetomium thermophilum MTHFR (cMTHFR) in both active (R) and inhibited (T) states. We reveal FAD occlusion by Tyr361 in the T-state, which prevents substrate interaction. Remarkably, the inhibited form of cMTHFR accommodates two AdoMet molecules per subunit. In addition, we conducted a detailed investigation of the phosphorylation sites in hMTHFR, three of which are novel. Based on the structural framework provided by our cMTHFR model, we propose a possible mechanism to explain the allosteric structural transition of MTHFR, including the impact of phosphorylation on AdoMet-dependent inhibition.

biochemistry↗

bioRxiv2023

Structure of full-length cobalamin-dependent methionine synthase and cofactor loading captured in crystallo

Cobalamin-dependent methionine synthase (MS) is a key enzyme in methionine and folate one-carbon metabolism. MS is a large multi-domain protein capable of binding and activating three substrates: homocysteine, folate, S-adenosylmethionine for methylation. Achieving three chemically distinct methylations necessitates significant domain rearrangements to facilitate substrate access to the cobalamin cofactor at the right time. The distinct conformations required for each reaction have eluded structural characterization as its inherently dynamic nature renders structural studies difficult. Here, we use a thermophilic MS homolog (tMS) as a functional MS model. Its exceptional stability enabled characterization of MS in the absence of cobalamin, marking the first studies of a cobalamin-binding protein in its apoenzyme state. More importantly, we report the first high-resolution full-length MS structure, ending a multi-decade quest. We also captured cobalamin loading in crystallo, providing structural insights into holoenzyme formation. Our work paves the way for unraveling how MS orchestrates large-scale domain rearrangements crucial for achieving challenging chemistries.

biochemistry↗

bioRxiv2022

SbCYP79A61 Produces Phenylacetaldoxime, a Precursor of Benzyl Cyanide and Phenylacetic Acid in Sorghum bicolor

Aldoximes are amino acid derivatives that serve as intermediates for numerous specialized metabolites including cyanogenic glycosides, glucosinolates, and auxins. Aldoxime formation is mainly catalyzed by cytochrome P450 monooxygenases of the 79 family (CYP79s) that can have broad or narrow substrate specificity. Aldoxime biosynthesis in the cereal sorghum (Sorghum bicolor) is not well characterized. This study identified nine CYP79-encoding genes in the genome of sorghum. A phylogenetic analysis of CYP79 showed that SbCYP79A61 formed a subclade with maize ZmCYP79A61, previously characterized to be involved in aldoxime biosynthesis. Functional characterization of this sorghum enzyme using transient expression in Nicotiana benthamiana and stable overexpression in Arabidopsis thaliana revealed that SbCYP79A61 catalyzes the production of phenylacetaldoxime (PAOx) from phenylalanine, but unlike the maize enzyme, displays no detectable activity against tryptophan. Additionally, targeted metabolite analysis after stable isotope feeding assays revealed that PAOx can serve as a precursor of phenylacetic acid (PAA) in sorghum and identified benzyl cyanide as an intermediate of PAOx-derived PAA biosynthesis in both sorghum and maize. Taken together, our results demonstrate that SbCYP79A61 produces PAOx in sorghum and may serve in the biosynthesis of other nitrogen-containing phenylalanine-derived metabolites involved in mediating biotic and abiotic stresses. HighlightIdentification of SbCYP79A61, an enzyme catalyzing the production of phenylacetaldoxime (PAOx) in sorghum, and identification of benzyl cyanide as an intermediate of PAOx-derived phenylacetic acid (PAA) biosynthesis in monocots.

plant biology↗