Biology subjects

Berndsen, C. E.

Publications and source records attributed to Berndsen, C. E..

11 recordsLinked to original sources

bioRxiv2026

Biophysical and enzymatic comparison of Bacillus safensis and Bacillus subtilis malate dehydrogenase (MDH) enzymes

Species of Bacillus bacteria including Bacillus safensis and Bacillus subtilis are finding increasing uses in biotechnology and bioremediation, thanks in part to their metabolic robustness. Malate dehydrogenase (MDH) is at the heart of central metabolism and thus a better understanding of Bacillus MDH proteins could aid in the optimization of these applications. MDH of Bacillus spp. belong to the lactate dehydrogenase (LDH)-like class of MDHs, otherwise known as the MDH3 class. Despite wide prevalence in nature among prokaryotes and archaea, this typically homotetrameric class is understudied compared to the MDH1 and MDH2 classes found in eukaryotes. We therefore recombinantly expressed and purified MDH proteins from two societally relevant Bacillus spp.-B. safensis and B. subtilis-and characterized them biophysically (via Size Exclusion Chromatography-Small Angle X-ray Scattering (SEC-SAXS) and Differential Scanning Fluorimetry (DSF)) and enzymatically (via spectroscopic activity assays). As expected based on their high sequence identity, the two MDH orthologs had similar properties in most regards, including a tetrameric structure and high susceptibility to substrate inhibition. However, we uncovered differences in conditional thermal stability, in addition to subtle differences in enzymatic activity that offer insight into the workings of LDH-like MDH. Summary statementMalate dehydrogenase (MDH) is a fundamental metabolic enzyme, from microbes to mammals, yet comparably little is known about microbial MDH, especially MDH of the tetrameric MDH3 class. We compare the biophysical and enzymatic properties of two such enzymes from the societally relevant bacterial species Bacillus subtilis and Bacillus safensis, offering useful insight with potential biotechnological implications.

biochemistry↗

bioRxiv2025

Molecular Architecture of the human Citrate Synthase-Malate Dehydrogenase 2 metabolon

Metabolons are transient biomolecular complexes that enhance the efficiency of metabolic pathways through substrate channeling. These complexes are difficult to study because of the transient nature, thus limiting our understanding of how they are formed and regulated. The citric acid cycle is proposed to contain many such complexes although few have been characterized structurally. Here, we provide direct structural evidence for the complex of human Citrate Synthase and human mitochondrial Malate Dehydrogenase 2, which is part of the larger proposed citric acid cycle metabolon. Our structural model supports previous crosslinking studies and suggests that hMDH2 can interact with each subunit of the hCS dimer, forming up to a hexameric complex. However, this complex appears transient as titration of hMDH2 into hCS in activity assays does not saturate. We further show that the interaction site with hCS is non-specific, as hCS could also stimulate oxaloacetate formation by cytosolic and plant MDH enzymes. This structural model will provide a base for understanding the structure and regulation of the broader citric acid cycle metabolon.

biochemistry↗

bioRxiv2025

Functional Characterization of Arabidopsis α-Amylase3 (AMY3): Amylose Specificity and Structural Insights into Its Duplex Carbohydrate-Binding Module

Flowering plants contain a plastid-localized [a]-amylase (AMY3) with several recognized domains: two N-terminal carbohydrate-binding modules, an alpha-alpha hairpin that binds the catalytically inactive activator {beta}-amylase9, and a C-terminal catalytic domain. Despite considerable research on leaf starch metabolism in Arabidopsis, little is known about the function of AMY3. Starch is composed of two glucose polymers: highly branched amylopectin (70-90%) and largely unbranched amylose (10-30%). Because [a]-amylases are endo-amylases, we predicted that AMY3 might prefer to hydrolyze amylose, in contrast to the exo-amylase {beta}-amylase1 (BAM1), which we predicted would prefer amylopectin. Using iodine spectroscopy, we observed that AMY3 caused the percent amylose of corn starch to decrease, whereas BAM1 caused the percent amylose to increase before a gradual decrease. Enzyme assays revealed that AMY3 has a higher affinity for amylose compared to amylopectin, whereas BAM1 has a higher affinity for amylopectin. Furthermore, we found that the starch in amy3 leaves had twice as much amylose as WT leaves at the end of the day which suggests potential health and industrial applications. We purified the AMY3 CBM region and studied its structure with small-angle X-ray scattering, seeking to understand its role in substrate specificity. The AMY3 CBMs form a unique duplex where two interlocking {beta}-strands connect CBM1 and CBM2. The duplex CBM forms a dimer in solution and can bind amylose and amylopectin. However, AMY3 without the CBMs still prefers amylose, indicating the duplex CBM is not essential for amylose specificity in vitro. However, the duplex CBM is important for AMY3 dimerization.

biochemistry↗

bioRxiv2025

Structural and enzymatic divergence between human MDH isoforms underlies their specialized regulatory roles in metabolism

Malate dehydrogenase (MDH: EC:1.1.1.37) catalyzes a key NAD+-dependent redox reaction integral to cellular metabolism. In humans, the cytosolic (hMDH1) and mitochondrial (hMDH2) isoforms operate in distinct compartments, suggesting potential differences in regulation. Here, we present a comparative analysis of hMDH1 and hMDH2 under physiologically relevant conditions, integrating enzymatic assays, ligand binding studies, small-angle X-ray scattering (SAXS), and molecular modeling. Our findings reveal that hMDH2 activity is inhibited by -ketoglutarate, glutamate, NAD+, ATP, and citrate at concentrations consistent with mitochondrial metabolic states characterized by elevated amino acid catabolism or redox stress. Conversely, hMDH1 exhibits minimal impact by these metabolites, with only modest inhibition observed in the presence of ATP and ADP. SAXS analyses confirm that both isoforms maintain stable dimeric structures upon ligand binding, indicating that regulation is not mediated by global conformational changes. Structural modeling and normal mode analyses identify increased flexibility in hMDH1, particularly within the active site loop, thumb loop, and a partially disordered C-terminal helix. In contrast, hMDH2 displays a more rigid architecture and a more electropositive active site environment, correlating with its heightened sensitivity to anionic metabolites. Fluorescence quenching experiments further support these distinctions, demonstrating stronger binding affinities for nucleotide-based ligands in hMDH2 compared to hMDH1. Collectively, these results suggest that isoform-specific regulation of human MDH arises from differences in local structural dynamics and electrostatics, rather than large-scale structural rearrangements. hMDH2 appears adapted to integrate mitochondrial metabolic signals, modulating malate oxidation in response to cellular conditions, while hMDH1 maintains consistent cytosolic function across diverse metabolic states.

biochemistry↗

bioRxiv2024

The pseudoenzyme β-amylase9 from Arabidopsis binds to and enhances the activity of α-amylase3: A possible mechanism to promote stress-induced starch degradation

Starch accumulation in plant tissues provides an important carbon source at night and for regrowth after periods of dormancy and in times of stress. Both [a]- and {beta}-amylases (AMYs and BAMs, respectively) catalyze starch hydrolysis, but their functional roles are unclear. Moreover, the presence of catalytically inactive amylases that show starch excess phenotypes when deleted presents an interesting series of questions on how starch degradation is regulated. Plants lacking one of these catalytically inactive {beta}-amylases, BAM9, were shown to have enhanced starch accumulation when combined with mutations in BAM1 and BAM3, the primary starch degrading BAMs in response to stress and at night, respectively. Importantly, BAM9 has been reported to be transcriptionally induced by stress through activation of SnRK1. Using yeast two-hybrid experiments, we identified the plastid-localized AMY3 as a potential interaction partner for BAM9. We found that BAM9 interacted with AMY3 in vitro and that BAM9 enhances AMY3 activity 3-fold. Modeling of the AMY3-BAM9 complex revealed a previously undescribed N-terminal structural feature in AMY3 that we call the alpha-alpha hairpin that could serve as a potential interaction site. Additionally, AMY3 lacking the alpha-alpha hairpin is unaffected by BAM9. Structural analysis of AMY3 showed that it can form a homodimer in solution and that BAM9 appears to replace one of the AMY3 monomers to form a heterodimer. Collectively these data suggest that BAM9 is a pseudoamylase that activates AMY3 in response to cellular stress, possibly facilitating starch degradation to provide an additional energy source for stress recovery.

biochemistry↗

bioRxiv2022

Structural and functional analysis of the human Cone-rod homeobox transcription factor

The cone-rod homeobox (CRX) protein is a critical K50 homeodomain transcription factor responsible for the differentiation and maintenance of photoreceptor neurons in the vertebrate retina. Mutant alleles in the human gene encoding CRX result in a variety of distinct blinding retinopathies, including retinitis pigmentosa, cone-rod dystrophy, and Leber congenital amaurosis. Despite the success of using in vitro biochemistry, animal models, and genomics approaches to study this clinically relevant transcription factor over the past 24 years since its initial characterization, there are no high-resolution structures in the published literature for the CRX protein. In this study, we use bioinformatic approaches and small-angle x-ray scattering (SAXS) structural analysis to further understand the biochemical complexity of the human CRX homeodomain (CRX-HD). We find that the CRX-HD is a compact, globular monomer in solution that can specifically bind functional cis-regulatory elements encoded upstream of retina -specific genes. This study presents the first structural analysis of CRX, paving the way for a new approach to studying the biochemistry of this protein and its disease-causing mutant protein variants.

biochemistry↗

bioRxiv2021

The β-amylase7 gene in Zea mays encodes a protein with structural and catalytic properties similar to Arabidopsis BAM2

Starch accumulates in the plastids of green plant tissue during the day to provide carbon for metabolism at night. Starch hydrolysis is catalyzed by members of the {beta}-amylase (BAM) family, which in Arabidopsis thaliana (At), includes nine structurally and functionally diverse members. One of these enzymes, AtBAM2, is a plastid-localized enzyme that is unique among characterized {beta}-amylases since it is tetrameric and exhibits sigmoidal kinetics. Sequence alignments show that the BAM domains of AtBAM7, a catalytically inactive, nuclear-localized transcription factor with an N-terminal DNA binding domain, and AtBAM2 are more closely related to each other than they are to any other AtBAM. Since BAM2 is found in more ancient lineages, it was hypothesized that BAM7 evolved from BAM2. However, analysis of the genomes of 48 flowering plants revealed 12 species that appear to have a BAM7 gene but lack a BAM2 gene. Upon closer inspection, these BAM7 proteins have a greater percent identity to AtBAM2 than to AtBAM7, and they share all of the AtBAM2 functional residues that BAM7 proteins normally lack. We hypothesize that these genes may encode a BAM2-like protein although they are currently annotated as BAM7-like genes. To test this hypothesis, we designed a cDNA of the short form of corn BAM7 (ZmBAM7-S) for expression in E. coli. Small Angle X-Ray Scattering data indicate that ZmBAM7-S has a tetrameric solution structure more similar to that of AtBAM2 than AtBAM1. In addition, partially purified ZmBAM7-S is catalytically active and exhibits sigmoidal kinetics. Together these data suggest that some BAM7 genes may encode a functional BAM2. Exploring and understanding {beta}-amylase gene structure could have impacts on the current annotation of genes.

biochemistry↗

bioRxiv2019

Solution structure and assembly of β-amylase 2 from Arabidopsis thaliana

Starch is a key energy storage molecule in plants that requires controlled synthesis and breakdown for effective plant growth. {beta}-amylases (BAMs) hydrolyze starch into maltose to help meet the metabolic needs of the plant. In the model plant, Arabidopsis thaliana, there are nine BAMs which have apparently distinct functional and domain structures, although the functions of only a few of the BAMs are known and there are no 3-D structures of BAMs from this organism. Recently, AtBAM2 was proposed to form a tetramer based on chromatography and activity assays of mutants, however there was no direct observation of this tetramer. We collected small-angle X-ray scattering data on AtBAM2 and N-terminal mutants to describe the structure and assembly of the tetramer. Comparison of the scattering of the AtBAM2 tetramer to data collected using the sweet potato (Ipomoea batatas) BAM5, which is also reported to form a tetramer, showed there were differences in the overall assembly. Analysis of N-terminal truncations of AtBAM2 identified a loop sequence found only in BAM2 orthologs that appears to be critical for AtBAM2 tetramer assembly as well as activity.

biochemistry↗

bioRxiv2018

Resilience of BST-2/Tetherin structure to single amino acid substitutions

Human Tetherin, also known as BST-2 or CD317, is a dimeric, extracellular membrane-bound protein that consists of N and C terminal membrane anchors connected by an extracellular domain. BST-2 is involved in binding enveloped viruses, such as HIV, and inhibiting viral release in addition to a role in NF-kB signaling. Viral tethering by Tetherin can be disrupted by the interaction with Vpu in HIV-1 in addition to other viral proteins. The structural mechanism of Tetherin function is not clear and the effects of human Tetherin mutations identified by sequencing consortiums are not known. To address this gap in the knowledge, we used data from the Ensembl database to construct and model known human missense mutations within the ectodomain to investigate how the structure of the ectodomain influences function. From the data, we identified an island of sequence stability within the ectodomain, which corresponds to functionally or structurally important region identified in previous biochemical and biophysical studies. Additionally, most mutations have little effect on structure, suggesting that they would not affect function. These findings are in agreement with biochemical and cellular studies which suggest that mutations that do not disrupt the alpha helices of Tetherin have little apparent effect on function. Thus, Tetherin sequence is likely less important than structure and this apparent flexibility may allow for greater anti-viral activities with a larger number of viruses.

biochemistry↗

bioRxiv2017

Modeling of Epigenetic Modification-Induced Changes in CRX-dependent Genes cis-Regulatory Elements

PurposeDNA methylation is a well characterized epigenetic repressor of mRNA transcription in many plant and vertebrate systems. However, the mechanism of this repression is not fully understood. The process of synthesizing a strand of RNA from DNA, or transcription, is controlled by proteins that regulate RNA polymerase activity by binding to specific gene regulatory sequences. Cone-rod homeobox (CRX) is a well-characterized mammalian transcription factor that controls photoreceptor cell specific gene expression. While much is known about the functions and DNA binding specificity of CRX, less is known about how DNA methylation modulates CRX binding affinity to genomic cis-regulatory elements.\n\nMethodsWe used bisulfite pyrosequencing of human ocular tissues to measure DNA methylation levels of the regulatory regions of RHO, PDE6B, PAX6, and LINE. To describe the molecular mechanism of repression, we used molecular modeling to illustrate the effect of DNA methylation on human RHO regulatory sequences.\n\nResultsIn this study, we demonstrate an inverse correlation between DNA methylation in regulatory regions adjacent to the human RHO and PDE6B genes and their subsequent transcription in human ocular tissues. Docking of CRX to our DNA models shows that CRX interacts with the grooves of these sequences, suggesting changes in groove structure could regulate binding. Molecular dynamics simulations of the RHO promoter and enhancer regions show changes in the flexibility and groove width upon epigenetic modification. Models also demonstrate that changes to the local dynamics of CRX binding sites within RHO regulatory sequences which may account for the repression of CRX dependent transcription.\n\nConclusionCollectively, these data demonstrate epigenetic regulation of CRX binding sites in human retinal tissue and provide insight into the mechanism of this mode of epigenetic regulation to be tested in future experiments.

molecular biology↗

bioRxiv2017

Conformational locking of Ufm1 upon binding to the Ufm1-interacting sequence of Uba5

Ubiquitin fold modifier 1 (Ufm1) is a ubiquitin-like protein (UBL) found in eukaryotic organisms which plays a crucial role in ER stress management and signal transduction. The crystal structure of UFM1 and its E1 (Uba5) in complex shows that Ufm1 binds to the adenylation domain of UBA5 and interacts with a separate Ufm1-interacting sequence (UIS) in the C-terminus of UBA5. The UIS interacts with Ufm1 on the opposite side of Ufm1 protein from the adenylation domain of Uba5 and the reason for this second interaction site is unclear. We analyzed Ufm1 bound to the UIS sequence through molecular dynamics simulations in order to identify additional functions for this interaction. We found that the residues in the adenylation interaction site of Ufm1 have less movement when the UIS peptide was bound to Ufm1 and formed a structure that aligns well with Ufm1 bound to the Uba5 adenylation domain. We further identified an amino acid that connects the UIS to the adenylation domain interacting site. Mutation of this amino acid decreases charging activity and shifts the Ufm1 conformation population toward the unlocked configuration even in the presence of the UIS peptide. These data suggest a role for the Uba5 UIS in stimulating activation of Ufm1.

biochemistry↗