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At least 523 records · Page 29
All sourcesNASA NTRSDOE OSTI
DOE OSTI2015

Structural Characterization of a Newly Identified Component of α-Carboxysomes: The AAA+ Domain Protein CsoCbbQ

Carboxysomes are bacterial microcompartments that enhance carbon fixation by concentrating ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its substrate CO 2 within a proteinaceous shell. They are found in all cyanobacteria, some purple photoautotrophs and many chemoautotrophic bacteria. Carboxysomes consist of a protein shell that encapsulates several hundred molecules of RuBisCO, and contain carbonic anhydrase and other accessory proteins. Genes coding for carboxysome shell components and the encapsulated proteins are typically found together in an operon. The α-carboxysome operon is embedded in a cluster of additional, conserved genes that are presumably related to its function. In many chemoautotrophs, products of the expanded carboxysome locus include CbbO and CbbQ, a member of the AAA+ domain superfamily. We bioinformatically identified subtypes of CbbQ proteins and show that their genes frequently co-occur with both Form IA and Form II RuBisCO. The α-carboxysome-associated ortholog, CsoCbbQ, from Halothiobacillus neapolitanus forms a hexamer in solution and hydrolyzes ATP. The crystal structure shows that CsoCbbQ is a hexamer of the typical AAA+ domain; the additional C-terminal domain, diagnostic of the CbbQ subfamily, structurally fills the inter-monomer gaps, resulting in a distinctly hexagonal shape. Finally, we show that CsoCbbQ interacts with CsoCbbO and is a component of the carboxysome shell, the first example of ATPase activity associated with a bacterial microcompartment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families

Metagenomics has made accessible an enormous reserve of global biochemical diversity. In order to tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. Here, we validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Metallomics of two microorganisms relevant to heavy metal bioremediation reveal fundamental differences in metal assimilation and utilization

Although as many as half of all proteins are thought to require a metal cofactor, the metalloproteomes of microorganisms remain relatively unexplored. Microorganisms from different environments are likely to vary greatly in the metals that they assimilate, not just among the metals with well-characterized roles but also those lacking any known function. Herein we investigated the metal utilization of two microorganisms that were isolated from very similar environments and are of interest because of potential roles in the immobilization of heavy metals, such as uranium and chromium. The metals assimilated and their concentrations in the cytoplasm of Desulfovibrio vulgaris strain Hildenborough (DvH) and Enterobacter cloacae strain Hanford (EcH) varied dramatically, with a larger number of metals present in Enterobacter. For example, a total of 9 and 19 metals were assimilated into their cytoplasmic fractions, respectively, and DvH did not assimilate significant amounts of zinc or copper whereas EcH assimilated both. However, bioinformatic analysis of their genome sequences revealed a comparable number of predicted metalloproteins, 813 in DvH and 953 in EcH. These allowed some rationalization of the types of metal assimilated in some cases (Fe, Cu, Mo, W, V) but not in others (Zn, Nd, Ce, Pr, Dy, Hf and Th). It was also shown that U binds an unknown soluble protein in EcH but this incorporation was the result of extracellular U binding to cytoplasmic components after cell lysis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

The influence of protein electrostatics on potential inversion in flavoproteins

Biology uses relatively few electron-transfer cofactors, tuning their potentials, electronic couplings, and reorganization energies to carry out the required chemistry. It is remarkable that the potential ordering of two-electron transfer active flavins can be normal (first oxidation at low potential and second oxidation at high potential) or inverted, and the gap between the potentials can be as large as one volt. Analysis based on structural bioinformatics and electrostatics indicates that the ordering of the flavin redox potential is influenced by protein electrostatics. In all 36 flavoproteins examined, the introduction of a negative charge near the flavin in silico increases the extent of potential inversion (by lowering the electrochemical potential of the second electron-transfer step); the introduction of a positive charge near the flavin favors normally ordered potentials. We also find that the addition of positive charges increases the electrochemical potential for the naturally occurring one-electron transition in flavodoxins (between deprotonated hydroquinone and neutral semiquinone) and also increases the second one-electron transition in bifurcating flavins (between anionic semiquinone and fully oxidized flavin). Finally, we find that proximity of a proton acceptor, notably conserved arginine, supports proton-coupled electron transfer because it may act as a proton acceptor, promoting potential inversion. This key arginine residue may enable two-electron transfer chemistry by promoting the proton-coupled electron transfer process over the pure electron transfer process, suggesting how a protein's flavin environment may influence one- or two-electron chemistry in flavoproteins.

Singh, Niven [Duke Univ., Durham, NC (United State↗
DOE OSTI2016

Methanobactin transport machinery

Methanotrophic bacteria use methane, a potent greenhouse gas, as their primary source of carbon and energy. The first step in methane metabolism is its oxidation to methanol. In almost all methanotrophs, this chemically challenging reaction is catalyzed by particulate methane monooxygenase (pMMO), a copper-dependent integral membrane enzyme. Methanotrophs acquire copper (Cu) for pMMO by secreting a small ribosomally produced, posttranslationally modified natural product called methanobactin (Mbn). Mbn chelates Cu with high affinity, and the Cu-loaded form (CuMbn) is reinternalized into the cell via an active transport process. Bioinformatic and gene regulation studies suggest that two proteins might play a role in CuMbn handling: the TonB-dependent transporter MbnT and the periplasmic binding protein MbnE. Disruption of the gene that encodes MbnT abolishes CuMbn uptake, as reported previously, and expression of MbnT in Escherichia coli confers the ability to take up CuMbn. Biophysical studies of MbnT and MbnE reveal specific interactions with CuMbn, and a crystal structure of apo MbnE is consistent with MbnE's proposed role as a periplasmic CuMbn transporter. Notably, MbnT and MbnE exhibit different levels of discrimination between cognate and noncognate CuMbns. Furthermore, these findings provide evidence for CuMbn–protein interactions and begin to elucidate the molecular mechanisms of its recognition and transport.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The SrrAB two-component system regulates Staphylococcus aureus pathogenicity through redox sensitive cysteines

Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. In this work, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A majority of Rhodobacter sphaeroides promoters lack a crucial RNA polymerase recognition feature, enabling coordinated transcription activation

Using an in vitro transcription system with purified RNA polymerase (RNAP) to investigate rRNA synthesis in the photoheterotrophic α-proteobacterium Rhodobacter sphaeroides, we identified a surprising feature of promoters recognized by the major holoenzyme. Transcription from R. sphaeroides rRNA promoters was unexpectedly weak, correlating with absence of 7T, the very highly conserved thymine found at the last position in –10 elements of promoters in most bacterial species. Thymine substitutions for adenine at position –7 in the three rRNA promoters strongly increased intrinsic promoter activity, indicating that R. sphaeroides RNAP can utilize –7T when present. rRNA promoters were activated by purified R. sphaeroides CarD, a transcription factor found in many bacterial species but not in β- and γ-proteobacteria. Overall, CarD increased the activity of 15 of 16 native R. sphaeroides promoters tested in vitro that lacked –7T, whereas it had no effect on three of the four native promoters that contained –7T. Genome-wide bioinformatic analysis of promoters from R. sphaeroides and two other α-proteobacterial species indicated that 30 to 43% contained –7T, whereas 90 to 99% of promoters from non–α-proteobacteria contained –7T. Thus, promoters lacking –7T appear to be widespread in α-proteobacteria and may have evolved away from consensus to enable their coordinated regulation by transcription factors like CarD. We observed a strong reduction in R. sphaeroides CarD levels when cells enter stationary phase, suggesting that reduced activation by CarD may contribute to inhibition of rRNA transcription when cells enter stationary phase, the stage of growth when bacterial ribosome synthesis declines.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Discovery of cyanophycin dipeptide hydrolase enzymes suggests widespread utility of the natural biopolymer cyanophycin

Cyanophycin is a bacterial polymer mainly used for nitrogen storage. It is composed of a peptide backbone of L-aspartate residues with L-arginines attached to their side chains through isopeptide bonds. Cyanophycin is degraded in two steps: Cyanophycinase cleaves the polymer into β-Asp-Arg dipeptides, which are hydrolyzed into free Asp and Arg by enzymes possessing isoaspartyl dipeptide hydrolase activity. Two unrelated enzymes with this activity, isoaspartyl dipeptidase (IadA) and isoaspartyl aminopeptidase (IaaA) have been shown to degrade β-Asp-Arg dipeptides, but bacteria which encode cyanophycin-metabolizing genes can lack iaaA and iadA genes. In this study, we investigate a previously uncharacterized enzyme whose gene can cluster with cyanophycin-metabolizing genes. This enzyme, which we name cyanophycin dipeptide hydrolase (CphZ), is specific for dipeptides derived from cyanophycin degradation. Accordingly, a co-complex structure of CphZ and β-Asp-Arg shows that CphZ, unlike IadA or IaaA, recognizes all portions of its β-Asp-Arg substrate. Bioinformatic analyses showed that CphZ is found in very many proteobacteria and is homologous to an uncharacterized protein encoded in the “arginine/ornithine transport” (aot) operon of many pseudomonas species, including Pseudomonas aeruginosa . In vitro assays show that AotO is indeed a CphZ, and in cellulo growth experiments show that this enzyme and the aot operon allow P. aeruginosa to take up and use β-Asp-Arg as a sole carbon and nitrogen source. Together the results establish the novel, highly specific enzyme subfamily of CphZs, suggesting that cyanophycin is potentially used by a much wider range of bacteria than previously appreciated.

Science & Technology - Other Topics↗
DOE OSTI2024

Electron transfer in polysaccharide monooxygenase catalysis

Polysaccharide monooxygenase (PMO) catalysis involves the chemically difficult hydroxylation of unactivated C–H bonds in carbohydrates. The reaction requires reducing equivalents and will utilize either oxygen or hydrogen peroxide as a cosubstrate. Two key mechanistic questions are addressed here: 1) How does the enzyme regulate the timely and tightly controlled electron delivery to the mononuclear copper active site, especially when bound substrate occludes the active site? and 2) How does this electron delivery differ when utilizing oxygen or hydrogen peroxide as a cosubstrate? Using a computational approach, potential paths of electron transfer (ET) to the active site copper ion were identified in a representative AA9 family PMO from Myceliophthora thermophila ( Mt PMO9E). When Y62, a buried residue 12 Å from the active site, is mutated to F, lower activity is observed with O 2 . However, a WT-level activity is observed with H 2 O 2 as a cosubstrate indicating an important role in ET for O 2 activation. To better understand the structural effects of mutations to Y62 and axial copper ligand Y168, crystal structures were solved of the wild type Mt PMO9E and the variants Y62W, Y62F, and Y168F. A bioinformatic analysis revealed that position 62 is conserved as either Y or W in the AA9 family. The Mt PMO9E Y62W variant has restored activity with O 2 . Overall, the use of redox-active residues to supply electrons for the reaction with O 2 appears to be widespread in the AA9 family. Furthermore, the results provide a molecular framework to understand catalysis with O 2 versus H 2 O 2 .

Sayler, Richard I. (ORCID:000000017252707X)↗
DOE OSTI2024

Electrochemical cofactor recycling of bacterial microcompartments

Bacterial microcompartments (BMCs) are prokaryotic organelles that consist of a protein shell which sequesters metabolic reactions in its interior. While most of the substrates and products are relatively small and can permeate the shell, many of the encapsulated enzymes require cofactors that must be regenerated inside. We have analyzed the occurrence of an enzyme previously assigned as a cobalamin (vitamin B 12 ) reductase and, curiously, found it in many unrelated BMC types that do not employ B 12 cofactors. We propose Nicotinamide adenine dinucleotide (NAD+) regeneration as the function of this enzyme and name it Metabolosome Nicotinamide Adenine Dinucleotide Hydrogen (NADH) dehydrogenase (MNdh). Its partner shell protein BMC-T SE (tandem domain BMC shell protein of the single layer type for electron transfer) assists in passing the generated electrons to the outside. We support this hypothesis with bioinformatic analysis, functional assays, Electron Paramagnetic Resonance spectroscopy, protein voltammetry, and structural modeling verified with X-ray footprinting. This finding represents a paradigm for the BMC field, identifying a new, widely occurring route for cofactor recycling and a new function for the shell as separating redox environments.

bacterial microcompartment↗
DOE OSTI2025

Leaky ribosomal scanning enables tunable translation of bicistronic ORFs in green algae

Advances in sequencing technology have unveiled examples of nucleus-encoded polycistrons, once considered rare. Exclusively polycistronic transcripts are prevalent in green algae, although the mechanism by which multiple polypeptides are translated from a single transcript is unknown. Here, we used bioinformatic and in vivo mutational analyses to evaluate competing mechanistic models for translation of bicistronic mRNAs in green algae. High-confidence manually curated datasets of bicistronic loci from two divergent green algae, Chlamydomonas reinhardtii and Auxenochlorella protothecoides, revealed a preference for weak Kozak-like sequences for ORF 1 and an underrepresentation of potential initiation codons before the ORF 2 start codon, which are suitable conditions for leaky ribosome scanning to allow ORF 2 translation. We used mutational analysis in A. protothecoides to test the mechanism. In vivo manipulation of the ORF 1 Kozak-like sequence and start codon altered reporter expression at ORF 2, with a weaker Kozak-like sequence enhancing expression and a stronger one diminishing it. A synthetic bicistronic dual reporter demonstrated inversely adjustable activity of green fluorescent protein expressed from ORF 1 and luciferase from ORF 2, depending on the strength of the ORF 1 Kozak-like sequence. Our findings demonstrate that translation of multiple ORFs in green algal bicistronic transcripts is consistent with episodic leaky scanning of ORF 1 to allow translation at ORF 2. This work has implications for the potential functionality of upstream open reading frames (uORFs) found across eukaryotic genomes and for transgene expression in synthetic biology applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Pyrodictium abyssi AbpX reveals a calcium-responsive family of microbial biomatrix proteins that form thermostable hydrogels

Evolutionary pressure on microbial communities propagating under extreme environmental conditions often results in unique structural adaptations to promote cell survival. In this work, we report an investigation of AbpX, a biomatrix protein identified in cultures of the hyperthermophilic archaeon Pyrodictium abyssi. Under ex vivo and in vitro conditions, AbpX assembles into a paracrystalline lattice composed of semiflexible fibrils. CryoEM analysis of recombinant AbpX fibrils reveals that the precursor protein polymerizes through donor strand complementation (DSC), a process previously reported for chaperone-usher fimbriae in Gram-negative bacteria. Unlike the latter DSC protein polymers, AbpX undergoes chaperone-free polymerization in the presence of calcium ions, which are sequestered at the donor strand-acceptor groove interface between protomers in the fibril. Using a combination of cryoEM and crystallographic information, a structural model is proposed for the AbpX lattice that provides insight into its potential role in biofilm formation. These findings suggest that calcium ion coordination may contribute to fibril assembly and preorganize fibrils for incorporation into the protein lattice. Bioinformatic analysis indicates that AbpX exemplifies a distinct and broadly distributed clade of calcium ion responsive biomatrix proteins within the TasA superfamily that can be fabricated into hydrogel biomaterials in vitro under environmentally benign conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Discrete and Structurally Unique Proteins (T$\bar{a}$pirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose

A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins (“t$\bar{a}$pirins,” origin from M$\bar{a}$ori: to join), not directly associated with cellulases, that mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two t$\bar{a}$pirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, t$\bar{a}$pirins are specific to these extreme thermophiles. T$\bar{a}$pirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the t$\bar{a}$pirins for cellulose. Crystallization of a cellulose-binding truncation from one t$\bar{a}$pirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face. In addition, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ t$\bar{a}$pirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Divergent Evolution of Nuclear Localization Signal Sequences in Herpesvirus Terminase Subunits

The tripartite terminase complex of herpesviruses assembles in the cytoplasm of infected cells and exploits the host nuclear import machinery to gain access to the nucleus, where capsid assembly and genome-packaging occur. Here we analyzed the structure and conservation of nuclear localization signal (NLS) sequences previously identified in herpes simplex virus 1 (HSV-1) large terminase and human cytomegalovirus (HCMV) small terminase. We found a monopartite NLS at the N terminus of large terminase, flanking the ATPase domain, that is conserved only in α-herpesviruses. In contrast, small terminase exposes a classical NLS at the far C terminus of its helical structure that is conserved only in two genera of the β-subfamily and absent in α- and γ-herpesviruses. In addition, we predicted a classical NLS in the third terminase subunit that is partially conserved among herpesviruses. Bioinformatic analysis revealed that both location and potency of NLSs in terminase subunits evolved more rapidly than the rest of the amino acid sequence despite the selective pressure to keep terminase gene products active and localized in the nucleus. Here, we propose that swapping NLSs among terminase subunits is a regulatory mechanism that allows different herpesviruses to regulate the kinetics of terminase nuclear import, reflecting a mechanism of virus:host adaptation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase–rhodanese fusion protein functions in sulfur assimilation

Hydrogen sulfide (H 2 S) is a signaling molecule that is toxic at elevated concentrations. In eukaryotes, it is cleared via a mitochondrial sulfide oxidation pathway, which comprises sulfide quinone oxidoreductase, persulfide dioxygenase (PDO), rhodanese, and sulfite oxidase and converts H 2 S to thiosulfate and sulfate. Natural fusions between the non-heme iron containing PDO and rhodanese, a thiol sulfurtransferase, exist in some bacteria. However, little is known about the role of the PDO–rhodanese fusion (PRF) proteins in sulfur metabolism. Herein, we report the kinetic properties and the crystal structure of a PRF from the Gram-negative endophytic bacterium Burkholderia phytofirmans. The crystal structures of wild-type PRF and a sulfurtransferase-inactivated C314S mutant with and without glutathione were determined at 1.8, 2.4, and 2.7 Å resolution, respectively. We found that the two active sites are distant and do not show evidence of direct communication. The B. phytofirmans PRF exhibited robust PDO activity and preferentially catalyzed sulfur transfer in the direction of thiosulfate to sulfite and glutathione persulfide; sulfur transfer in the reverse direction was detectable only under limited turnover conditions. Together with the kinetic data, our bioinformatics analysis reveals that B. phytofirmans PRF is poised to metabolize thiosulfate to sulfite in a sulfur assimilation pathway rather than in sulfide stress response as seen, for example, with the Staphylococcus aureus PRF or sulfide oxidation and disposal as observed with the homologous mammalian proteins.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2019

Insights into an unusual Auxiliary Activity 9 family member lacking the histidine brace motif of lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are redox-enzymes involved in biomass degradation. All characterized LPMOs possess an active site of two highly conserved histidine residues coordinating a copper ion (the histidine brace), which are essential for LPMO activity. However, some protein sequences that belong to the AA9 LPMO family display a natural N-terminal His to Arg substitution (Arg-AA9). These are found almost entirely in the phylogenetic fungal class Agaricomycetes, associated with wood decay, but no function has been demonstrated for any Arg-AA9. Through bioinformatics, transcriptomic, and proteomic analyses we present data, which suggest that Arg-AA9 proteins could have a hitherto unidentified role in fungal degradation of lignocellulosic biomass in conjunction with other secreted fungal enzymes. We present the first structure of an Arg-AA9, LsAA9B, a naturally occurring protein from Lentinus similis The LsAA9B structure reveals gross changes in the region equivalent to the canonical LPMO copper-binding site, whereas features implicated in carbohydrate binding in AA9 LPMOs have been maintained. We obtained a structure of LsAA9B with xylotetraose bound on the surface of the protein although with a considerably different binding mode compared with other AA9 complex structures. In addition, we have found indications of protein phosphorylation near the N-terminal Arg and the carbohydrate-binding site, for which the potential function is currently unknown. Finally, our results are strong evidence that Arg-AA9s function markedly different from canonical AA9 LPMO, but nonetheless, may play a role in fungal conversion of lignocellulosic biomass.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗