Structure Function Studies of the Phenylalanine Hydroxylase Active Site and a Summary of Structural Features

Immunoglobulin A (IgA) plays a key role in defending mucosal surfaces against attack by infectious microorganisms. Such sites present a major site of susceptibility due to their vast surface area and their constant exposure to ingested and inhaled material. The importance of IgA to effective immune defence is signalled by the fact that more IgA is produced than all the other immunoglobulin classes combined. Indeed, IgA is not just the most prevalent antibody class at mucosal sites, but is also present at significant concentrations in serum. The unique structural features of the IgA heavy chain allow IgA to polymerise, resulting in mainly dimeric forms, along with some higher polymers, in secretions. Both serum IgA, which is principally monomeric, and secretory forms of IgA are capable of neutralising and removing pathogens through a range of mechanisms, including triggering the IgA Fc receptor known as FcαRI or CD89 on phagocytes. The effectiveness of these elimination processes is highlighted by the fact that various pathogens have evolved mechanisms to thwart such IgA-mediated clearance. As the structure–function relationships governing the varied capabilities of this immunoglobulin class come into increasingly clear focus, and means to circumvent any inherent limitations are developed, IgA-based monoclonal antibodies are set to emerge as new and potent options in the therapeutic arena.

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Structural insights into dihydroxylation of terephthalate, a product of polyethylene terephthalate degradation

Journal of Bacteriology ◽

10.1128/jb.00543-21 ◽

2022 ◽

Author(s):

Jai Krishna Mahto ◽

Neetu Neetu ◽

Monica Sharma ◽

Monika Dubey ◽

Bhanu Prakash Vellanki ◽

...

Keyword(s):

Crystal Structure ◽

Substrate Specificity ◽

Polyethylene Terephthalate ◽

Active Site ◽

Catalytic Domain ◽

Structural Features ◽

Comamonas Testosteroni ◽

Plastic Pollution ◽

Microbial Utilization ◽

Steady State Kinetics

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

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Recent insights into lytic polysaccharide monooxygenases (LPMOs)

Biochemical Society Transactions ◽

10.1042/bst20170549 ◽

2018 ◽

Vol 46 (6) ◽

pp. 1431-1447 ◽

Cited By ~ 39

Author(s):

Tobias Tandrup ◽

Kristian E. H. Frandsen ◽

Katja S. Johansen ◽

Jean-Guy Berrin ◽

Leila Lo Leggio

Keyword(s):

Active Site ◽

Room Temperature ◽

Experimental Studies ◽

Binding Mode ◽

Structural Features ◽

Oxygen Species ◽

Animal Kingdom ◽

X Ray ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered within the last 10 years. By degrading recalcitrant substrates oxidatively, these enzymes are major contributors to the recycling of carbon in nature and are being used in the biorefinery industry. Recently, two new families of LPMOs have been defined and structurally characterized, AA14 and AA15, sharing many of previously found structural features. However, unlike most LPMOs to date, AA14 degrades xylan in the context of complex substrates, while AA15 is particularly interesting because they expand the presence of LPMOs from the predominantly microbial to the animal kingdom. The first two neutron crystallography structures have been determined, which, together with high-resolution room temperature X-ray structures, have putatively identified oxygen species at or near the active site of LPMOs. Many recent computational and experimental studies have also investigated the mechanism of action and substrate-binding mode of LPMOs. Perhaps, the most significant recent advance is the increasing structural and biochemical evidence, suggesting that LPMOs follow different mechanistic pathways with different substrates, co-substrates and reductants, by behaving as monooxygenases or peroxygenases with molecular oxygen or hydrogen peroxide as a co-substrate, respectively.

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Electronic and geometric structure-function correlations of the coupled binuclear copper active site

Pure and Applied Chemistry ◽

10.1351/pac198355071069 ◽

1983 ◽

Vol 55 (7) ◽

pp. 1069-1088 ◽

Cited By ~ 33

Author(s):

E. I. Solomon

Keyword(s):

Structure Function ◽

Geometric Structure ◽

Active Site ◽

Binuclear Copper

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Complexity and Dynamics of Semi-Arid Vegetation Structure, Function and Diversity Across Spatial Scales from Full Waveform Lidar

10.18122/td/1658/boisestate ◽

2020 ◽

Author(s):

Ginikanda Yapa Mudiyanselage Nayani Thanuja Ilangakoon

Keyword(s):

Structure Function ◽

Functional Diversity ◽

Vegetation Structure ◽

Structural Features ◽

Airborne Lidar ◽

Arid Ecosystems ◽

Full Waveform ◽

Waveform Lidar ◽

Full Waveform Lidar ◽

Semi Arid

Semi-arid ecosystems cover approximately 40% of the earth's terrestrial landscape and show high dynamicity in ecosystem structure and function. These ecosystems play a critical role in global carbon dynamics, productivity, and habitat quality. Semi-arid ecosystems experience a high degree of disturbance that can severely alter ecosystem services and processes. Understanding the structure-function relationships across spatial extents are critical in order to assess their demography, response to disturbance, and for conservation management. In this research, using state-of-the-art full waveform lidar (airborne and spaceborne) and field observations, I developed a framework to assess the complexity and dynamics of vegetation structure, function and diversity across spatial scales in a semi-arid ecosystem. Difficulty in differentiating low stature vegetation from bare ground is the key remote sensing challenge in semi-arid ecosystems. In this study, I developed a workflow to differentiate key plant functional types (PFTs) using both structural and biophysical variables derived from the full waveform lidar and an ensemble random forest technique. The results revealed that waveform lidar pulse width can clearly distinguish shrubs from bare ground. The models showed PFT classification accuracy of 0.81-0.86% and 0.60-0.70% at 10 m and 1 m spatial resolutions, respectively. I found that structural variables were more important than the biophysical variables to differentiate the PFTs in this study area. The study further revealed an overlap between the structural features of different PFTs (e.g. shrubs from trees). Using structural features, I derived three main functional traits (canopy height, plant area index and foliage height diversity) of shrubs and trees that describe canopy architecture and light use efficiency of the ecosystem. I evaluated the trends and patterns of functional diversity and their relationship with non-climatic abiotic factors and fire disturbance. In addition to the fine resolution airborne lidar, I used simulated large footprint spaceborne lidar representing the newly launched Global Ecosystem Dynamics Investigation system (GEDI, a lidar sensor on the International Space Station) to evaluate the potential of capturing functional diversity trends of semi-arid ecosystems at global scales. The consistency of diversity trends between the airborne lidar and GEDI confirmed GEDI's potential to capture functional diversity. I found that the functional diversity in this ecosystem is mainly governed by the local elevation gradient, soil type, and slope. All three functional diversity indices (functional richness, functional evenness and functional divergence) showed a diversity breakpoint near elevations of 1500 m - 1700 m. Functional diversity of fire-disturbed areas revealed that the fires in our study area resulted in a more even and less divergent ecosystem state. Finally, I quantified aboveground biomass using the structural features derived from both the airborne lidar and GEDI data. Regional estimates of biomass can indicate whether an ecosystem is a net carbon sink or source as well as the ecosystem's health (e.g. biodiversity). Further, the potential of large footprint lidar data to estimate biomass in semi-arid ecosystems are not yet fully explored due to the inherent overlapping vegetation responses in the ground signals that can be affected by the ground slope. With a correction to the slope effect, I found that large footprint lidar can explain 42% of variance of biomass with a RMSE of 351 kg/ha (16% RMSE). The model estimated 82% of the study area with less than 50% uncertainty in biomass estimates. The cultivated areas and the areas with high functional richness showed the highest uncertainties. Overall, this dissertation establishes a novel framework to assess the complexity and dynamics of vegetation structure and function of a semi-arid ecosystem from space. This work enhances our understanding of the present state of an ecosystem and provides a foundation for using full waveform lidar to understand the impact of these changes to ecosystem productivity, biodiversity and habitat quality in the coming decades. The methods and algorithms in this dissertation can be directly applied to similar ecosystems with relevant corrections for the appropriate sensor. In addition, this study provides insights to related NASA missions such as ICESat-2 and future NASA missions such as NISAR for deriving vegetation structure and dynamics related to disturbance.

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Mitochondrial ClpX activates an essential biosynthetic enzyme through partial unfolding

eLife ◽

10.7554/elife.54387 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 3

Author(s):

Julia R Kardon ◽

Jamie A Moroco ◽

John R Engen ◽

Tania A Baker

Keyword(s):

Active Site ◽

Autonomous System ◽

Aminolevulinic Acid ◽

Structural Features ◽

Biosynthetic Enzyme ◽

Cofactor Binding ◽

Partial Unfolding ◽

Chaperone Interactions ◽

Aaa Protein ◽

Insight Into

Mitochondria control the activity, quality, and lifetime of their proteins with an autonomous system of chaperones, but the signals that direct substrate-chaperone interactions and outcomes are poorly understood. We previously discovered that the mitochondrial AAA+ protein unfoldase ClpX (mtClpX) activates the initiating enzyme for heme biosynthesis, 5-aminolevulinic acid synthase (ALAS), by promoting cofactor incorporation. Here, we ask how mtClpX accomplishes this activation. Using S. cerevisiae proteins, we identified sequence and structural features within ALAS that position mtClpX and provide it with a grip for acting on ALAS. Observation of ALAS undergoing remodeling by mtClpX revealed that unfolding is limited to a region extending from the mtClpX-binding site to the active site. Unfolding along this path is required for mtClpX to gate cofactor binding to ALAS. This targeted unfolding contrasts with the global unfolding canonically executed by ClpX homologs and provides insight into how substrate-chaperone interactions direct the outcome of remodeling.

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Structural Features of a Bacteroidetes-Affiliated Cellulase Linked with a Polysaccharide Utilization Locus

Scientific Reports ◽

10.1038/srep11666 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 14

Author(s):

A.E. Naas ◽

A.K. MacKenzie ◽

B. Dalhus ◽

V.G.H. Eijsink ◽

P.B. Pope

Keyword(s):

Active Site ◽

Three Dimensional ◽

Structural Features ◽

Structure And Function ◽

Dimensional Structure ◽

Active Site Cleft ◽

Polysaccharide Utilization Locus ◽

And Function ◽

Rumen Metagenome

Abstract Previous gene-centric analysis of a cow rumen metagenome revealed the first potentially cellulolytic polysaccharide utilization locus, of which the main catalytic enzyme (AC2aCel5A) was identified as a glycoside hydrolase (GH) family 5 endo-cellulase. Here we present the 1.8 Å three-dimensional structure of AC2aCel5A and characterization of its enzymatic activities. The enzyme possesses the archetypical (β/α)8-barrel found throughout the GH5 family and contains the two strictly conserved catalytic glutamates located at the C-terminal ends of β-strands 4 and 7. The enzyme is active on insoluble cellulose and acts exclusively on linear β-(1,4)-linked glucans. Co-crystallization of a catalytically inactive mutant with substrate yielded a 2.4 Å structure showing cellotriose bound in the −3 to −1 subsites. Additional electron density was observed between Trp178 and Trp254, two residues that form a hydrophobic “clamp”, potentially interacting with sugars at the +1 and +2 subsites. The enzyme’s active-site cleft was narrower compared to the closest structural relatives, which in contrast to AC2aCel5A, are also active on xylans, mannans and/or xyloglucans. Interestingly, the structure and function of this enzyme seem adapted to less-substituted substrates such as cellulose, presumably due to the insufficient space to accommodate the side-chains of branched glucans in the active-site cleft.

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Mouse Gut Microbiome-Encoded β-Glucuronidases Identified Using Metagenome Analysis Guided by Protein Structure

mSystems ◽

10.1128/msystems.00452-19 ◽

2019 ◽

Vol 4 (4) ◽

Cited By ~ 5

Author(s):

Benjamin C. Creekmore ◽

Josh H. Gray ◽

William G. Walton ◽

Kristen A. Biernat ◽

Michael S. Little ◽

...

Keyword(s):

Protein Structure ◽

Active Site ◽

Human Microbiome ◽

Drug Efficacy ◽

Human Microbiome Project ◽

Structural Features ◽

Model Organisms ◽

Mouse Strains ◽

Sequencing Data ◽

Metagenome Analysis

ABSTRACT Gut microbial β-glucuronidase (GUS) enzymes play important roles in drug efficacy and toxicity, intestinal carcinogenesis, and mammalian-microbial symbiosis. Recently, the first catalog of human gut GUS proteins was provided for the Human Microbiome Project stool sample database and revealed 279 unique GUS enzymes organized into six categories based on active-site structural features. Because mice represent a model biomedical research organism, here we provide an analogous catalog of mouse intestinal microbial GUS proteins—a mouse gut GUSome. Using metagenome analysis guided by protein structure, we examined 2.5 million unique proteins from a comprehensive mouse gut metagenome created from several mouse strains, providers, housing conditions, and diets. We identified 444 unique GUS proteins and organized them into six categories based on active-site features, similarly to the human GUSome analysis. GUS enzymes were encoded by the major gut microbial phyla, including Firmicutes (60%) and Bacteroidetes (21%), and there were nearly 20% for which taxonomy could not be assigned. No differences in gut microbial gus gene composition were observed for mice based on sex. However, mice exhibited gus differences based on active-site features associated with provider, location, strain, and diet. Furthermore, diet yielded the largest differences in gus composition. Biochemical analysis of two low-fat-associated GUS enzymes revealed that they are variable with respect to their efficacy of processing both sulfated and nonsulfated heparan nonasaccharides containing terminal glucuronides. IMPORTANCE Mice are commonly employed as model organisms of mammalian disease; as such, our understanding of the compositions of their gut microbiomes is critical to appreciating how the mouse and human gastrointestinal tracts mirror one another. GUS enzymes, with importance in normal physiology and disease, are an attractive set of proteins to use for such analyses. Here we show that while the specific GUS enzymes differ at the sequence level, a core GUSome functionality appears conserved between mouse and human gastrointestinal bacteria. Mouse strain, provider, housing location, and diet exhibit distinct GUSomes and gus gene compositions, but sex seems not to affect the GUSome. These data provide a basis for understanding the gut microbial GUS enzymes present in commonly used laboratory mice. Further, they demonstrate the utility of metagenome analysis guided by protein structure to provide specific sets of functionally related proteins from whole-genome metagenome sequencing data.

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Structure-Function Relationships in [FeFe]-Hydrogenase Active Site Maturation

Journal of Biological Chemistry ◽

10.1074/jbc.r111.310797 ◽

2012 ◽

Vol 287 (17) ◽

pp. 13532-13540 ◽

Cited By ~ 61

Author(s):

Yvain Nicolet ◽

Juan C. Fontecilla-Camps

Keyword(s):

Structure Function ◽

Active Site

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