M. Sutter, Matthew R Melnicki, Frederik Schulz et al.

Nature Communications • 2021

Bacterial microcompartments (BMCs) are organelles that segregate segments of metabolic pathways which are incompatible with surrounding metabolism. BMCs consist of a selectively permeable shell, composed of three types of structurally conserved proteins, together with sequestered enzymes that vary among functionally distinct BMCs. Genes encoding shell proteins are typically clustered with those for the encapsulated enzymes. Here, we report that the number of identifiable BMC loci has increased twenty-fold since the last comprehensive census of 2014, and the number of distinct BMC types has doubled. The new BMC types expand the range of compartmentalized catalysis and suggest that there is more BMC biochemistry yet to be discovered. Our comprehensive catalog of BMCs provides a framework for their identification, correlation with bacterial niche adaptation, experimental characterization, and development of BMC-based nanoarchitectures for biomedical and bioengineering applications. Bacterial microcompartments (BMCs) are organelles consisting of a protein shell in which certain metabolic reactions take place separated from the cytoplasm. Here, Sutter et al. present a comprehensive catalog of BMC loci, substantially expanding the number of known BMCs and describing distinct types and compartmentalized reactions.

L. Hor, R. Dobson, M. Downton et al.

Journal of Biological Chemistry • 2013

Background: Diaminopimelate epimerase catalyzes a key step in the synthesis of meso-diaminopimelate and lysine. Results: Solution and crystal studies show that diaminopimelate epimerase exists as an active dimer, whereas a monomeric mutant is catalytically inactive. Conclusion: The diaminopimelate epimerase dimer is essential for function with evidence suggesting that dimerization attenuates subunit dynamics. Significance: Structural insights into the design of antimicrobial agents to disrupt diaminopimelate epimerase dimerization are provided. Diaminopimelate (DAP) epimerase is involved in the biosynthesis of meso-DAP and lysine, which are important precursors for the synthesis of peptidoglycan, housekeeping proteins, and virulence factors in bacteria. Accordingly, DAP epimerase is a promising antimicrobial target. Previous studies report that DAP epimerase exists as a monomeric enzyme. However, we show using analytical ultracentrifugation, X-ray crystallography, and enzyme kinetic analyses that DAP epimerase from Escherichia coli exists as a functional dimer in solution and the crystal state. Furthermore, the 2.0-Å X-ray crystal structure of the E. coli DAP epimerase dimer shows for the first time that the enzyme exists in an open, active conformation. The importance of dimerization was subsequently probed by using site-directed mutagenesis to generate a monomeric mutant (Y268A). Our studies show that Y268A is catalytically inactive, thus demonstrating that dimerization of DAP epimerase is essential for catalysis. Molecular dynamics simulations indicate that the DAP epimerase monomer is inherently more flexible than the dimer, suggesting that dimerization optimizes protein dynamics to support function. Our findings offer insight into the development of novel antimicrobial agents targeting the dimeric antibiotic target DAP epimerase.

Xia Hua, Jian Han, Xin Zhou et al.

AIChE Journal • 2022

Oxygen, as a terminal electron acceptor, is an essential substrate in the aerobic bio-oxidation process, affecting bacterial vitality and bio-oxidation performance. In this study, a new and smart platform biotechnology of sealed-oxygen supply bioreactor (SOS-BR) was developed by improving gas pressure to significantly intensify oxygen transfer rate and resolving the formidable barriers of aerobic catalysis. In virtue of SOS-BR, the bio-productivity was greatly improved for three representative substrates (xylose, furfural, glycerol) bio-oxidation with the whole-cell catalysis of Gluconobacter oxydans. The determination of oxygen transfer coefficient (KL α ) established an upgraded theoretical dynamic model for gas pressure intersification biosystem. Additionally, viscosity measurement and combined pressure control strategy explained the inflection point phenomenon of productivity and confirmed the intensify mechanism. The new strategy of significantly intensifying oxygen transfer provided insightful ideas for overcoming the subbon obstacle of obligate aerobic catalysis, and further promoted industrial practicability of bio-oxidation.

Ping-Yi Li, P. Ji, Chunyang Li et al.

Journal of Biological Chemistry • 2014

Background: Catalytic mechanisms of GTSAG motif subfamily enzymes of the bacterial hormone-sensitive lipases (HSLs) family are largely unknown. Results: E25, a GTSAG motif subfamily esterase, adopts a novel dimerization pattern. Dimerization keeps the catalytic Asp282 orientation for E25 catalysis. Conclusion: Dimerization and some catalytic profiles of E25 are distinctive from other HSLs. Significance: Our study sheds light on protein folding and evolution of HSLs. Hormone-sensitive lipases (HSLs) are widely distributed in microorganisms, plants, and animals. Microbial HSLs are classified into two subfamilies, an unnamed new subfamily and the GDSAG motif subfamily. Due to the lack of structural information, the detailed catalytic mechanism of the new subfamily is not yet clarified. Based on sequence analysis, we propose to name the new subfamily as the GTSAG motif subfamily. We identified a novel HSL esterase E25, a member of the GTSAG motif subfamily, by functional metagenomic screening, and resolved its structure at 2.05 Å. E25 is mesophilic (optimum temperature at 50 °C), salt-tolerant, slightly alkaline (optimum pH at 8.5) for its activity, and capable of hydrolyzing short chain monoesters (C2–C10). E25 tends to form dimers both in the crystal and in solution. An E25 monomer contains an N-terminal CAP domain, and a classical α/β hydrolase-fold domain. Residues Ser186, Asp282, and His312 comprise the catalytic triad. Structural and mutational analyses indicated that E25 adopts a dimerization pattern distinct from other HSLs. E25 dimer is mainly stabilized by an N-terminal loop intersection from the CAP domains and hydrogen bonds and salt bridges involving seven highly conserved hydrophilic residues from the catalytic domains. Further analysis indicated that E25 also has some catalytic profiles different from other HSLs. Dimerization is essential for E25 to exert its catalytic activity by keeping the accurate orientation of the catalytic Asp282 within the catalytic triad. Our results reveal the structural basis for dimerization and catalysis of an esterase from the GTSAG motif subfamily of the HSL family.

D. Parsonage, K. Nelson, G. Ferrer-Sueta et al.

Biochemistry • 2015

Peroxiredoxins make up a ubiquitous family of cysteine-dependent peroxidases that reduce hydroperoxide or peroxynitrite substrates through formation of a cysteine sulfenic acid (R-SOH) at the active site. In the 2-Cys peroxiredoxins, a second (resolving) cysteine reacts with the sulfenic acid to form a disulfide bond. For all peroxiredoxins, structural rearrangements in the vicinity of the active site cysteine(s) are necessary to allow disulfide bond formation and subsequent reductive recycling. In this study, we evaluated the rate constants for individual steps in the catalytic cycle of Salmonella typhimurium AhpC. Conserved Trp residues situated close to both peroxidatic and resolving cysteines in AhpC give rise to large changes in fluorescence during the catalytic cycle. For recycling, AhpF very efficiently reduces the AhpC disulfide, with a single discernible step and a rate constant of 2.3 × 10(7) M(-1) s(-1). Peroxide reduction was more complex and could be modeled as three steps, beginning with a reversible binding of H2O2 to the enzyme (k1 = 1.36 × 10(8) M(-1) s(-1), and k-1 = 53 s(-1)), followed by rapid sulfenic acid generation (620 s(-1)) and then rate-limiting disulfide bond formation (75 s(-1)). Using bulkier hydroperoxide substrates with higher Km values, we found that different efficiencies (kcat/Km) for turnover of AhpC with these substrates are primarily caused by their slower rates of binding. Our findings indicate that this bacterial peroxiredoxin exhibits rates for both reducing and oxidizing parts of the catalytic cycle that are among the fastest observed so far for this diverse family of enzymes.

Yonghai Feng, Yi Yu, Hui Shi et al.

Science Advances • 2024

The unique bacterial infection microenvironment (IME) usually requires complicated design of nanomaterials to adapt to IME for enhancing antibacterial therapy. Here, an alternative IME adaptative nitrite reductase–mimicking nanozyme is constructed by in situ growth of ultrasmall copper sulfide clusters on the surface of a nanofibrillar lysozyme assembly (NFLA/CuS NHs), which can temporally regulate nitric oxide (NO) gradient concentration to kill bacteria initially and promote tissue regeneration subsequently. Benefiting from a copper nitrite reductase (CuNIR)–inspired structure with CuS cluster as active center and NFLA as skeleton, NFLA/CuS NHs efficiently boost the catalytic reduction of nitrite to NO. The inherent supramolecular fibrillar networks displays excellent bacterial capture capability, facilitating initial high-concentration NO attacks on the bacteria. The subsequent catalytic release of low-concentration NO by NFLA/CuS NHs–mediated nitrite reduction remarkably promotes cell migration and angiogenesis. This work paves the way for dynamically eliminating MDR bacterial infection and promoting tissue regeneration in a simple and smart way through CuNIR-mimicking catalysis.

Martin G. Peverelli, T. P. Soares da Costa, N. Kirby et al.

Journal of Biological Chemistry • 2016

Diaminopimelate decarboxylase (DAPDC) catalyzes the final step in the diaminopimelate biosynthesis pathway of bacteria. The product of the reaction is the essential amino acid l-lysine, which is an important precursor for the synthesis of the peptidoglycan cell wall, housekeeping proteins, and virulence factors of bacteria. Accordingly, the enzyme is a promising antibacterial target. Previous structural studies demonstrate that DAPDC exists as monomers, dimers, and tetramers in the crystal state. However, the active oligomeric form has not yet been determined. We show using analytical ultracentrifugation, small angle x-ray scattering, and enzyme kinetic analyses in solution that the active form of DAPDC from Bacillus anthracis, Escherichia coli, Mycobacterium tuberculosis, and Vibrio cholerae is a dimer. The importance of dimerization was probed further by generating dimerization interface mutants (N381A and R385A) of V. cholerae DAPDC. Our studies indicate that N381A and R385A are significantly attenuated in catalytic activity, thus confirming that dimerization of DAPDC is essential for function. These findings provide scope for the development of new antibacterial agents that prevent DAPDC dimerization.

Blair Ney, Carlo R. Carere, R. Sparling et al.

Frontiers in Microbiology • 2017

F420 is a microbial cofactor that mediates a wide range of physiologically important and industrially relevant redox reactions, including in methanogenesis and tetracycline biosynthesis. This deazaflavin comprises a redox-active isoalloxazine headgroup conjugated to a lactyloligoglutamyl tail. Here we studied the catalytic significance of the oligoglutamate chain, which differs in length between bacteria and archaea. We purified short-chain F420 (two glutamates) from a methanogen isolate and long-chain F420 (five to eight glutamates) from a recombinant mycobacterium, confirming their different chain lengths by HPLC and LC/MS analysis. F420 purified from both sources was catalytically compatible with purified enzymes from the three major bacterial families of F420-dependent oxidoreductases. However, long-chain F420 bound to these enzymes with a six- to ten-fold higher affinity than short-chain F420. The cofactor side chain also significantly modulated the kinetics of the enzymes, with long-chain F420 increasing the substrate affinity (lower Km) but reducing the turnover rate (lower kcat) of the enzymes. Molecular dynamics simulations and comparative structural analysis suggest that the oligoglutamate chain of F420 makes dynamic electrostatic interactions with conserved surface residues of the oxidoreductases while the headgroup binds the catalytic site. In conjunction with the kinetic data, this suggests that electrostatic interactions made by the oligoglutamate tail result in higher-affinity, lower-turnover catalysis. Physiologically, we propose that bacteria have selected for long-chain F420 to better control cellular redox reactions despite tradeoffs in catalytic rate. Conversely, this suggests that industrial use of shorter-length F420 will greatly increase the rates of bioremediation and biocatalysis processes relying on purified F420-dependent oxidoreductases.

Lihua Wu, Yue Luo, Chaofeng Wang et al.

ACS Nano • 2023

In this work, a biomimetic nanozyme catalyst with rapid and efficient self-bacteria-killing and wound-healing performances was synthesized. Through an in situ reduction reaction, a PCN-222 metal organic framework (MOF) was doped with bismuth nanoparticles (Bi NPs) to form Bi-PCN-222, an interfacial Schottky heterojunction biomimetic nanozyme catalyst, which can kill 99.9% of Staphylococcus aureus (S. aureus). The underlying mechanism was that Bi NP doping can endow Bi-PCN-222 MOF with self-driven charge transfer through the Schottky interface and the capability of oxidase-like and peroxidase-like activity, because a large number of free electrons can be captured by surrounding oxygen species to produce radical oxygen species (ROS). Furthermore, once bacteria contact Bi-PCN-222 in a physiological environment, its appropriate redox potential can trigger electron transfer through the electron transport pathway in bacterial membranes and then the interior of the bacteria, which disturbs the bacterial respiration process and subsequent metabolism. Additionally, Bi-PCN-222 can also accelerate tissue regeneration by upregulating fibroblast proliferation and angiogenesis genes (bFGF, VEGF, and HIF-1α), thereby promoting wound healing. This biomimetic enzyme-catalyzed strategy will bring enlightenment to the design of self-bacterial agents for efficient disinfection and tissue reconstruction simultaneously.

Hacksung Kim, P. Pastén, J. Gaillard et al.

Journal of the American Chemical Society • 2003

We describe the characterization of an unknown and difficult to identify but geochemically and environmentally significant MnOx structure produced by a freshwater bacterium, Leptothrix discophora SP-6, using combined transmission electron microscopy (TEM), extended X-ray absorption fine structure (EXAFS), and UV Raman spectroscopy. The large surface-to-volume ratio of the needle-shaped nanocrystalline MnO2 formed around the bacterial cells coupled to the porous, zeolite-like structure has the potential to catalyze reactions and oxidize and adsorb metals.

V. Karuppiah, K. Ranaghan, Nicole G H Leferink et al.

ACS Catalysis • 2017

Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS), and we show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains, use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterized sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle, making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes toward alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids.

Yong Wang, Qi Zhan, Xinlu Wang et al.

Nature Communications • 2020

The bacterial effector MavC catalyzes non-canonical ubiquitination of host E2 enzyme UBE2N without engaging any of the conventional ubiquitination machinery, thereby abolishing UBE2N’s function in forming K63-linked ubiquitin (Ub) chains and dampening NF-кB signaling. We now report the structures of MavC in complex with conjugated UBE2N~Ub and an inhibitor protein Lpg2149, as well as the structure of its ortholog, MvcA, bound to Lpg2149. Recognition of UBE2N and Ub depends on several unique features of MavC, which explains the inability of MvcA to catalyze ubiquitination. Unexpectedly, MavC and MvcA also possess deubiquitinase activity against MavC-mediated ubiquitination, highlighting MavC as a unique enzyme possessing deamidation, ubiquitination, and deubiquitination activities. Further, Lpg2149 directly binds and inhibits both MavC and MvcA by disrupting the interactions between enzymes and Ub. These results provide detailed insights into catalysis and regulation of MavC-type enzymes and the molecular mechanisms of this non-canonical ubiquitination machinery. The bacterial effector MavC can ubiquitinate the host E2 enzyme UBE2N to dampen the host immune response. Here, the authors provide mechanistic insight into this non-canonical ubiquitination machinery and reveal the structural basis for the functional differences between MavC and its close homolog MvcA.

Jian Yang, Yuqun Xu, T. Miyakawa et al.

Applied and Environmental Microbiology • 2020

Heterotrophic bacterial communities are key players in marine biogeochemical cycling due to their ability to remineralize organic carbon. Processing of complex organic matter requires heterotrophic bacteria to produce extracellular enzymes with precise specificity to depolymerize substrates to sizes sufficiently small for uptake. Thus, extracellular enzymatic hydrolysis initiates microbe-driven heterotrophic carbon cycling. In this study, based on biochemical and structural analyses, we revealed the depolymerization mechanism of β-1,3-glucan, a carbon reserve in algae, by laminarinase from an alga-associated marine Flavobacterium. The findings provide new insights into the substrate recognition and catalysis of bacterial laminarinase and promote a better understanding of how extracellular enzymes are involved in organic matter cycling. ABSTRACT Laminarin is an abundant algal polysaccharide that serves as carbon storage and fuel to meet the nutrition demands of heterotrophic microbes. Laminarin depolymerization catalyzed by microbial extracellular enzymes initiates remineralization, a key process in ocean biogeochemical cycles. Here, we described a glycoside hydrolase 16 (GH16) family laminarinase from a marine alga-associated Flavobacterium at the biochemical and structural levels. We found that the endolytic enzyme cleaved laminarin with a preference for β-1,3-glycoside linkages and showed transglycosylation activity across a broad range of acceptors. We also solved and compared high-resolution crystal structures of laminarinase in the apo form and in complex with β-1,3-tetrasaccharides, revealing an expanded catalytic cleft formed following substrate binding. Moreover, structure and mutagenesis studies identified multiple specific contacts between the enzyme and glucosyl residues essential for the substrate specificity for β-1,3-glucan. These results provide novel insights into the structural requirements for substrate binding and catalysis of GH16 family laminarinase, enriching our understanding of bacterial utilization of algal laminarin. IMPORTANCE Heterotrophic bacterial communities are key players in marine biogeochemical cycling due to their ability to remineralize organic carbon. Processing of complex organic matter requires heterotrophic bacteria to produce extracellular enzymes with precise specificity to depolymerize substrates to sizes sufficiently small for uptake. Thus, extracellular enzymatic hydrolysis initiates microbe-driven heterotrophic carbon cycling. In this study, based on biochemical and structural analyses, we revealed the depolymerization mechanism of β-1,3-glucan, a carbon reserve in algae, by laminarinase from an alga-associated marine Flavobacterium. The findings provide new insights into the substrate recognition and catalysis of bacterial laminarinase and promote a better understanding of how extracellular enzymes are involved in organic matter cycling.

Godwin E. Oyiwona, J. Ogbonna, C. Anyanwu

Bio-Research • 2019

Two H-type dual-chambered glucose-fed MFC reactors, each with a working volume of 250 ml designated as GPWW (enriched from garri wastewater) and the other as DWW (enriched from domestic wastewater) were constructed under same conditions and the anodes were continuously fed with synthetic medium (SM) at a flow rate of 0.7 ml min -1 . An average power density (Pd) of 1.5 ± 1.39 W/m 3 for GPWW and 0.6 ± 0.6 W/m 3 for DWW was observed in 150 days of operation. The peak biomass density for DWW was significantly (P < 0.05 T-test) higher than that of GPWW by a factor of 2 (8.99mg versus 3.4 BSA/total anode respectively) on day 120 suggesting that more biomass was formed on DWW than GPWW reactor. This indicates that exo-electrogens in DWW reactor directed more of electron flow to cell synthesis rather than to current. The archaea footprints detected on the anodes were limiting factors in current generation. One vital milestone in this study is the discovery of the potential of garri-processing wastewater as a veritable substrate in microbial fuel cell technology. Keywords: Biomass density, Dissimilatory metal reducing bacteria, Current density, Microbial fuel cell, Power density

Jessica A Gable, T. Poulos, A. Follmer

Journal of the American Chemical Society • 2023

Despite being one of the most well-studied aspects of cytochrome P450 chemistry, important questions remain regarding the nature and ubiquity of allosteric regulation of catalysis. The crystal structure of a bacterial P450, P450terp, in the presence of substrate reveals two binding sites, one above the heme in position for regioselective hydroxylation and another in the substrate access channel. Unlike many bacterial P450s, P450terp does not exhibit an open to closed conformational change when substrate binds; instead, P450terp uses the second substrate molecule to hold the first substrate molecule in position for catalysis. Spectral titrations clearly show that substrate binding to P450terp is cooperative with a Hill coefficient of 1.4 and is supported by isothermal titration calorimetry. The importance of the allosteric site was explored by a series of mutations that weaken the second site and that help hold the first substrate in position for proper catalysis. We further measured the coupling efficiency of both the wild-type (WT) enzyme and the mutant enzymes. While the WT enzyme exhibits 97% efficiency, each of the variants showed lower catalytic efficiency. Additionally, the variants show decreased spin shifts upon binding of substrate. These results are the first clear example of positive homotropic allostery in a class 1 bacterial P450 with its natural substrate. Combined with our recent results from P450cam showing complex substrate allostery and conformational dynamics, our present study with P450terp indicates that bacterial P450s may not be as simple as once thought and share complex substrate binding properties usually associated with only mammalian P450s.

Yanli Huang, Xufeng Wan, Qiang Su et al.

Nature Communications • 2024

Implant-associated infections due to the formation of bacterial biofilms pose a serious threat in medical healthcare, which needs effective therapeutic methods. Here, we propose a multifunctional nanoreactor by spatiotemporal ultrasound-driven tandem catalysis to amplify the efficacy of sonodynamic and chemodynamic therapy. By combining piezoelectric barium titanate with polydopamine and copper, the ultrasound-activated piezo-hot carriers transfer easily to copper by polydopamine. It boosts reactive oxygen species production by piezoelectrics, and facilitates the interconversion between Cu2+ and Cu+ to promote hydroxyl radical generation via Cu+ -catalyzed chemodynamic reactions. Finally, the elevated reactive oxygen species cause bacterial membrane structure loosening and DNA damage. Transcriptomics and metabolomics analysis reveal that intracellular copper overload restricts the tricarboxylic acid cycle, promoting bacterial cuproptosis-like death. Therefore, the polyetherketoneketone scaffold engineered with the designed nanoreactor shows excellent antibacterial performance with ultrasound stimulation and promotes angiogenesis and osteogenesis on-demand in vivo.

Yan Ni, Chun‐Xiu Li, Jie Zhang et al.

Advanced Synthesis &amp; Catalysis • 2011

Abstract A β‐ketoacyl‐ACP reductase (FabG) gene from Bacillus sp. ECU0013 was heterologously overexpressed in Escherichia coli and the encoded protein was purified to homogeneity. The recombinant reductase could reduce a broad spectrum of prochiral ketones including aromatic ketones and keto esters and showed the highest activity in the asymmetric reduction of ethyl 2‐oxo‐4‐phenylbutyrate (OPBE). Using E. coli cells coexpressing both FabG and glucose dehydrogenase (GDH) genes, as much as 620 g⋅L −1 of OPBE was almost stoichiometrically converted to ethyl ( S )‐2‐hydroxy‐4‐phenylbutyrate [( S )‐HPBE] with excellent (&gt;99%) enantiomeric excess. More importantly, the process could be performed smoothly without external addition of an expensive cofactor as usually done and could be scaled up very easily. All these positive features demonstrate the applicability of this reductase for the large‐scale production of optically active α‐hydroxy acids/esters.

C. J. Jackson, J.-L. Foo, N. Tokuriki et al.

Proceedings of the National Academy of Sciences • 2009

To efficiently catalyze a chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex, the chemical reaction and product release. These distinct demands could be satisfied via fluctuation between different conformational substates (CSs) with unique configurations and catalytic properties. However, there is debate as to how these rapid conformational changes, or dynamics, exactly affect catalysis. As a model system, we have studied bacterial phosphotriesterase (PTE), which catalyzes the hydrolysis of the pesticide paraoxon at rates limited by a physical barrier—either substrate diffusion or conformational change. The mechanism of paraoxon hydrolysis is understood in detail and is based on a single, dominant, enzyme conformation. However, the other aspects of substrate turnover (substrate binding and product release), although possibly rate-limiting, have received relatively little attention. This work identifies “open” and “closed” CSs in PTE and dominant structural transition in the enzyme that links them. The closed state is optimally preorganized for paraoxon hydrolysis, but seems to block access to/from the active site. In contrast, the open CS enables access to the active site but is poorly organized for hydrolysis. Analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.

Pratima Bajpai

Bacterial Nanocellulose for Papermaking and Packaging • 2024

Bacterial cellulose is recognized as a multifaceted, versatile biomaterial with abundant applications. It is a completely biodegradable, ecological, non-toxic, chemically stable, and biocompatible material. Unlike plant cellulose, it is characterized by high crystallinity, a higher degree of polymerization, and higher tensile strength and Young’s modulus. In addition, bacterial cellulose, unlike vegetable cellulose, has a smaller diameter of fibres and hence possesses higher hydrophilicity. The properties of bacterial cellulose depend on multiple factors, such as culture conditions, the type of microorganisms, and nutrients present in the growth medium. These factors have a huge impact on the properties of the polymer, such as strength, crystallinity, degree of polymerization, or hygroscopicity.

Teunie van Herk, Aloysius F. Hartog, Alida M. van der Burg et al.

Advanced Synthesis &amp; Catalysis • 2005

Abstract Bacterial non‐specific acid phosphatases normally catalyze the dephosphorylation of a variety of substrates. As shown previously the enzymes from Shigella flexneri and Salmonella enterica are also able to catalyze the phosphorylation of inosine to inosine monophosphate and D ‐glucose to D ‐glucose 6‐phosphate (D‐G6P) using cheap pyrophosphate as the phosphate donor. After optimization high yields (95%) are achieved in the latter reaction and we show here that it is possible to use these enzymes in a preparative manner. This prompted us to investigate by using 31 P NMR and HPLC also the phosphorylation of a broad range of carbohydrates and alcohols. Many cyclic carbohydrates are phosphorylated in a regioselective manner. Non‐cyclic carbohydrates are phosphorylated as well. Phosphorylation of linear alcohols, cyclic and aromatic alcohols is also possible. In all cases the acid phosphatase from Shigella prefers a primary alcohol function above a secondary one. We conclude that these enzymes are an attractive alternative to existing chemical and enzymatic methods in the phosphorylation of a broad range of compounds.

luyan ma

• 2020

&amp;lt;p&amp;gt;Microbial nanowires are nanofilaments that could offer an extracellular electron transfer (EET) pathway linking the bacterial respiratory chain to external surfaces, such as oxidized metals in the environment and engineered electrodes in renewable energy devices. Filaments proposed to function as nanowires have been reported in multiple bacteria, yet it remains largely unclear about the composition and electron transfer mechanism of bacterial nanowires. Pseudomonas aeruginosa is an environmental and electrochemically active bacterium. In this study, we found nanotube-like extracellular filaments in P. aeruginosa biofilms, which were bacterial membrane extensions similar to the nanowires reported in Shewanella oneidensis. Remarkably, conductive probe atomic force microscope showed measurable conductivity of these extracellular filaments, suggesting that they may function as nanowires in P. aeruginosa. Our results also indicated that the electron shuttle pyocyanin significantly affected the conductivity of P. aeruginosa nanowires, suggesting that the electron transfer mechanism of P. aeruginosa nanowires was different from S. oneidensis. Furthermore, factors that impact biofilm formation, such as flagella, type IV pili, and exopolysaccharides, were not essential for nanowires formation, while affect the formation and length of nanowires of P. aeruginosa. Taken together, this is the first report that investigated the role of electron shuttle on the conductivity of nanowires and factors that affected nanowires formation.&amp;lt;/p&amp;gt;

Pratima Bajpai

Bacterial Nanocellulose for Papermaking and Packaging • 2024

Bacterial nanocellulose (BNC) biosynthesis is a well-organized and strictly controlled process and has two stages: first, the formation of 1,4-glucan linkages, and subsequently the assembly and cellulose crystallization. The process starts with the carbon source, such as glucose and fructose, being transported into the cell, where the cellulose precursor UDPG is produced. Bcs then polymerizes glucose from UDPG into 1,4-glucan strands. Finally, cellulose chains are secreted as sub-fibrils through pores in the cell membrane and then combined into ribbons in a 3D nanofiber network supported by hydrogen bonds.

Norzawani Jaffar

Focus on Bacterial Biofilms • 2022

Most pathogenic bacteria species form biofilm as their protective mode of growth, which helps them survive from the bactericidal effect of the antimicrobials or the killing activity of the host immune cells. The bacteria cells’ survivability via biofilm formation creates challenges in the medical field in terms of the device and also disease-related to biofilm. The impact of the bacterial biofilm issue is worsening over time, and the association to the high tolerance to the antimicrobial agents leads to increased morbidity and mortality worldwide. This review will highlight the main characteristics of the biofilm, the issue of biofilm in clinical practice, which also covered the pertinence of the biofilm in clinical practice, device-related biofilm disease, oral disease, and the significant bacterial species involved in the biofilm-related infections. Knowledge about the vital role of bacterial biofilm in related disorders will give new insight into the best approaches and alternative treatments for biofilm-related disease.

Hammam Antar, Young-Min Soh, Stefano Zamuner et al.

Science Advances • 2021

Three-component ParABS systems are widely distributed factors for plasmid partitioning and chromosome segregation in bacteria. ParB acts as adaptor protein between the 16–base pair centromeric parS DNA sequences and the DNA segregation proteins ParA and Smc (structural maintenance of chromosomes). Upon cytidine triphosphate (CTP) and parS DNA binding, ParB dimers form DNA clamps that spread onto parS -flanking DNA by sliding, thus assembling the so-called partition complex. We show here that CTP hydrolysis is essential for efficient chromosome segregation by ParABS but largely dispensable for Smc recruitment. Our results suggest that CTP hydrolysis contributes to partition complex assembly via two mechanisms. It promotes ParB unloading from DNA to limit the extent of ParB spreading, and it recycles off-target ParB clamps to allow for parS retargeting, together superconcentrating ParB near parS . We also propose a model for clamp closure involving a steric clash when binding ParB protomers to opposing parS half sites.

Roland Weis, Margit Winkler, Matthias Schittmayer et al.

Advanced Synthesis &amp; Catalysis • 2009

Abstract Innovative biohydroxylation catalysts for the preparation of drug metabolites were developed from scratch. A set of bacterial and fungal sequences of putative and already known bifunctional P450 enzymes was identified by protein sequence alignments, expressed in Escherichia coli and characterised. Notably, a fungal self‐sufficient cytochrome P450 (CYP) from Aspergillus fumigatus turned out to be especially stable during catalyst preparation and application and also in presence of organic co‐solvents. To enhance the catalytic activity and broaden the substrate specificity of those variants with high expression levels prominent single mutations were introduced. Selected improved variants were then used as lyophilised bacterial lysates for the synthesis of 4′‐hydroxydiclofenac and 6‐hydroxychlorzoxazone, the two metabolites of active pharmaceutical compounds diclofenac and chlorzoxazone representing the same metabolites as generated by human P450s.

Jacqueline N. Watson, Simon Newstead, Veedeeta Dookhun et al.

FEBS Letters • 2004

A recombinant D92G mutant sialidase from Micromonospora viridifaciens has been cloned, expressed and purified. Kinetic studies reveal that the replacement of the conserved aspartic acid with glycine results in a catalytically competent retaining sialidase that possesses significant activity against activated substrates. The contribution of this aspartate residue to the free energy of hydrolysis for natural substrates is greater than 19 kJ/mol. The three dimensional structure of the D92G mutant shows that the removal of aspartic acid 92 causes no significant re‐arrangement of the active site, and that an ordered water molecule substitutes for the carboxylate group of D92.

Atsuki Nakano, Jun-ichi Kishikawa, Atsuko Nakanishi et al.

PNAS Nexus • 2022

Abstract Adenosine triphosphate (ATP) synthases (F0F1-ATPases) are crucial for all aerobic organisms. F1, a water-soluble domain, can catalyze both the synthesis and hydrolysis of ATP with the rotation of the central γε rotor inside a cylinder made of α3β3 in three different conformations (referred to as βE, βTP, and βDP). In this study, we determined multiple cryo-electron microscopy structures of bacterial F0F1 exposed to different reaction conditions. The structures of nucleotide-depleted F0F1 indicate that the ε subunit directly forces βTP to adopt a closed form independent of the nucleotide binding to βTP. The structure of F0F1 under conditions that permit only a single catalytic β subunit per enzyme to bind ATP is referred to as unisite catalysis and reveals that ATP hydrolysis unexpectedly occurs on βTP instead of βDP, where ATP hydrolysis proceeds in the steady-state catalysis of F0F1. This indicates that the unisite catalysis of bacterial F0F1 significantly differs from the kinetics of steady-state turnover with continuous rotation of the shaft.

Florian Rudroff, Joanna Rydz, Freek H. Ogink et al.

Advanced Synthesis &amp; Catalysis • 2007

Abstract Microbial Baeyer–Villiger oxidation of representative prochiral ketones with a cyclobutanone structural motif was investigated using a collection of eight monooxygenases of different bacterial origin. This platform of enzymes is able to perform stereoselective biotransformations on an array of structurally diverse substrates. With several ketone precursors, biooxidations yielded enantiocomplementary butyrolactones as key intermediates for the synthesis of natural products and bioactive compounds. The microbial Baeyer–Villiger oxidation allows a facile and rapid entry to several compound classes in a desymmetrization reaction upon de novo generation of chirality.

María Maneiro, Antonio Peón, Emilio Lence et al.

Biochemical Journal • 2014

Structural, biochemical and computational studies to study substrate binding and the role of the conserved residues of the DHQ1 (type I dehydroquinase) enzyme active site are reported in the present paper. The crystal structure of DHQ1 from Salmonella typhi in complex with (2R)-2-methyl-3-dehydroquinic acid, a substrate analogue, was solved at 1.5 Å. The present study reveals a previously unknown key role for conserved Glu46, Phe145 and Met205 and Gln236, Pro234 and Ala233 residues, with the latter three being located in the flexible substrate-covering loop. Gln236 was shown to be responsible for the folding of this loop and for the dramatic reduction of its flexibility, which triggers active site closure. Glu46 was found to be key in bringing the substrate close to the lysine/histidine catalytic pocket to initiate catalysis. The present study could be useful in the rational design of inhibitors of this challenging and recognized target for the development of novel herbicides and antimicrobial agents.

Ronja Driller, Sophie Janke, Monika Fuchs et al.

Nature Communications • 2018

Abstract Terpenes constitute the largest and structurally most diverse natural product family. Most terpenoids exhibit a stereochemically complex macrocyclic core, which is generated by C–C bond forming of aliphatic oligo-prenyl precursors. This reaction is catalysed by terpene synthases (TPSs), which are capable of chaperoning highly reactive carbocation intermediates through an enzyme-specific reaction. Due to the instability of carbocation intermediates, the proteins’ structural dynamics and enzyme:substrate interactions during TPS catalysis remain elusive. Here, we present the structure of the diterpene synthase CotB2, in complex with an in crystallo cyclised abrupt reaction product and a substrate-derived diphosphate. We captured additional snapshots of the reaction to gain an overview of CotB2’s catalytic mechanism. To enhance insights into catalysis, structural information is augmented with multiscale molecular dynamic simulations. Our data represent fundamental TPS structure dynamics during catalysis, which ultimately enable rational engineering towards tailored terpene macrocycles that are inaccessible by conventional chemical synthesis.

D Ralt, J S Wishnok, R Fitts et al.

Journal of Bacteriology • 1988

We have developed a rapid and sensitive fluorimetric method, based on the formation of a fluorescent product from nitrosation of 2,3-diaminonaphthalene, for measuring the ability of bacteria to catalyze nitrosation of amines. We have shown in Escherichia coli that nitrosation can be induced under anaerobic conditions by nitrite and nitrate, that formate is the most efficient electron donor for this reaction, and that nitrosation may be catalyzed by nitrate reductase (EC 1.7.99.4). The narG mutants defective in nitrate reductase do not catalyze nitrosation, and the fnr gene is essential for nitrosation. Induction by nitrite or nitrate of nitrosation, N2O production, and nitrate reductase activity all require the narL gene.

Carolyn M.C. Lam, María Suárez Diez, Miguel Godinho et al.

FEBS Letters • 2012

Bacteria have long been used for the synthesis of a wide range of useful proteins and compounds. The developments of new bioprocesses and improvements of existing strategies for syntheses of valuable products in various bacterial cell hosts have their own challenges and limitations. The field of synthetic biology has combined knowledge from different science and engineering disciplines and facilitated the advancement of novel biological components which has inspired the design of targeted biosynthesis. Here we discuss recent advances in synthetic biology with relevance to biosynthesis in bacteria and the applications of computational algorithms and tools for manipulation of cellular components. Continuous improvements are necessary to keep up with increasing demands in terms of complexity, scale, and predictability of biosynthesis products.

Hammam Antar, Young-Min Soh, Stefano Zamuer et al.

bioRxiv (Cold Spring Harbor Laboratory) • 2021

Abstract Three-component ParABS systems are widely distributed factors for plasmid partitioning and chromosome segregation in bacteria. ParB protein acts as an adaptor between the 16 bp centromeric parS DNA sequences and the DNA segregation ATPase ParA. It accumulates at high concentrations at and near a parS site by assembling a partition complex. ParB dimers form a DNA sliding clamp whose closure at parS requires CTP binding. The mechanism underlying ParB loading and the role of CTP hydrolysis however remain unclear. We show that CTP hydrolysis is dispensable for Smc recruitment to parS sites in Bacillus subtilis but is essential for chromosome segregation by ParABS in the absence of Smc. Our results suggest that CTP hydrolysis contributes to partition complex assembly via two mechanisms. It recycles off-target ParB clamps to allow for new attempts at parS targeting and it limits the extent of spreading from parS by promoting DNA unloading. We also propose a model for how parS DNA catalyzes ParB clamp closure involving a steric clash between ParB protomers binding to opposing parS half sites.

J. Kristin Smith, John Hsieh, Carol A. Fierke

Biopolymers • 2007

Abstract Ribonuclease P (RNase P) is a ribonucleoprotein (RNP) complex that catalyzes the metal‐dependent maturation of the 5′ end of precursor tRNAs (pre‐tRNAs) in all organisms. RNase P is comprised of a catalytic RNA (P RNA), and at least one essential protein (P protein). Although P RNA is the catalytic subunit of the enzyme and is active in the absence of P protein under high salt concentrations in vitro, the protein is still required for enzyme activity in vivo. Therefore, the function of the P protein and how it interacts with both P RNA and pre‐tRNA have been the focus of much ongoing research. RNA‐protein interactions in RNase P serve a number of critical roles in the RNP including stabilizing the structure, and enhancing the affinity for substrates and metal ions. This review examines the role of RNA‐protein interactions in bacterial RNase P from both structural and mechanistic perspectives. © 2007 Wiley Periodicals, Inc. Biopolymers 87: 329–338, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Aloysius F. Hartog, Ron Wever

Advanced Synthesis &amp; Catalysis • 2015

Abstract The use of substrate engineering allowed the enzymatic sulfation by an arylsulfotransferase from Desulfitobacterium hafniense of a number of carbohydrate derivatives. Specific sulfation of carbohydrates chemically or enzymatically is notoriously difficult or complex. As we have shown previously, the arylsulfotransferase is able to sulfate a variety of phenolic alcohols as well as non‐phenolic alcohols, albeit the conversion in the latter case is limited and in general less than a few percent. Here we used the strategy of substrate engineering to increase the conversion by attaching a hydrophobic group to the carbohydrate. The formed sulfated intermediates may be used as building blocks in the formation of more complex sulfated carbohydrates via a transglycosylation reaction. magnified image

Henning Kirst, Cheryl A. Kerfeld

BMC Biology • 2019

Abstract Bacterial cells have long been thought to be simple cells with little spatial organization, but recent research has shown that they exhibit a remarkable degree of subcellular differentiation. Indeed, bacteria even have organelles such as magnetosomes for sensing magnetic fields or gas vesicles controlling cell buoyancy. A functionally diverse group of bacterial organelles are the bacterial microcompartments (BMCs) that fulfill specialized metabolic needs. Modification and reengineering of these BMCs enable innovative approaches for metabolic engineering and nanomedicine.

S. Kambourakis, K. Draths, J. W. Frost

Journal of the American Chemical Society • 2000

Among a spectrum of uses, 1 gallic acid and pyrogallol are often incorporated into chemical syntheses to provide the trihydrox-ylated aromatic ring of biologically active molecules such as the antibiotic trimethoprim 1 , the muscle relaxant gallamine tri-ethiodide 2 , and the insecticide bendiocarb 3 . The availability of gallic acid is restricted by its current isolation from insect carapices (gall nuts) harvested in China, and an isolate (tara powder) derived from the ground seed pod of a tree found in Peru. 1 Thermal decarboxylation of gallic acid in copper autoclaves affords pyrogallol. 1 As part of a larger effort 2 to supplant isolation of natural products from scarce natural sources, gallic acid and pyrogallol were targeted for microbe-catalyzed synthesis from abundant glucose. In lieu of an elaborated biosynthetic pathway leading to gallic acid, a pathway had to be created. The result is Escherichia coli KL7/pSK6.161, which synthesizes 20 g/L of gallic acid in 12% yield from glucose. E. coli RB791 serA :: aroB / pSK6.234 then converts gallic acid into pyrogallol in yields of 93 - 97%

A. Ugo, Arazu Vivian Amara, Igwe Cn et al.

Fermentation Technology • 2017

Microbes have been novel hosts for suitable industrial enzymes including lipases. With the rapid increase in biotechnological industries, there is need for improvement of bioproducts, enhance environmental safety and product yield. Microbes are qualified biological arsenal for the achievement of the above mentioned targets in the industrial sectors. Lipases as versatile biological catalyst has given a promising prospect in meeting the needs for most industries such as biodiesel, foods and drinks, leather, textile, detergents, pharmaceuticals and medicals. Catalytic importance of lipases includes hydrolysis, esterification and transesterification. Each of the mentioned reactions has their industrial applications. Lipases exhibit various properties, with respect to its source. Immobilization has made the use of microbial lipases attain its optimum performance and hence suitable for various reaction and the need to add flavor to the immobilization processes.

R. Jha, C. Strauss

ACS Synthetic Biology • 2020

Enzyme engineering for gain-of-function requires navigating a large combinatorial sequence space efficiently. Typically, many mutations are needed to get significant improvements while a single 'bad' mutation can inactivate the enzyme. To establish high throughput screening and achieve enhanced resolution between two variants, genetic libraries of organophos-phate hydrolase enzyme (PON1) were rapidly screened via an engineered positive feedback circuit: a PNP-specific transcrip-tion factor (TF) regulated expression of PON1, which catalyzed paraoxon breakdown and PNP production. Rare active mu-tant colonies, picked by simple visual fluorescence of a PON1-GFP (Green Fluorescent Protein) fusion, were characterized. In a single screening round, high (library-scale) throughput enabled discovery of enhanced paraoxon degradation activity in PON1 including structurally unexpected mutations.

L. Wackett, McKnight

Microbial Biotechnology • 2023

Polymer biof i lms for biocatalysis https://www.ncbi.nlm.nih.gov/pmc/artic les/PMC95 28183/ In some cases, the most catalytically useful bacteria are not adept at forming biofilms naturally. This study addressed that issue by demonstrating different methods of making polymerinduced biofilms. Anammox biof i lm reactor https://pubs.acs.org/doi/10.1021/acsestwater.0c00107 Anammox microbes have been shown to be very useful in industrial nitrogen removal from wastewaters. This paper describes the development and operation of a flowthrough Anammox biofilm reactor.