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DOE OSTI2025

Role of Ammonium Hydroxide on Glucose Oxidase Immobilized in Metal–Azolate Framework-7 Enzyme Activity

Enzymes are nature’s catalysts, but their instability outside native environments limits practical applications. Metal–organic frameworks (MOFs) offer a promising platform for enzyme immobilization, enhancing stability, and reusability through their tunable porosity and crystallinity. While ZIF-based MOFs have been extensively studied, metal azolate framework-7 (MAF-7) remains largely unexplored for biomimetic mineralization. Its hydrophilic character makes it a promising alternative for enzyme encapsulation; however, the role of essential basic modulators such as ammonium hydroxide (NH 4 OH) on enzyme structure and function has not been investigated. Here, in this study, we examine the NH 4 OH mediated synthesis of glucose oxidase within MAF-7 using powder X-ray diffraction (PXRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), cryogenic transmission electron microscopy (cryo-TEM), and enzyme assays. Our results show that NH 4 OH modulates crystal morphology, enzyme activity, and nucleation behavior, with higher concentrations leading to partial enzyme denaturation and reduced catalytic performance. By integrating time-resolved cryo-TEM with activity assays, we uncover how modulator concentration impacts early stage crystallization and enzyme folding, a mechanistic insight not previously demonstrated in MOF systems. These findings highlight modulator chemistry as a critical, underexplored variable in MOF synthesis, advancing rational design strategies for enzyme@MOFs (E@MOF) beyond ZIFs and toward more tunable, biocompatible frameworks like MAF-7.

36 MATERIALS SCIENCE↗

DOE OSTI2021

Near-complete depolymerization of polyesters with nano-dispersed enzymes

Successfully interfacing enzymes and biomachinery with polymers affords on-demand modification and/or programmable degradation during the manufacture, utilization and disposal of plastics, but requires controlled biocatalysis in solid matrices with macromolecular substrates. Embedding enzyme microparticles speeds up polyester degradation, but compromises host properties and unintentionally accelerates the formation of microplastics with partial polymer degradation. Here we show that by nanoscopically dispersing enzymes with deep active sites, semi-crystalline polyesters can be degraded primarily via chain-end-mediated processive depolymerization with programmable latency and material integrity, akin to polyadenylation-induced messenger RNA decay. It is also feasible to achieve processivity with enzymes that have surface-exposed active sites by engineering enzyme-protectant-polymer complexes. Poly(caprolactone) and poly(lactic acid) containing less than 2 weight per cent enzymes are depolymerized in days, with up to 98 per cent polymer-to-small-molecule conversion in standard soil composts and household tap water, completely eliminating current needs to separate and landfill their products in compost facilities. Furthermore, oxidases embedded in polyolefins retain their activities. However, hydrocarbon polymers do not closely associate with enzymes, as their polyester counterparts do, and the reactive radicals that are generated cannot chemically modify the macromolecular host. This study provides molecular guidance towards enzyme-polymer pairing and the selection of enzyme protectants to modulate substrate selectivity and optimize biocatalytic pathways. Furthermore, the results also highlight the need for in-depth research in solid-state enzymology, especially in multi-step enzymatic cascades, to tackle chemically dormant substrates without creating secondary environmental contamination and/or biosafety concerns.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

Local Environment Affects the Activity of Enzymes on a 3D Molecular Scaffold

The ability to coordinate and confine enzymes presents an opportunity to affect their performance and to create chemically active materials. Recent studies show that polymers and biopolymers can be used to scaffold enzymes, and that can lead to the modulated biocatalytic efficiency. In this work, we investigated the role of microenvironments on enzyme activity using a well-defined molecular scaffold. An enzyme, glucose oxidase (GO x ), was positioned at different locations of a three-dimensional (3D) octahedral DNA scaffold (OS), allowing the enzyme’s polyanionic environments to be altered. Using electrical sensing, based on a bipolar junction transistor, we measured directly and in real-time the enzyme’s proton generation at these different microenvironments. We found a 200% enhancement of immobilized enzyme over free GOx and about a 30% increase in catalytic rates when the enzyme was moved on the same molecular scaffold to a microenvironment with a higher local concentration of polyanions, which suggests a role of local pH on the enzymatic activity.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

DOE OSTI2022

On Single-Cell Enzyme Assays in Marine Microbial Ecology and Biogeochemistry

Extracellular enzyme activity is a well-established parameter for evaluating microbial biogeochemical roles in marine ecosystems. The presence and activity of extracellular enzymes in seawater provide insights into the quality and quantity of organic matter being processed by the present microorganisms. A key challenge in our understanding of these processes is to decode the extracellular enzyme repertoire and activities of natural communities at the single-cell level. Current measurements are carried out on bulk or size-fractionated samples capturing activities of mixed populations. This approach – even with size-fractionation – cannot be used to trace enzymes back to their producers, nor distinguish the active microbial members, leading to a disconnect between measured activities and the producer cells. By targeting extracellular enzymes and resolving their activities at the single-cell level, we can investigate underlying phenotypic heterogeneity among clonal or closely related organisms, characterize enzyme kinetics under varying environmental conditions, and resolve spatio-temporal distribution of individual enzyme producers within natural communities. In this perspective piece, we discuss state-of-the-art technologies in the fields of microfluidic droplets and functional screening of prokaryotic cells for measuring enzyme activity in marine seawater samples, one cell at a time. We further elaborate on how this single-cell approach can be used to address research questions that cannot be answered with current methods, as pertinent to the enzymatic degradation of organic matter by marine microorganisms.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Spray Drying Enzyme-Treated Cellulose Nanofibrils

Enzyme-treated cellulose nanofibrils (CNFs) were produced via a lab-scale mass colloider using bleached kraft pulp (BKP) to evaluate their processability and power requirements during refining and spray-drying operations. To evaluate the energy efficiency in the CNF refining process, the net energy consumption, degree of polymerization (DP), and viscosity were determined. Less energy was consumed to attain a given fines level by using the endoglucanase enzymes. The DP and viscosity were also decreased using the enzymes. The morphological properties of the enzyme-pretreated spray-dried CNF powders (SDCNFs) were measured. Subsequently, the enzyme-pretreated SDCNFs were added to a PP matrix with MAPP as a coupling agent. The mixture was then compounded through a co-rotating twin-screw extruder to determine whether the enzyme treatment of the CNFs affects the mechanical properties of the composites. Compared to earlier studies on enhancing PMCs with SDCNF powders, this research investigates the use of enzyme-pretreated SDCNF powders. It was confirmed that the strength properties of PP increased by adding SDCNFs, and the strength properties were maintained after adding enzyme-pretreated SDCNFs.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

BETO 2021 Peer Review - Enzyme Engineering and Optimization (EEO)

The FY2020 SOT economic model for the DMR BDO process indicates that the production and use of biomass degrading enzymes represents ~10% of the MFSP of BDO. We are thus working to enhance the performance of the dominant cellulase enzyme, Cel7A. In Fy2018-19, we selected 100 promising genes from the "Cel7A wheel of life" and cloned them into T. reesei using a constitutive promotor. This natural diversity screening resulted in the discovery of several enzymes exhibiting improved characteristics relative to the industry standard Cel7A from T. reesei (Tr). Growth of the transformed host on glucose insured that the Cre1 induced cellulase expression cascade was suppressed, thus enabling purification of the target gene product. The first top performing enzyme found was from P. funiculosum (Pf). Other top performing enzymes were identified from T. aculeatus, T. terrestris, and A. oryzae. Computational modeling predicted the structural subsites responsible for the improvements, which were cloned into the sequence of PfCel7A. The recombinant enzymes were expressed in T. reesei, purified, and tested. Testing was not possible with SOT relevant substrates, so the best-case substrate was used; solids from the DMR process subjected to a novel dilute alkaline wash to remove the "lignin shield." Two chimeric Cel7A enzymes show improved performance (1.2 to 1.35x) relative to PfCel7A, which in turn showed considerable improvement (1.6x) relative to T. reesei Cel7A. These new enzymes have been provided to our industrial partner for evaluation.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗

DOE OSTI2021

The Moderately (D)efficient Enzyme: Catalysis-Related Damage In Vivo and Its Repair

Enzymes have in vivo life spans. Analysis of life spans, i.e., lifetime totals of catalytic turnovers, suggests that nonsurvivable collateral chemical damage from the very reactions that enzymes catalyze is a common but underdiagnosed cause of enzyme death. Analysis also implies that many enzymes are moderately deficient in that their active-site regions are not naturally as hardened against such collateral damage as they could be, leaving room for improvement by rational design or directed evolution. Enzyme life span might also be improved by engineering systems that repair otherwise fatal active-site damage, of which a handful are known and more are inferred to exist. Unfortunately, the data needed to design and execute such improvements are lacking: there are too few measurements of in vivo life span, and existing information about the extent, nature, and mechanisms of active-site damage and repair during normal enzyme operation is too scarce, anecdotal, and speculative to act on. Fortunately, advances in proteomics, metabolomics, cheminformatics, comparative genomics, and structural biochemistry now empower a systematic, data-driven approach for identifying, predicting, and validating instances of active-site damage and its repair. These capabilities would be practically useful in enzyme redesign and improvement of in-use stability and could change our thinking about which enzymes die young in vivo, and why.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

Characterization and engineering of a two-enzyme system for plastics depolymerization

Significance Deconstruction of recalcitrant polymers, such as cellulose or chitin, is accomplished in nature by synergistic enzyme cocktails that evolved over millions of years. In these systems, soluble dimeric or oligomeric intermediates are typically released via interfacial biocatalysis, and additional enzymes often process the soluble intermediates into monomers for microbial uptake. The recent discovery of a two-enzyme system for polyethylene terephthalate (PET) deconstruction, which employs one enzyme to convert the polymer into soluble intermediates and another enzyme to produce the constituent PET monomers (MHETase), suggests that nature may be evolving similar deconstruction strategies for synthetic plastics. This study on the characterization of the MHETase enzyme and synergy of the two-enzyme PET depolymerization system may inform enzyme cocktail-based strategies for plastics upcycling.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Strangers in a foreign land: ‘Yeastizing’ plant enzymes

Abstract Expressing plant metabolic pathways in microbial platforms is an efficient, cost‐effective solution for producing many desired plant compounds. As eukaryotic organisms, yeasts are often the preferred platform. However, expression of plant enzymes in a yeast frequently leads to failure because the enzymes are poorly adapted to the foreign yeast cellular environment. Here, we first summarize the current engineering approaches for optimizing performance of plant enzymes in yeast. A critical limitation of these approaches is that they are labour‐intensive and must be customized for each individual enzyme, which significantly hinders the establishment of plant pathways in cellular factories. In response to this challenge, we propose the development of a cost‐effective computational pipeline to redesign plant enzymes for better adaptation to the yeast cellular milieu. This proposition is underpinned by compelling evidence that plant and yeast enzymes exhibit distinct sequence features that are generalizable across enzyme families. Consequently, we introduce a data‐driven machine learning framework designed to extract ‘yeastizing’ rules from natural protein sequence variations, which can be broadly applied to all enzymes. Additionally, we discuss the potential to integrate the machine learning model into a full design‐build‐test cycle.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Increasing thermostability of the key photorespiratory enzyme glycerate 3‐kinase by structure‐based recombination

As global temperatures rise, improving crop yields will require enhancing the thermotolerance of crops. One approach for improving thermotolerance is using bioengineering to increase the thermostability of enzymes catalysing essential biological processes. Photorespiration is an essential recycling process in plants that is integral to photosynthesis and crop growth. The enzymes of photorespiration are targets for enhancing plant thermotolerance as this pathway limits carbon fixation at elevated temperatures. We explored the effects of temperature on the activity of the photorespiratory enzyme glycerate kinase (GLYK) from various organisms and the homologue from the thermophilic alga Cyanidioschyzon merolae was more thermotolerant than those from mesophilic plants, including Arabidopsis thaliana. To understand enzyme features underlying the thermotolerance of C. merolae GLYK (CmGLYK), we performed molecular dynamics simulations using AlphaFold-predicted structures, which revealed greater movement of loop regions of mesophilic plant GLYKs at higher temperatures compared to CmGLYK. Based on these simulations, hybrid proteins were produced and analysed. These hybrid enzymes contained loop regions from CmGLYK replacing the most mobile corresponding loops of AtGLYK. Two of these hybrid enzymes had enhanced thermostability, with melting temperatures increased by 6 °C. One hybrid with three grafted loops maintained higher activity at elevated temperatures. Whilst this hybrid enzyme exhibited enhanced thermostability and a similar Km for ATP compared to AtGLYK, its Km for glycerate increased threefold. This study demonstrates that molecular dynamics simulation-guided structure-based recombination offers a promising strategy for enhancing the thermostability of other plant enzymes with possible application to increasing the thermotolerance of plants under warming climates.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Liquefaction of different corn stover fractions assisted by enzyme-biomass deconstruction

Recalcitrant properties of different sources of lignocellulosic biomass represent challenges in materials processing within a biorefinery, as well as enzyme efficient deconstruction to fermentable sugars. Limitations include lignin derived enzyme inhibitors, enzyme inhibition by hydrolysis products, and resistance to mixing due to rheological properties of lignocellulosic particulates at high solids loadings. Consequently, we examined conditions that might be used to achieve liquid slurries (i.e., liquefaction) at solids loadings of 300 g/L before the material enters the biorefinery through a pretreatment step. This work explores enzyme-assisted liquefaction in a fed-batch process using the commercial enzymes Celluclast 1.5L or Ctec-2 at 1FPU/g or 3 FPU/g of dry solids, basis. Corn stover pellets were fed into a 1 L stirred bioreactor containing enzyme solution over a 5-hour period until reaching 30% solids loading (dry wgt / vol basis). After 6, 24 and 96 hours from the start of the run, samples were taken and characterized with respect to their sugar composition, rheology, water absorption and enzyme activity. Slurries with dramatically reduced yield stresses were achieved for corn stover. Yield stresses of 178±7 Pa (3 FPU, Celluclast 1.5L) and 79±6 Pa (3FPU, Ctec-2) were measured for corn stover at 24 hours, compared to 6,000 Pa for samples without enzyme.Yield stress was 155± 29 Pa (3FPU, Ctec-2) and 257 ± 72 Pa (1 FPU, Celluclast 1.5L) for corn cobs at 24 hours.A profile based on 6, 24 and 96h of yield stress measurements and sugar conversion is presented. Enzyme activity is measured and the impact of liquefaction on an integrated processing in a biorefinery operation is discussed.

Gutierrez, Diana↗

DOE OSTI2021

The moderately defficient enzyme: Catalysis-related damage in vivo and its repair

Enzymes have in vivo lifespans. Analysis of lifespans – lifetime totals of catalytic turnovers – suggests that non-survivable collateral chemical damage from the very reactions that enzymes catalyze is a common but underdiagnosed cause of enzyme death. Analysis also implies that many enzymes are moderately deficient in that their active-site regions are not naturally as hardened against such collateral damage as they could be, leaving room for improvement by rational design or directed evolution. Enzyme lifespan might also be improved by engineering systems that repair otherwise fatal active-site damage, of which a handful are known and more are inferred to exist. Unfortunately, the data needed to design and execute such improvements is lacking: there are too few measurements of in vivo lifespan, and existing information on the extent, nature, and mechanisms of active-site damage and repair during normal enzyme operation is too scarce, anecdotal, and speculative to act on. Fortunately, advances in proteomics, metabolomics, cheminformatics, comparative genomics, and structural biochemistry now empower a systematic, data-driven approach to identify, predict, and validate instances of active-site damage and its repair. These capabilities would be practically useful in enzyme redesign and improvement of in-use stability, and could change thinking about which enzymes die young in vivo, and why.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

The moderately defficient enzyme: Catalysis-related damage in vivo and its repair

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study

Plant evolution has produced enzymes that may not be optimal for maximizing yield and quality in today’s agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized more quickly than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cell’s growth rate to the target gene’s function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in Saccharomyces cerevisiae and EvolvR in Escherichia coli, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

The anaerobic fungus Caecomyces churrovis produces H 2 via a non-bifurcating NADH-dependent enzyme complex

ABSTRACT Hydrogenosomes are mitochondria-derived organelles that produce ATP and H 2 to support energy metabolism in anaerobic eukaryotes. H 2 production allows reoxidation of reduced cofactors generated during fermentative metabolism; however, the metabolic mechanisms for H 2 production in anaerobic eukaryotes remains incompletely understood. In particular, it remains unclear whether anaerobic fungi (AF) hydrogenosomes use a ferredoxin-dependent pathway or a distinct mechanism to regenerate NAD(P) + and link electron transfer to H 2 formation. Here, by combining genomic search, proteomic analysis, and enzymology, we reveal the molecular mechanism for H 2 production in the AF strain Caecomyces churrovis . Our enzyme assays on the organelle fraction of C. churrovis revealed the activity of H 2 :NAD + oxidoreductase but not pyruvate:ferredoxin oxidoreductase, which is usually linked to H 2 formation. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE, NuoF) in C. churrovis , and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified enzymes, we found Hyd and NuoEF proteins formed H 2 directly from NADH independently of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is commonly shared among the AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex in eukaryotes. Moreover, this complex could potentially be exploited as a target for controlling AF H 2 production and altering fungal metabolism. IMPORTANCE H 2 production is a prominent feature of anaerobic energy metabolism, yet our understanding of eukaryotic mechanisms remains limited. Anaerobic fungi (AF) are key decomposers of lignocellulose and contribute to hydrogen flux in anaerobic environments. Although it has been more than 40 years since the H 2 production from AF was first reported, the molecular mechanism for hydrogenosomal H 2 production and redox balance remains unclear. We demonstrate that AF produce H 2 from NADH utilizing a non-bifurcating NADH-dependent enzyme complex rather than an electron-bifurcating, ferredoxin-dependent variant. We show that this enzyme complex is conserved across multiple AF lineages and thus demonstrate the occurrence of a non-bifurcating NADH-dependent enzyme in eukaryotes. This discovery expands our understanding of eukaryotic hydrogenosomal metabolism, reveals a previously unknown strategy for redox balancing, and highlights potential targets for manipulating H 2 production. These insights have broad implications for microbial energy metabolism, anaerobic ecosystems, and bioengineering of H 2 -producing systems.

Zhang, Bo [Department of Chemical Engineering, Uni↗

NASA NTRS2002

RNA-Cleaving DNA Enzymes with Altered Regio- or Enantioselectivity

In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5' - phosphodiester following a wibonucleotide or a 3',5' -phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5' -phosphodiester exhibits a k(sub cat) of approx. 0.01/ min and catalytic efficiency, k(sub cat)/k(sub m) of approx. 10(exp 5)/ M min. The enzyme that cleaves an L-ribonudeotide is about 10-fold slower and has a catalytic efficiency of approx. 4 x 10(exp 5)/ M min. Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 C. In a comparison of each enzyme s activity with either its corresponding substrate that contains an unnatural ribonudeotide or a substrate that instead contains a standard ribonucleotide, the 2',5' -phosphodiester-deaving DNA enzyme exhibited a regioselectivity of 6000- fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 50-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.

Ordoukhanian, Phillip↗

NASA NTRS1994

A DNA enzyme that cleaves RNA

BACKGROUND: Several types of RNA enzymes (ribozymes) have been identified in biological systems and generated in the laboratory. Considering the variety of known RNA enzymes and the similarity of DNA and RNA, it is reasonable to imagine that DNA might be able to function as an enzyme as well. No such DNA enzyme has been found in nature, however. We set out to identify a metal-dependent DNA enzyme using in vitro selection methodology. RESULTS: Beginning with a population of 10(14) DNAs containing 50 random nucleotides, we carried out five successive rounds of selective amplification, enriching for individuals that best promote the Pb(2+)-dependent cleavage of a target ribonucleoside 3'-O-P bond embedded within an otherwise all-DNA sequence. By the fifth round, the population as a whole carried out this reaction at a rate of 0.2 min-1. Based on the sequence of 20 individuals isolated from this population, we designed a simplified version of the catalytic domain that operates in an intermolecular context with a turnover rate of 1 min-1. This rate is about 10(5)-fold increased compared to the uncatalyzed reaction. CONCLUSIONS: Using in vitro selection techniques, we obtained a DNA enzyme that catalyzes the Pb(2+)-dependent cleavage of an RNA phosphoester in a reaction that proceeds with rapid turnover. The catalytic rate compares favorably to that of known RNA enzymes. We expect that other examples of DNA enzymes will soon be forthcoming.

Non-NASA Center↗

NASA NTRS2005

Conservation of Fold and Topology of Functional Elements in Thiamin Pyrophosphate Enzymes

Thiamin pyrophosphate (TPP)-dependent enzymes are a highly divergent family of proteins binding both TPP and metal ions. They perform decarboxylation-hydroxyaldehydes. Prior -ketoacids and of a common - (O=)C-C(OH)- fragment of to knowledge of three-dimensional structures of these enzmes, the GDGY25-30NN sequence was used to identify these enzymes. Subsequently, a number of structural studies on those enzymes revealed multi-subunit organization and the features of the two duplicate cofactor binding sites. Analyzing the structures of 44 structurally known enzymes, we found that the common structure of these enzymes is reduced to 180-220 amino acid long fragments of two PP and two PYR domains that form the [PP:PYR]2 binding center of two cofactor molecules. The structures of PP and PYR are arranged in a similar fold-sheet with triplets of helices on both sides.Dconsisting of a six-stranded Residues surrounding the cofactors are not strictly conserved, but they provide the same interatomic contacts required for the catalytic functions that these enzymes perform while maintaining interactive structural integrity. These structural and functional amino acids are topological counterparts located in the same positions of the conserved fold of sets of PP and PYR domains. Additional parallels include short fragments of sequences that link these amino acids to the fold and function. This report on the structural commonalities amongst TPP dependent enzymes is thought to contribute new approaches to annotation that may assist in advancing the functional proteomics of TPP dependent enzymes, and trace their complexity within evolutionary context.

Dominiak, P.↗