Cofactor Specificity Research Articles

Structural insights into the mechanism underlying the dual cofactor specificity of glyoxylate reductase from Acetobacter aceti in the β-hydroxyacid dehydrogenase family

The β-hydroxyacid dehydrogenase family exhibits diverse cofactor preferences: some enzymes favor NAD, others favor NADP, and a subset can utilize both NAD and NADPH. Glyoxylate reductase from Acetobacter aceti JCM 20276 (AacGR) exhibits a dual cofactor specificity for NADPH and NADH in its catalytic reduction of glyoxylate to glycolate. In contrast to conventional cofactor-discriminating motifs, NRX and DXX, found in NADP- and NAD-specific enzymes, respectively, AacGR has a TPS motif in the equivalent position. Here we report X-ray crystallographic analysis of AacGR in its ligand-free form, and in complexes with NADPH and NADH, revealing critical interactions: Ser41 of the TPS motif interacted with the 2′-phosphate group of NADPH, while no analogous interaction occurred with the ribose hydroxy groups of NADH. Moreover, the TPS motif resided within a characteristic β-turn-like structure adjacent to a long flexible loop. Site-directed mutagenesis and kinetic analyses suggest that Ser41 facilitates NADPH binding, while the lack of a direct interaction of the TPS motif with NADH may allow for NADH utilization. The conformational dynamics of the TPS-containing β-turn-like structure along with the flexible loop likely govern the dual cofactor specificity and catalytic turnover of AacGR.

The INSIGHT platform: Enhancing NAD(P)-dependent specificity prediction for co-factor specificity engineering

Enzyme specificity towards cofactors like NAD(P)H is crucial for applications in bioremediation and eco-friendly chemical synthesis. Despite their role in converting pollutants and creating sustainable products, predicting enzyme specificity faces challenges due to sparse data and inadequate models. To bridge this gap, we developed the cutting-edge INSIGHT platform to enhance the prediction of coenzyme specificity in NAD(P)-dependent enzymes. INSIGHT integrates extensive data from principal bioinformatics resources, concentrating on both NADH and NADPH specificities, and utilizes advanced protein language models to refine the predictions. This integration not only strengthens computational predictions but also meets the practical demands of high-throughput screening and optimization. Experimental validation confirms INSIGHT's effectiveness, boosting our ability to engineer enzymes for efficient, sustainable industrial and environmental processes. This work advances the practical use of computational tools in enzyme research, addressing industrial needs and offering scalable solutions for environmental challenges.

Biochemical and structural elucidation of the L-carnitine degradation pathway of the human pathogen Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic human pathogen which can use host-derived L-carnitine as sole carbon and energy source. Recently, an L-carnitine transporter (Aci1347) and a specific monooxygense (CntA/CntB) for the intracellular cleavage of L-carnitine have been characterized. Subsequent conversion of the resulting malic semialdehyde into the central metabolite L-malate was hypothesized. Alternatively, L-carnitine degradation via D-malate with subsequent oxidation into pyruvate was proposed. Here we describe the in vitro and in vivo reconstitution of the entire pathway, starting from the as yet uncharacterized gene products of the carnitine degradation gene operon. Using recombinantly purified enzymes, enantiomer-specific formation of D-malate by the NAD(P)+-dependent malic semialdehyde dehydrogenase (MSA-DH) is demonstrated. The solved X-ray crystal structure of tetrameric MSA-DH reveals the key catalytic residues Cys290 and Glu256, accessible through opposing substrate and cofactor funnels. Specific substrate binding is enabled by Arg166, Arg284 and Ser447 while dual cofactor specificity for NAD+ and NADP+ is mediated by Asn184. The subsequent conversion of the unusual D-malate reaction product by an uncharacterized NAD+-dependent malate dehydrogenase (MDH) is shown. Tetrameric MDH is a β-decarboxylating dehydrogenase that synthesizes pyruvate. MDH experiments with alternative substrates showed a high degree of substrate specificity. Finally, the entire A. baumannni pathway was heterologously reconstituted, allowing E. coli to grow on L-carnitine as a carbon and energy source. Overall, the metabolic conversion of L-carnitine via malic semialdehyde and D-malate into pyruvate, CO2 and trimethylamine was demonstrated. Trimethylamine is also an important gut microbiota-dependent metabolite that is associated with an increased risk of cardiovascular disease. The pathway reconstitution experiments allowed us to assess the TMA forming capacity of gut microbes which is related to human cardiovascular health.

Shifting redox reaction equilibria on demand using an orthogonal redox cofactor.

Nature's two redox cofactors, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), are held at different reduction potentials, driving catabolism and anabolism in opposite directions. In biomanufacturing, there is a need to flexibly control redox reaction direction decoupled from catabolism and anabolism. We established nicotinamide mononucleotide (NMN+) as a noncanonical cofactor orthogonal to NAD(P)+. Here we present the development of Nox Ortho, a reduced NMN+ (NMNH)-specific oxidase, that completes the toolkit to modulate NMNH:NMN+ ratio together with an NMN+-specific glucose dehydrogenase (GDH Ortho). The design principle discovered from Nox Ortho engineering and modeling is facilely translated onto six different enzymes to create NMN(H)-orthogonal biocatalysts with a consistent ~103-106-fold cofactor specificity switch from NAD(P)+ to NMN+. We assemble these enzymes to produce stereo-pure 2,3-butanediol in cell-free systems and in Escherichia coli, enabled by NMN(H)'s distinct redox ratio firmly set by its designated driving forces, decoupled from both NAD(H) and NADP(H).

The role of AdhE on ethanol tolerance and production in Clostridium thermocellum

Many anaerobic microorganisms use the bifunctional aldehyde and alcohol dehydrogenase, AdhE, to produce ethanol. One such organism is Clostridium thermocellum, which is of interest for cellulosic biofuel production. In the course of engineering this organism for improved ethanol tolerance and production, we observed that AdhE was a frequent target of mutations. Here, we characterized those mutations to understand their effects on enzymatic activity, as well ethanol tolerance and product formation in the organism. We found that there is a strong correlation between NADH-linked alcohol dehydrogenase (ADH) activity and ethanol tolerance. Mutations that decrease NADH-linked ADH activity increase ethanol tolerance; correspondingly, mutations that increase NADH-linked ADH activity decrease ethanol tolerance. We also found that the magnitude of ADH activity did not play a significant role in determining ethanol titer. Increasing ADH activity had no effect on ethanol titer. Reducing ADH activity had indeterminate effects on ethanol titer, sometimes increasing and sometimes decreasing it. Finally, this study shows that the cofactor specificity of ADH activity was found to be the primary factor affecting ethanol yield. We expect that these results will inform efforts to use AdhE enzymes in metabolic engineering approaches.

Functional and structural characterization of a thermostable flavin reductase from Geobacillus mahadii Geo-05

Flavin reductases play a vital role in catalyzing the reduction of flavin through NADH or NADPH oxidation. The gene encoding flavin reductase from the thermophilic bacterium Geobacillus mahadii Geo-05 (GMHpaC) was cloned, overexpressed in Escherichia coli BL21 (DE3) pLysS, and purified to homogeneity. The purified recombinant GMHpaC (Class II) contains chromogenic cofactors, evidenced by maximal absorbance peaks at 370 nm and 460 nm. GMHpaC stands out as the most thermostable and pH-tolerant flavin reductase reported to date, retaining up to 95 % catalytic activity after incubation at 70 °C for 30 min and maintaining over 80 % activity within a pH range of 2–12 for 30 min. Furthermore, GMHpaC's catalytic activity increases by 52 % with FMN as a co-factor compared to FAD and riboflavin. GMHpaC, coupled with 4-hydroxyphenylacetate-3-monooxygenase (GMHpaB) from G. mahadii Geo-05, enhances the hydroxylation of 4-hydroxyphenylacetate (HPA) by 85 %. The modeled structure of GMHpaC reveals relatively conserved flavin and NADH binding sites. Modeling and docking studies shed light on structural features and amino acid substitutions that determine GMHpaC's co-factor specificity. The remarkable thermostability, high catalytic activity, and general stability exhibited by GMHpaC position it as a promising enzyme candidate for various industrial applications.

A pH-dependent shift of redox cofactor specificity in a benzyl alcohol dehydrogenase of aromatoleum aromaticum EbN1

We characterise a reversible bacterial zinc-containing benzyl alcohol dehydrogenase (BaDH) accepting either NAD+ or NADP+ as a redox cofactor. Remarkably, its redox cofactor specificity is pH-dependent with the phosphorylated cofactors favored at lower and the dephospho-forms at higher pH. BaDH also shows different steady-state kinetic behavior with the two cofactor forms. From a structural model, the pH-dependent shift may affect the charge of a histidine in the 2′-phosphate-binding pocket of the redox cofactor binding site. The enzyme is phylogenetically affiliated to a new subbranch of the Zn-containing alcohol dehydrogenases, which share this conserved residue. BaDH appears to have some specificity for its substrate, but also turns over many substituted benzyl alcohol and benzaldehyde variants, as well as compounds containing a conjugated C=C double bond with the aldehyde carbonyl group. However, compounds with an sp3-hybridised C next to the alcohol/aldehyde group are not or only weakly turned over. The enzyme appears to contain a Zn in its catalytic site and a mixture of Zn and Fe in its structural metal-binding site. Moreover, we demonstrate the use of BaDH in an enzyme cascade reaction with an acid-reducing tungsten enzyme to reduce benzoate to benzyl alcohol.Key points•Zn-containing BaDH has activity with either NAD+or NADP+at different pH optima.•BaDH converts a broad range of substrates.•BaDH is used in a cascade reaction for the reduction of benzoate to benzyl alcohol.

A pseudoautosomal glycosylation disorder prompts the revision of dolichol biosynthesis

Dolichol is a lipid critical for N-glycosylation as a carrier for activated sugars and nascent oligosaccharides. It is commonly thought to be directly produced from polyprenol by the enzyme SRD5A3. Instead, we found that dolichol synthesis requires a three-step detour involving additional metabolites, where SRD5A3 catalyzes only the second reaction. The first and third steps are performed by DHRSX, whose gene resides on the pseudoautosomal regions of the X and Y chromosomes. Accordingly, we report a pseudoautosomal-recessive disease presenting as a congenital disorder of glycosylation in patients with missense variants in DHRSX (DHRSX-CDG). Of note, DHRSX has a unique dual substrate and cofactor specificity, allowing it to act as a NAD+-dependent dehydrogenase and as a NADPH-dependent reductase in two non-consecutive steps. Thus, our work reveals unexpected complexity in the terminal steps of dolichol biosynthesis. Furthermore, we provide insights into the mechanism by which dolichol metabolism defects contribute to disease.

Engineering of cofactor preference and catalytic activity of methanol dehydrogenase by growth-coupled directed evolution

Methanol, produced from carbon dioxide, natural gas, and biomass, has drawn increasing attention as a promising green carbon feedstock for biomanufacturing due to its sustainable and energy-rich properties. Nicotinamide adenine dinucleotide (NAD+)-dependent methanol dehydrogenase (MDH) catalyzes the oxidation of methanol to formaldehyde via NADH generation, providing a highly active C1 intermediate and reducing power for subsequent biosynthesis. However, the unsatisfactory catalytic efficiency and cofactor bias of MDH significantly impede methanol valorization, especially in nicotinamide adenine dinucleotide phosphate (NADP+)-dependent biosynthesis. Herein, we employed synthetic NADH and NADPH auxotrophic Escherichia coli strains as growth-coupled selection platforms for the directed evolution of MDH from Bacillus stearothermophilus DSM 2334. NADH or NADPH generated by MDH-catalyzed methanol oxidation enabled the growth of synthetic cofactor auxotrophs, establishing a positive correlation between the cell growth rate and MDH activity. Using this principle, MDH mutants exhibiting a 20-fold improvement in catalytic efficiency (kcat/Km) and a 90-fold cofactor specificity switch from NAD+ to NADP+ without a decrease in specific enzyme activity, were efficiently screened from random and semi-rationally designed libraries. We envision that these mutants will advance methanol valorization and that the synthetic cofactor auxotrophs will serve as versatile selection platforms for the evolution of NAD(P)+-dependent enzymes.

Poly-γ-glutamylation of biomolecules

Poly-γ-glutamate tails are a distinctive feature of archaeal, bacterial, and eukaryotic cofactors, including the folates and F420. Despite decades of research, key mechanistic questions remain as to how enzymes successively add glutamates to poly-γ-glutamate chains while maintaining cofactor specificity. Here, we show how poly-γ-glutamylation of folate and F420 by folylpolyglutamate synthases and γ-glutamyl ligases, non-homologous enzymes, occurs via processive addition of L-glutamate onto growing γ-glutamyl chain termini. We further reveal structural snapshots of the archaeal γ-glutamyl ligase (CofE) in action, crucially including a bulged-chain product that shows how the cofactor is retained while successive glutamates are added to the chain terminus. This bulging substrate model of processive poly-γ-glutamylation by terminal extension is arguably ubiquitous in such biopolymerisation reactions, including addition to folates, and demonstrates convergent evolution in diverse species from archaea to humans.

Multi-objective machine learning for designing enzyme cofactor specificity

Multi-objective machine learning for designing enzyme cofactor specificity

Loop 6 and the β-hairpin flap are structural hotspots that determine cofactor specificity in the FMN-dependent family of ene-reductases.

Flavin mononucleotide (FMN)-dependent ene-reductases constitute a large family of oxidoreductases that catalyze the enantiospecific reduction of carbon-carbon double bonds. The reducing equivalents required for substrate reduction are obtained from reduced nicotinamide by hydride transfer. Most ene-reductases significantly prefer, or exclusively accept, either NADPH or NADH. Despite their usefulness in biocatalytic applications, the structural determinants for cofactor preference remain elusive. We employed the NADPH-preferring 12-oxophytodienoic acid reductase 3 from Solanum lycopersicum (SlOPR3) as a model enzyme of the ene-reductase family and applied computational and structural methods to investigate the binding specificity of the reducing coenzymes. Initial docking results indicated that the arginine triad R283, R343, and R366 residing on and close to a critical loop at the active site (loop 6) are the main contributors to NADPH binding. In contrast, NADH binds unfavorably in the opposite direction toward the β-hairpin flap within a largely hydrophobic region. Notably, the crystal structures of SlOPR3 in complex witheither NADPH4 or NADH4 corroborated these different binding modes. Molecular dynamics simulations confirmed NADH binding near the β-hairpin flap and provided structural explanations for the low binding affinity of NADH to SlOPR3. We postulate that cofactor specificity is determined by the arginine triad/loop 6 and the residue(s) controlling access to a hydrophobic cleft formed by the β-hairpin flap. Thus, NADPH preference depends on a properly positioned arginine triad, whereas granting access to the hydrophobic cleft at the β-hairpin flap favors NADH binding.

Structure-Guided Protein Engineering of Glyceraldehyde-3-phosphate Dehydrogenase from Corynebacterium glutamicum for Dual NAD/NADP Cofactor Specificity.

Since the discovery of l-glutamate-producing Corynebacterium glutamicum, it has evolved to be an industrial workhorse. For biobased chemical production, suppling sufficient amounts of the NADPH cofactor is crucial. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme that converts glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate and produces NADH, is a major prospective solution for the cofactor imbalance issue. In this study, we determined the crystal structure of GAPDH from C. glutamicum ATCC13032 (CgGAPDH). Based on the structural information, we generated six CgGAPDH variants, CgGAPDHL36S, CgGAPDHL36S/T37K, CgGAPDHL36S/T37K/P192S, CgGAPDHL36S/T37K/F100V/P192S, CgGAPDHL36S/T37K/F100L/P192S, and CgGAPDHL36S/T37K/F100I/P192S, that can produce both NADH and NAPDH. The final CgGAPDHL36S/T37K/F100V/P192S variant showed a 212-fold increase in enzyme activity for NADP as well as 200% and 30% increased activity for the G3P substrate under NAD and NADP cofactor conditions, respectively. In addition, crystal structures of CgGAPDH variants in complex with NAD(P) permit the elucidation of differences between wild-type CgGAPDH and variants in relation to cofactor stabilization.

Creating NADP+ -Specific Formate Dehydrogenases from Komagataella phaffii by Enzymatic Engineering.

Most natural formate dehydrogenases (FDHs) exhibit NAD+ specificity, making it imperative to explore the engineering of FDH cofactor specificity for NADPH regeneration systems. The endogenous FDH of Komagataella phaffii (K. phaffii), termed KphFDH, is a typical NAD+ -specific FDH. However, investigations into engineering the cofactor specificity of KphFDH have yet to be conducted. To develop an NADP+ -specific variant of KphFDH, we selected D195, Y196, and Q197 as mutation sites and generated twenty site-directed variants. Through kinetic characterization, KphFDH/V19 (D195Q/Y196R/Q197H) was identified as the variant with the highest specificity towards NADP+ , with a ratio of catalytic efficiency (kcat /KM )NADP+ /(kcat /KM )NAD+ of 129.226. Studies of enzymatic properties revealed that the optimal temperature and pH for the reduction reaction of NADP+ catalyzed by KphFDH/V19 were 45 °C and 7.5, respectively. The molecular dynamics (MD) simulation was performed to elucidate the mechanism of high catalytic activity of KphFDH/V19 towards NADP+ . Finally, KphFDH/V19 was applied to an in vitro NADPH regeneration system with Meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum (StDAPDH/H227V). This study successfully created a KphFDH variant with high NADP+ specificity and demonstrated its practical applicability in an in vitro NADPH regeneration system.

Nucleoside-Driven Specificity of DNA Methyltransferase.

We have studied the adenosine binding specificities of two bacterial DNA methyltransferases, Taq methyltransferase (M.TaqI), and HhaI methyltransferase (M.HhaI). While they have similar cofactor binding pocket interactions, experimental data showed different specificity for novel S-nucleobase-l-methionine cofactors (SNMs; N=guanosyl, cytidyl, uridyl). Protein dynamics corroborate the experimental data on the cofactor specificities. For M.TaqI the specificity for S-adenosyl-l-methionine (SAM) is governed by the tight binding on the nucleoside part of the cofactor, while for M.HhaI the degree of freedom of the nucleoside chain allows the acceptance of other bases. The experimental data prove catalytically productive methylation by the M.HhaI binding pocket for all the SNMs. Our results suggest a new route for successful design of unnatural SNM analogues for methyltransferases as a tool for cofactor engineering.

Structure-based enzyme characterization and rational engineering of CgGapDH toward dual NAD/NADP cofactor specificity

Structure-based enzyme characterization and rational engineering of <i>Cg</i>GapDH toward dual NAD/NADP cofactor specificity

Network-wide thermodynamic constraints shape NAD(P)H cofactor specificity of biochemical reactions

The ubiquitous coexistence of the redox cofactors NADH and NADPH is widely considered to facilitate an efficient operation of cellular redox metabolism. However, it remains unclear what shapes the NAD(P)H specificity of specific redox reactions. Here, we present a computational framework to analyze the effect of redox cofactor swaps on the maximal thermodynamic potential of a metabolic network and use it to investigate key aspects of redox cofactor redundancy in Escherichia coli. As one major result, our analysis suggests that evolved NAD(P)H specificities are largely shaped by metabolic network structure and associated thermodynamic constraints enabling thermodynamic driving forces that are close or even identical to the theoretical optimum and significantly higher compared to random specificities. Furthermore, while redundancy of NAD(P)H is clearly beneficial for thermodynamic driving forces, a third redox cofactor would require a low standard redox potential to be advantageous. Our approach also predicts trends of redox-cofactor concentration ratios and could facilitate the design of optimal redox cofactor specificities.

Identification and characterization of archaeal and bacterial F420 -dependent thioredoxin reductases.

The thioredoxin pathway is an antioxidant system present in most organisms. Electrons flow from a thioredoxin reductase to thioredoxin at the expense of a specific electron donor. Most known thioredoxin reductases rely on NADPH as a reducing cofactor. Yet, in 2016, a new type of thioredoxin reductase was discovered in Archaea which utilize instead a reduced deazaflavin cofactor (F420 H2 ). For this reason, the respective enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). To have a broader understanding of the biochemistry of DFTRs, we identified and characterized two other archaeal representatives. A detailed kinetic study, which included pre-steady state kinetic analyses, revealed that these two DFTRs are highly specific for F420 H2 while displaying marginal activity with NADPH. Nevertheless, they share mechanistic features with the canonical thioredoxin reductases that are dependent on NADPH (NTRs). A detailed structural analysis led to the identification of two key residues that tune cofactor specificity of DFTRs. This allowed us to propose a DFTR-specific sequence motif that enabled for the first time the identification and experimental characterization of a bacterial DFTR.

Rank-ordering of known enzymes as starting points for re-engineering novel substrate activity using a convolutional neural network

Retro-biosynthetic approaches have made significant advances in predicting synthesis routes of target biofuel, bio-renewable or bio-active molecules. The use of only cataloged enzymatic activities limits the discovery of new production routes. Recent retro-biosynthetic algorithms increasingly use novel conversions that require altering the substrate or cofactor specificities of existing enzymes while connecting pathways leading to a target metabolite. However, identifying and re-engineering enzymes for desired novel conversions are currently the bottlenecks in implementing such designed pathways. Herein, we present EnzRank, a convolutional neural network (CNN) based approach, to rank-order existing enzymes in terms of their suitability to undergo successful protein engineering through directed evolution or de novo design towards a desired specific substrate activity. We train the CNN model on 11,800 known active enzyme-substrate pairs from the BRENDA database as positive samples and data generated by scrambling these pairs as negative samples using substrate dissimilarity between an enzyme's native substrate and all other molecules present in the dataset using Tanimoto similarity score. EnzRank achieves an average recovery rate of 80.72% and 73.08% for positive and negative pairs on test data after using a 10-fold holdout method for training and cross-validation. We further developed a web-based user interface (available at https://huggingface.co/spaces/vuu10/EnzRank) to predict enzyme-substrate activity using SMILES strings of substrates and enzyme sequence as input to allow convenient and easy-to-use access to EnzRank. In summary, this effort can aid de novo pathway design tools to prioritize starting enzyme re-engineering candidates for novel reactions as well as in predicting the potential secondary activity of enzymes in cell metabolism.

Biochemical and structural insight into the chemical resistance and cofactor specificity of the formate dehydrogenase from Starkeya novella.

Formate dehydrogenases (Fdhs) mediate the oxidation of formate to carbon dioxide and concomitant reduction of nicotinamide adenine dinucleotide (NAD+ ). The low cost of the substrate formate and importance of the product NADH as a cellular source of reducing power make this reaction attractive for biotechnological applications. However, the majority of Fdhs are sensitive to inactivation by thiol-modifying reagents. In this study, we report a chemically resistant Fdh (FdhSNO ) from the soil bacterium Starkeya novella strictly specific for NAD+ . We present its recombinant overproduction, purification and biochemical characterization. The mechanistic basis of chemical resistance was found to be a valine in position 255 (rather than a cysteine as in other Fdhs) preventing the inactivation by thiol-modifying compounds. To further improve the usefulness of FdhSNO as for generating reducing power, we rationally engineered the protein to reduce the coenzyme nicotinamide adenine dinucleotide phosphate (NADP+ ) with better catalytic efficiency than NAD+ . The single mutation D221Q enabled the reduction of NADP+ with a catalytic efficiency kCAT /KM of 0.4 s-1 ·mm-1 at 200 mm formate, while a quadruple mutant (A198G/D221Q/H379K/S380V) resulted in a fivefold increase in catalytic efficiency for NADP+ compared with the single mutant. We determined the cofactor-bound structure of the quadruple mutant to gain mechanistic evidence behind the improved specificity for NADP+ . Our efforts to unravel the key residues for the chemical resistance and cofactor specificity of FdhSNO may lead to wider use of this enzymatic group in a more sustainable (bio)manufacture of value-added chemicals, as for instance the biosynthesis of chiral compounds.