Sequence-based bioprospecting of myo-inositol oxygenase (Miox) reveals new homologues that increase glucaric acid production in Saccharomyces cerevisiae.

myo-Inositol oxygenase (Miox) is a rate-limiting enzyme for glucaric acid production via microbial fermentation. The enzyme converts myo-inositol to glucuronate, which is further converted to glucaric acid, a natural compound with industrial uses that range from detergents to pharmaceutical synthesis to polymeric materials. More than 2,000 Miox sequences are available in the Uniprot database but only thirteen are classified as reviewed in Swiss-Prot (August 2019). In this study, sequence similarity networks were used to identify new homologues to be expressed in Saccharomyces cerevisiae for glucaric acid production. The expression of four homologues did not lead to product formation. Some of these enzymes may have a defective "dynamic lid" - a structural feature important to close the reaction site - which might explain the lack of activity. Thirty-one selected Miox sequences did allow for product formation, of which twenty-five were characterized for the first time. Expression of Talaromyces marneffei Miox led to the accumulation of 1.76 ±â€¯0.33 g glucaric acid/L from 20 g glucose/L and 10 g/L myo-inositol. Specific glucaric acid titer with TmMiox increased 44 % compared to the often-used Arabidopsis thaliana variant AtMiox4 (0.258 vs. 0.179 g glucaric acid/g biomass). AtMiox4 activity decreased from 12.47 to 0.40 nmol/min/mg protein when cells exited exponential phase during growth on glucose, highlighting the importance of future research on Miox stability in order to further improve microbial production of glucaric acid.

Genome-wide analysis of Myo-inositol oxygenase gene family in tomato reveals their involvement in ascorbic acid accumulation.

BACKGROUND: Ascorbic acid (Vitamin C, AsA) is an antioxidant metabolite involved in plant development and environmental stimuli. AsA biosynthesis has been well studied in plants, and MIOX is a critical enzyme in plants AsA biosynthesis pathway. However, Myo-inositol oxygenase (MIOX) gene family members and their involvement in AsA biosynthesis and response to abiotic stress remain unclear. RESULTS: In this study, five tomato genes encoding MIOX proteins and possessing MIOX motifs were identified. Structural analysis and distribution mapping showed that 5 MIOX genes contain different intron/exon patterns and unevenly distributed among four chromosomes. Besides, expression analyses indicated the remarkable expression of SlMIOX genes in different plant tissues. Furthermore, transgenic lines were obtained by over-expression of the MIOX4 gene in tomato. The overexpression lines showed a significant increase in total ascorbate in leaves and red fruits compared to control. Expression analysis revealed that increased accumulation of AsA in MIOX4 overexpression lines is possible as a consequence of the multiple genes involved in AsA biosynthesis. Myo inositol (MI) feeding in leaf and fruit implied that the Myo-inositol pathway improved the AsA biosynthesis in leaves and fruits. MIOX4 overexpression lines exhibited a better light response, abiotic stress tolerance, and AsA biosynthesis capacity. CONCLUSIONS: These results showed that MIOX4 transgenic lines contribute to AsA biosynthesis, evident as better light response and improved oxidative stress tolerance. This study provides the first comprehensive analysis of the MIOX gene family and their involvement in ascorbate biosynthesis in tomato.

[Construction of a glucaric acid biosensor for screening myo-inositol oxygenase variants].

Glucaric acid (GA), a top value-added chemical from biomass, has been widely used for prevention and control of diseases and the production of polymer materials. In GA biosynthesis pathway, the conversion of inositol to glucuronic acid that catalyzed by myo-inositol oxygenase is the limiting step. It is necessary to improve MIOX activity. In the present study, we constructed a high-throughput screening system through combing the concentration of GA with the green fluorescent protein fluorescence intensity. By applying this screening system, three positive variants (K59V/R60A, R171S and D276A) screened from the mutant library. In comparison, the recombinant strain Escherichia coli BL21(DE3)/MU-R171S accumulated more GA, 136.5% of that of the parent strain.

Organophosphonate-degrading PhnZ reveals an emerging family of HD domain mixed-valent diiron oxygenases.

The founding members of the HD-domain protein superfamily are phosphohydrolases, and newly discovered members are generally annotated as such. However, myo-inositol oxygenase (MIOX) exemplifies a second, very different function that has evolved within the common scaffold of this superfamily. A recently discovered HD protein, PhnZ, catalyzes conversion of 2-amino-1-hydroxyethylphosphonate to glycine and phosphate, culminating a bacterial pathway for the utilization of environmentally abundant 2-aminoethylphosphonate. Using Mössbauer and EPR spectroscopies, X-ray crystallography, and activity measurements, we show here that, like MIOX, PhnZ employs a mixed-valent Fe(II)/Fe(III) cofactor for the O2-dependent oxidative cleavage of its substrate. Phylogenetic analysis suggests that many more HD proteins may catalyze yet-unknown oxygenation reactions using this hitherto exceptional Fe(II)/Fe(III) cofactor. The results demonstrate that the catalytic repertoire of the HD superfamily extends well beyond phosphohydrolysis and suggest that the mechanism used by MIOX and PhnZ may be a common strategy for oxidative C-X bond cleavage.

Circular dichroism, magnetic circular dichroism, and variable temperature variable field magnetic circular dichroism studies of biferrous and mixed-valent myo-inositol oxygenase: insights into substrate activation of O2 reactivity.

myo-Inositol oxygenase (MIOX) catalyzes the 4e(-) oxidation of myo-inositol (MI) to D-glucuronate using a substrate activated Fe(II)Fe(III) site. The biferrous and Fe(II)Fe(III) forms of MIOX were studied with circular dichroism (CD), magnetic circular dichroism (MCD), and variable temperature variable field (VTVH) MCD spectroscopies. The MCD spectrum of biferrous MIOX shows two ligand field (LF) transitions near 10000 cm(-1), split by ~2000 cm(-1), characteristic of six coordinate (6C) Fe(II) sites, indicating that the modest reactivity of the biferrous form toward O2 can be attributed to the saturated coordination of both irons. Upon oxidation to the Fe(II)Fe(III) state, MIOX shows two LF transitions in the ~10000 cm(-1) region, again implying a coordinatively saturated Fe(II) site. Upon MI binding, these split in energy to 5200 and 11200 cm(-1), showing that MI binding causes the Fe(II) to become coordinatively unsaturated. VTVH MCD magnetization curves of unbound and MI-bound Fe(II)Fe(III) forms show that upon substrate binding, the isotherms become more nested, requiring that the exchange coupling and ferrous zero-field splitting (ZFS) both decrease in magnitude. These results imply that MI binds to the ferric site, weakening the Fe(III)-µ-OH bond and strengthening the Fe(II)-µ-OH bond. This perturbation results in the release of a coordinated water from the Fe(II) that enables its O2 activation.

Structural, EPR, and Mössbauer characterization of (µ-alkoxo)(µ-carboxylato)diiron(II,III) model complexes for the active sites of mixed-valent diiron enzymes.

To obtain structural and spectroscopic models for the diiron(II,III) centers in the active sites of diiron enzymes, the (µ-alkoxo)(µ-carboxylato)diiron(II,III) complexes [Fe(II)Fe(III)(N-Et-HPTB)(O(2)CPh)(NCCH(3))(2)](ClO(4))(3) (1) and [Fe(II)Fe(III)(N-Et-HPTB)(O(2)CPh)(Cl)(HOCH(3))](ClO(4))(2) (2) (N-Et-HPTB = N,N,N',N'-tetrakis(2-(1-ethyl-benzimidazolylmethyl))-2-hydroxy-1,3-diaminopropane) have been prepared and characterized by X-ray crystallography, UV-visible absorption, EPR, and Mössbauer spectroscopies. Fe1-Fe2 separations are 3.60 and 3.63 Å, and Fe1-O1-Fe2 bond angles are 128.0° and 129.4° for 1 and 2, respectively. Mössbauer and EPR studies of 1 show that the Fe(III) (S(A) = 5/2) and Fe(II) (S(B) = 2) sites are antiferromagnetically coupled to yield a ground state with S = 1/2 (g= 1.75, 1.88, 1.96); Mössbauer analysis of solid 1 yields J = 22.5 ± 2 cm(-1) for the exchange coupling constant (H = JS(A)·S(B) convention). In addition to the S = 1/2 ground-state spectrum of 1, the EPR signal for the S = 3/2 excited state of the spin ladder can also be observed, the first time such a signal has been detected for an antiferromagnetically coupled diiron(II,III) complex. The anisotropy of the (57)Fe magnetic hyperfine interactions at the Fe(III) site is larger than normally observed in mononuclear complexes and arises from admixing S > 1/2 excited states into the S = 1/2 ground state by zero-field splittings at the two Fe sites. Analysis of the "D/J" mixing has allowed us to extract the zero-field splitting parameters, local g values, and magnetic hyperfine structural parameters for the individual Fe sites. The methodology developed and followed in this analysis is presented in detail. The spin Hamiltonian parameters of 1 are related to the molecular structure with the help of DFT calculations. Contrary to what was assumed in previous studies, our analysis demonstrates that the deviations of the g values from the free electron value (g = 2) for the antiferromagnetically coupled diiron(II,III) core in complex 1 are predominantly determined by the anisotropy of the effective g values of the ferrous ion and only to a lesser extent by the admixture of excited states into ground-state ZFS terms (D/J mixing). The results for 1 are discussed in the context of the data available for diiron(II,III) clusters in proteins and synthetic diiron(II,III) complexes.

The effects of protein environment and dispersion on the formation of ferric-superoxide species in myo-inositol oxygenase (MIOX): a combined ONIOM(DFT:MM) and energy decomposition analysis.

The catalytic reaction of myo-inositol oxygenase, a nonheme diiron enzyme, is initiated by the binding of an O(2) molecule to the ferrous center of a mixed-valence Fe(II)Fe(III) intermediate. This generates a (superoxo)Fe(III)Fe(III) reactive species that abstracts a hydrogen atom from the myo-inositol substrate. To understand the effects of protein environment and intracluster dispersion on this O(2)-binding process, we undertook a combined ONIOM(B3LYP:AMBER) and energy decomposition analysis. The interaction energy between the active site and the thousands of atoms present in the protein environment was decomposed into electrostatic, van der Waals (vdW) and polarization terms. These terms were further decomposed into contributions from individual amino acid residues. The dispersion effect, which is not adequately accounted for by the B3LYP method, was estimated in an empirical manner. The results show that the electrostatic, vdW, and polarization effects slightly enhance the O(2) binding process. The dispersion effect enhances O(2) binding more significantly than these effects. Despite these stabilizing effects, the entropy effect disfavors O(2) binding, making the process almost thermoneutral.

[Recent progress in the theoretical studies of structure, function, and reaction of biological molecules].

Essential biomolecular functions often involve electron-related events such as chemical reactions and photoluminescence phenomena. Theoretical description of such electronic processes requires the use of quantum mechanics (QM), but the number of atoms that can be handled with QM is usually smaller than the number of atoms present in a single protein. A reasonable strategy is therefore to give priority to a few tens or hundreds of atoms in the system and deal with them quantum mechanically. Lower-priority atoms influence the event occurring in the higher-priority area; therefore, their effect should also be taken into account. Under these circumstances, a reasonable approach is to apply two or more different theoretical methods to differently prioritized subsystems. QM can be combined, for example, with less accurate yet much less demanding molecular mechanics (MM). Our own N-layered integrated molecular orbital and molecular mechanics (ONIOM) method allows for such hybrid calculations, and our group has been applying it to a wide range of biology-related problems. In this paper, we briefly explain the theoretical background and the procedure for the theoretical investigation of biological systems. Subsequently, we provide an overview of some of our recent studies of metalloenzymes and photobiology-related problems.

Cloning of Glucuronokinase from Arabidopsis thaliana, the last missing enzyme of the myo-inositol oxygenase pathway to nucleotide sugars.

Nucleotide sugars are building blocks for carbohydrate polymers in plant cell walls. They are synthesized from sugar-1-phosphates or epimerized as nucleotide sugars. The main precursor for primary cell walls is UDP-glucuronic acid, which can be synthesized via two independent pathways. One starts with the ring cleavage of myo-inositol into glucuronic acid, which requires a glucuronokinase and a pyrophosphorylase for activation into UDP-glucuronate. Here we report on the purification of glucuronokinase from Lilium pollen. A 40-kDa protein was purified combining six chromatographic steps and peptides were de novo sequenced. This allowed the cloning of the gene from Arabidopsis thaliana and the expression of the recombinant protein in Escherichia coli for biochemical characterization. Glucuronokinase is a novel member of the GHMP-kinase superfamily having an unique substrate specificity for d-glucuronic acid with a K(m) of 0.7 mm. It requires ATP as phosphate donor (K(m) 0.56 mm). In Arabidopsis, the gene is expressed in all plant tissues with a preference for pollen. Genes for glucuronokinase are present in (all) plants, some algae, and a few bacteria as well as in some lower animals.

Insights into the (superoxo)Fe(III)Fe(III) intermediate and reaction mechanism of myo-inositol oxygenase: DFT and ONIOM(DFT:MM) study.

The (superoxo)Fe(III)Fe(III) reactive species and the catalytic reaction mechanism of a diiron enzyme, myo-inositol oxygenase (MIOX), were theoretically investigated by means of density functional theory (DFT) and ONIOM quantum mechanical/molecular mechanical (QM/MM) approaches. The ground state of the (superoxo)Fe(III)Fe(III) intermediate was shown to have a side-on coordination geometry and an S = 1/2 spin state, wherein the two iron sites are antiferromagnetically coupled while the superoxide site and the nearest iron are ferromagnetically coupled. A full reaction pathway leading to a D-glucuronate product from myo-inositol was proposed based on ONIOM computational results. Two major roles of the enzyme surrounding during the catalytic reaction were identified. One is to facilitate the initial H-abstraction step, and the other is to restrict the movement of the substrate via H-bonding interactions in order to avoid unwanted side reactions. In our proposed mechanism, O-O bond cleavage has the highest barrier, thus constituting the rate-limiting step of the reaction. The unique role of the bridging hydroxide ligand as a catalytic base was also identified.

myo-Inositol oxygenase: a radical new pathway for O(2) and C-H activation at a nonheme diiron cluster.

The enzyme myo-inositol oxygenase (MIOX) catalyzes conversion of myo-inositol (cyclohexan-1,2,3,5/4,6-hexa-ol or MI) to d-glucuronate (DG), initiating the only known pathway in humans for catabolism of the carbon skeleton of cell-signaling inositol (poly)phosphates and phosphoinositides. Recent kinetic, spectroscopic and crystallographic studies have shown that the enzyme activates its substrates, MI and O(2), at a carboxylate-bridged nonheme diiron(ii/iii) cluster, making it the first of many known nonheme diiron oxygenases to employ the mixed-valent form of its cofactor. Evidence suggests that: (1) the Fe(iii) site coordinates MI via its C1 and C6 hydroxyl groups; (2) the Fe(ii) site reversibly coordinates O(2) to produce a superoxo-diiron(iii/iii) intermediate; and (3) the pendant oxygen atom of the superoxide ligand abstracts hydrogen from C1 to initiate the unique C-C-bond-cleaving, four-electron oxidation reaction. This review recounts the studies leading to the recognition of the novel cofactor requirement and catalytic mechanism of MIOX and forecasts how remaining gaps in our understanding might be filled by additional experiments.

Computational modeling and in silico analysis of differential regulation of myo-inositol catabolic enzymes in Cryptococcus neoformans.

BACKGROUND: Inositol is a key cellular metabolite for many organisms. Cryptococcus neoformans is an opportunistic pathogen which primarily infects the central nervous system, a region of high inositol concentration, of immunocompromised individuals. Through the use of myo-inositol oxygenase C. neoformans can catabolize inositol as a sole carbon source to support growth and viability. RESULTS: Three myo-inositol oxygenase gene sequences were identified in the C. neoformans genome. Differential regulation was suggested by computational analyses of the three gene sequences. This included examination of the upstream regulatory regions, identifying ORE/TonE and UASINO sequences, conserved introns/exons, and in frame termination sequences. Homology modeling of the proteins encoded by these genes revealed key differences in the myo-inositol active site. CONCLUSION: The results suggest there are two functional copies of the myo-inositol oxygenase gene in the C. neoformans genome. The functional genes are differentially expressed in response to environmental inositol concentrations. Both the upstream regulatory regions of the genes and the structure of the specific proteins suggest that MIOX1 would function when inositol concentrations are low, whereas MIOX2 would function when inositol concentrations are high.

Crystal structure of a substrate complex of myo-inositol oxygenase, a di-iron oxygenase with a key role in inositol metabolism.

Altered metabolism of the inositol sugars myo-inositol (MI) and d-chiro-inositol is implicated in diabetic complications. In animals, catabolism of MI and D-chiro-inositol depends on the enzyme MI oxygenase (MIOX), which catalyzes the first committed step of the glucuronate-xylulose pathway, and is found almost exclusively in the kidneys. The crystal structure of MIOX, in complex with MI, has been determined by multiwavelength anomalous diffraction methods and refined at 2.0-A resolution (R=0.206, Rfree=0.253). The structure reveals a monomeric, single-domain protein with a mostly helical fold that is distantly related to the diverse HD domain superfamily. Five helices form the structural core and provide six ligands (four His and two Asp) for the di-iron center, in which the two iron atoms are bridged by a putative hydroxide ion and one of the Asp ligands, Asp-124. A key loop forms a lid over the MI substrate, which is coordinated in bidentate mode to one iron atom. It is proposed that this mode of iron coordination, and interaction with a key Lys residue, activate MI for bond cleavage. The structure also reveals the basis of substrate specificity and suggests routes for the development of specific MIOX inhibitors.

Purification, crystallization and preliminary crystallographic analysis of mouse myo-inositol oxygenase.

Myo-inositol oxygenase (MIOX) catalyzes the novel oxidative cleavage of myo-inositol (MI) and its epimer D-chiro inositol (DCI) to D-glucuronate. MIOX utilizes an Fe(II)/Fe(III) binuclear iron centre for the dioxygen-dependent cleavage of the C1-C6 bond in MI. Despite its key role in inositol metabolism, the structural basis of its unique four-electron oxidation mechanism and its substrate specificity remain unknown. In order to answer these questions and to facilitate the use of this key enzyme for the development of new therapeutic strategies for diabetes, the mouse enzyme has been cloned, expressed in Escherichia coli, purified and crystallized from 4.4 M sodium formate. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 44.87, b = 77.26, c = 84.84 angstroms, and diffract to 2.8 angstroms resolution.

Demonstration by 2H ENDOR spectroscopy that myo-inositol binds via an alkoxide bridge to the mixed-valent diiron center of myo-inositol oxygenase.

myo-Inositol oxygenase (MIOX) is a non-heme diiron oxygenase that cleaves cyclohexane-(1,2,3,5/4,6-hexa)-ol (myo-inositol, MI) to d-glucuronate. Here, we use 2H ENDOR spectroscopy to demonstrate that MI binds to the diiron(II/III) cofactor of MIOX via an alkoxide bridge, most likely involving O1. Analysis shows that MI adopts a symmetrical geometry in which the O-C-2H plane of the bridge is approximately orthogonal to the Fe-O-Fe plane.

Evidence for C-H cleavage by an iron-superoxide complex in the glycol cleavage reaction catalyzed by myo-inositol oxygenase.

myo-Inositol oxygenase (MIOX) activates O2 at a mixed-valent nonheme diiron(II/III) cluster to effect oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate [myo-inositol (MI)] by four electrons to d-glucuronate. Abstraction of hydrogen from C1 by a formally (superoxo)diiron(III/III) intermediate was previously proposed. Use of deuterium-labeled substrate, 1,2,3,4,5,6-[2H]6-MI (D6-MI), has now permitted initial characterization of the C-H-cleaving intermediate. The MIOX.1,2,3,4,5,6-[2H]6-MI complex reacts rapidly and reversibly with O2 to form an intermediate, G, with a g = (2.05, 1.98, 1.90) EPR signal. The rhombic g-tensor and observed hyperfine coupling to 57Fe are rationalized in terms of a (superoxo)diiron(III/III) structure with coordination of the superoxide to a single iron. G decays to H, the intermediate previously detected in the reaction with unlabeled substrate. This step is associated with a kinetic isotope effect of > or =5, showing that the superoxide-level complex does indeed cleave a C-H(D) bond of MI.

A coupled dinuclear iron cluster that is perturbed by substrate binding in myo-inositol oxygenase.

myo-Inositol oxygenase (MIOX) uses iron as its cofactor and dioxygen as its cosubstrate to effect the unique, ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate to d-glucuronate. The nature of the iron cofactor and its interaction with the substrate, myo-inositol (MI), have been probed by electron paramagnetic resonance (EPR) and Mössbauer spectroscopies. The data demonstrate the formation of an antiferromagnetically coupled, high-spin diiron(III/III) cluster upon treatment of solutions of Fe(II) and MIOX with excess O(2) or H(2)O(2) and the formation of an antiferromagnetically coupled, valence-localized, high-spin diiron(II/III) cluster upon treatment with either limiting O(2) or excess O(2) in the presence of a mild reductant (e.g., ascorbate). Marked changes to the spectra of both redox forms upon addition of MI and analogy to changes induced by binding of phosphate to the diiron(II/III) cluster of the protein phosphatase, uteroferrin, suggest that MI coordinates directly to the diiron cluster, most likely in a bridging mode. The addition of MIOX to the growing family of non-heme diiron oxygenases expands the catalytic range of the family beyond the two-electron oxidation (hydroxylation and dehydrogenation) reactions catalyzed by its more extensively studied members such as methane monooxygenase and stearoyl acyl carrier protein Delta(9)-desaturase.