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Results show that 3-chlorocatechol degradation follows a dechlorination or dehalogenation step catalyzed by a muconolactone isomerase-related enzyme by Rhodococcus opacus 1CP. Data also point out to a genetic background for the degradation pathway.

Research has been conducted on the monofluorophenols. Results demonstrate that fluoropyrogallols are involved in the monofluorophenol biotransformation in Rhodococcus opacus 1cp.

A study was conducted to describe the gene macA encoding the microbial enzyme maleylacetate reductases, in the degradation of chlorocatechols or 5-chlorohydroxyquinol through an analysis of cloned chloroaromatic compound-utilizing proteobacteria, such as the chlorophenol-degrading strain Rhodococcus opacus. Genetic encodings of maleylacetate reductases were found in specialized chlorocatechol gene clusters. Analysis showed that maleylacetate reductase macA in R opacus was not a part of the gene cluster.

Cloning and sequence analyses were performed on a DNA fragment from Rhodococcus opacus, which comprised the gene for the 3-oxoadipate enol-lactone-hydrolyzing enzyme of strain 1CP. The results revealed that the gene was part of a protocatechuate catabolic gene cluster. The gene was also found to be connected to the pcaC gene, which codes for a different 4-carboxymuconolactone decarboxylase in proteobacteria. Moreover, the transcription of the pcal, coding for a subgroup of succinyl-CoA:3-oxoadipate CoA-transferase, was divergent from pcaH.

Polymerase chain reaction with primers obtained from sequences of N termini and peptides of purified chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase was used to clone the chlorocatechol catabolic enzymes from Rhodococcus opacus 1CP. Sequencing analysis showed that the clones were from different areas of the same gene cluster. The results further revealed that the transcription of clcB gene for chloromuconate was divergent from a gene coding a LysR-type regulatory protein.

A fragment of the Rhodococcus erythropolis 1CP catechol gene cluster was cloned and sequenced to characterize the pathways of catechol and chlorocatechol catabolism. DNA sequence analysis of the Rhodococcus erythropolis 1CP muconate cycloisomerase-encoding region indicated the presence of five open reading frames. However, the cloned DNA did not contain the complete catA gene for catechol 1,2-dioxygenase. Furthermore, the catechol 1,2-dioxygenase of the microbe was similar to the chlorocatechol dioxygenases of gram-negative strains.

Standard procedures enable the purification of 6-chlorohydroxyquinol 1,2-dioxygenase from 2,4,6-trichlorophenol-grown cells of Streptomyces rochei 303. A comparative study of enzymatic activity of the S. rochei enzyme with that of Azotobacter sp. strain GP1 reveals that S. rochei enzyme prefers 6-chlorohydroxyquinol over hydroxyquinol. Sequence analysis reveals significant similarity between these enzymes in the NH2-terminal amino acid sequences. The enzyme has 1 mol of iron per mol of enzyme and contains of two identical 31-kDa subunits.

Research has been conducted on 1,2-dioxygenase, chloromuconolactone dehalogenase and chloromuconate cycloisomerase from Rhodococcus opacus 1cp grown on 2-chlorophenol. Results demonstrate that 2-fluoromuconate can be converted to 5-fluoromuconolactone by chloromuconate cycloisomerase, and that 5-fluoromuconolactone can be dehalogenated to cis-dienelactone by chloromuconolactone dehalogenase.

This work was aimed at studying the response of soil non-spore-forming actinobacterial strainArthrobacter agilisLush 13 to changing natural conditions, such as nutrient availability and the presence of degradable and recalcitrant aliphatic and aromatic substrates. TheA. agilisstrain Lush13 was able to degrade octane, nonane, hexadecane, benzoate, phenol, and 2,3-, 2,4-, 2,5-, 2,6-dichlorophenols, but not grew on 3,4-dichlorophenol, 2,3,4-, 2,4,5-, 2,4,6-trichlorophenol (TCP), pentachlorophenol (PCP), 2-chlorobenzoate, 3-chlorobenzoate, 3,5-dichlorobenzoate, 2,4-dichlorobenzoate. Under growth-arresting conditions due to nitrogen- or multiple starvation or recalcitrant (non-utilizable) carbon source, the studied strain preserved viability for prolonged periods (4–24 months) due to transition to dormancy in the form of conglomerated small and ultrasmall cyst-like dormant cells (CLC). Dormant cells were shown to germinate rapidly (30 min or later) after removal of starvation stress, and this process was followed by breakdown of conglomerates with the eliberation and further division of small multiple actively growing daughter cells. Results of this study shed some light to adaptive capabilities of soil arthrobacters in pure and polluted environments. [ABSTRACT FROM PUBLISHER]

The process of benzoate degradation by strainRhodococcus opacus1CP after a five-year dormancy was investigated and its peculiarities were revealed. The strain was shown to be capable of growth on benzoate at a concentration of up to 10 g L−1. The substrate specificity of benzoate dioxygenase (BDO) during the culture growth on a medium with a low (200–250 mg L−1) and high (4 g L−1) concentration of benzoate was assessed. BDO ofR. opacus1CP was shown to be an extremely narrow specificity enzyme. Out of 31 substituted benzoates, only with one, 3-chlorobenzoate, its activity was higher than 9% of that of benzoate. Two dioxygenases, catechol 1,2-dioxygenase (Cat 1,2-DO) and protocatechuate 3,4-dioxygenase (PCA 3,4-DO), were identified in a cell-free extract, purified and characterized. The substrate specificity of Cat 1,2-DO isolated from cells of strain 1CP after the dormancy was found to differ significantly from that of Cat 1,2-DO isolated earlier from cells of this strain grown on benzoate. By its substrate specificity, the described Cat 1,2-DO was close to the Cat 1,2-DO from strain 1CP grown on 4-methylbenzoate. Neither activity nor inhibition by protocatechuate was observed during the reaction of Cat 1,2-DO with catechol, and catechol had no inhibitory effect on the reaction of PCA 3,4-DO with protocatechuate. [ABSTRACT FROM AUTHOR]

This work investigated the distribution of theclcFgene in actinobacteria isolated from different ecotopes. The gene encodes chloromuconolactone dehalogenase (CMLD) ClcF, the enzyme found to date in only one representative of Gram‐positive bacteria,Rhodococcus opacus1CP, adapted to 2‐chlorophenol (2CP). Using primers specific to theclcFgene, from the DNA matrix of rhodococcal strains closely related to speciesRhodococcus wratislaviensis(P1, P12, P13, P20, G10, KT112, KT723, BO1) we obtained PCR products whose nucleotide sequences were 100% identical to that of theclcFgene from strainR. opacus1CP. CMLDs isolated from the biomass of strainsRhodococcusspp. G10 and P1 grown on 2CP did not differ by their subunit molecular mass deduced from the known amino acid sequence of theclcFgene from the ClcF of strainR. opacus1CP. Matrix‐assisted laser dissociation/ionization time‐of‐flight mass spectrometry showed the presence of a peak withm/z11,194–11,196 Da both in whole cells and in protein solutions with a ClcF activity. Thus, we have first time shown the distribution of ClcF among actinobacteria isolated from geographically distant habitats. [ABSTRACT FROM PUBLISHER]

Abstract: Biodegradation of hydrophobic organic contaminants is often accompanied by the accumulation of hydrophobic intermediates. This paper is devoted to the experimental evaluation of electron balance between the hydrophobic intermediates and the other biodegradation products. The ratio of the fraction of electrons that are not held in the hydrophobic intermediates to the total fraction of electrons released from the substrate during its conversion is called a degradation level (DL). Under certain conditions, the DL value is equal to the angular coefficient of the plot in the coordinates , where S and COD are current values of substrate concentration and chemical oxygen demand of the ethylacetate extract from the culture; S 0 and COD0 are the respective initial values. This approach has been used for analysis of the conversion of PAHs by Sphingomonas sp. VKM B-2434. The DL values obtained correspond well to most published data on PAH metabolism by this strain. The DL values significantly increased as a result of addition of the strains consuming the products of conversion of PAHs by Sphingomonas sp. VKM B-2434. The proposed method seems useful for characterizing biodegradation of hydrophobic pollutants by both pure and mixed microbial cultures. [Copyright &y& Elsevier]

Chlorophenols belong to the group of toxic and persistent to microbial attack xenobiotics. Nevertheless, due to the adaptation microorganisms acquire the ability to use chlorophenols as the sole source of carbon and energy. The present review describes the diversity of aerobic pathways for the utilization of halogenated phenols by bacteria with the emphasis on the main reactions and intermediates formed, enzymes responsible for these reactions and their genetic basis. Taking into account (i) the fact that enzymes degrading chlorophenols are similar to the ones involved in the conversion of other (chloro)aromatic compounds and (ii) that present numerous publications describing the properties of separated enzymes or encoding their genes are published, this review was planned as the attempt to present both, the most general and specific aspects in chlorophenols degradation with the emphasis on the literature of the last ten years. [ABSTRACT FROM AUTHOR]

In this paper we report the isolation and characterization of an anaerobic enrichment culture as well as of a Rhodococcus sp. strain 2 capable of degrading 3,4-dihaloanilines under nitrate reducing conditions. Using mass spectrometry several of the intermediates formed in the process of 3,4-dichloroaniline conversion were identified. Most interesting is the observation of reductive deamination and the formation of 1,2-dichlorobenzene as one of the intermediates. Using 19F NMR and fluorinated 3,4-dihaloaniline model substrates it was corroborated that reductive deamination of the anilines to give dihalobenzene intermediates represents a new initial step in the anaerobic microbial degradation of these halogenated anilines. [Copyright &y& Elsevier]

Analyzes the biochemical characterization of the muconate and the chloromuconate chcloisomerases of the chlorophenol-utilizing rhodoccus erythropolis. Details on aerobic degradation of aromatic compounds; Components of the degradation of chlorinated aromatic compounds; Purification of genomic and plasmid DNAs; Cloning of the muconate cycloisomeras-encoding region; Status of genes from erythropolis.

Chlorocatechol 1,2-dioxygenase from Rhodococcus erythropolis 1CP was purified to homogeneity. In contrast to chlorocatechol 1,2-dioxygenase from Gram-negative strains which have a very broad substrate tolerance, the Rhodococcus enzyme was relatively more specific and had a distinct preference for 4-substituted catechols. Protein and metal analysis indicate an unusual stoichiometry of one atom each of iron and manganese/64-kDa homodimer. The N-terminal amino acid sequence (27 residues) of the Rhodococcus chlorocatechol 1,2-dioxygenase was determined and exhibited 15-22% identity to the published sequences of catechol 1,2-dioxygenases and other chlorocatechol 1,2-dioxygenases. Antiserum was raised in rabbits and antibodies against Rhodococcus chlorocatechol 1,2-dioxygenase were affinity purified. Dot-blot analysis revealed a very weak reaction between the antibodies and partially purified chlorocatechol 1,2-dioxygenases from Alcaligenes eutrophus JMP134 and Pseudomonas putida 87. No reaction between these antibodies and above enzymes was observed using Western blotting. Kinetic and immunochemical data as well as comparison of subunit molecular mass and suggest that the Rhodococcus enzyme differs significantly from the known highly similar chlorocatechol 1,2-dioxygenases of Gram-negative strains and seems to be only distantly related to them. [ABSTRACT FROM AUTHOR]

The blue laccase from the white-rot basidiomycete fungus Panus tigrinus, an enzyme involved in lignin biodegradation, has been crystallized. P. tigrinus laccase crystals grew within one week at 296 K using the sitting-drop vapour- diffusion method in 22%(w/v) PEG 4000, 0.2 M CaCl2, 100 mM Tris-HCl pH 7.5. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 54.2, b = 111.6, c = 97.1, β = 97.7°, and contain 46% solvent. A complete native data set was collected to 1.4 Å resolution at the copper edge. Molecular replacement using the Coprinus cinereus laccase structure (PDB code 1 hfu) as a starting model was performed and initial electron-density maps revealed the presence of a full complement of copper ions. Model refinement is in progress. The P. tigrinus laccase structural model exhibits the highest resolution available to date and will assist in further elucidation of the catalytic mechanism and electron-transfer processes for this class of enzymes. [ABSTRACT FROM AUTHOR]

Of all NMR observable isotopes 19F is the one perhaps most convenient for studies on biodegradation of environmental pollutants. The reasons underlying this potential of 19F NMR are discussed and illustrated on the basis of a study on the biodegradationof fluorophenols by four Rhodococcus strains. The results indicate marked differences between the biodegradation pathways of fluorophenols among the various Rhodococcus species. This holds not only for the level and nature of the fluorinated biodegradation pathway intermediates that accumulate, but also for the regioselectivity of the initialhydroxylation step. Several of the Rhodococcus species contain a phenol hydroxylase that catalyses the oxidative defluorination of ortho-fluorinated di- and trifluorophenols. Furthermore, it is illustrated how the 19F NMR technique can be used as a tool in the process of identification of an accumulated unknown metabolite, in this case most likely 5-fluoromaleylacetate. Altogether, the 19F NMR technique proved valid to obtain detailed information on the microbial biodegradation pathways of fluorinated organics, but also to provide information on the specificity of enzymes generally considered unstable and, for this reason, not much studied so far. [ABSTRACT FROM AUTHOR]

Abstract: 5-Chloromuconolactone dehalogenase (5-CMLD) is a unique enzyme that catalyzes the conversion of 5-chloromuconolactone into cis-dienelactone in the new modified ortho-pathway of the 3-chlorocatechol degradation by Rhodococcus opacus 1CP. In all other known chlorocatechol pathways the dehalogenation is a spontaneous secondary reaction of the unstable chloromuconate intermediate following the lactonization process catalyzed by the muconate cycloisomerases. The crystallographic structure of the decameric 5-CMLD was solved by Molecular Replacement, using the coordinates of the low resolution structure of the highly homologous muconolactone isomerase, an enzyme of the conventional ortho-pathway. Muconolactone isomerase catalyzes the endocyclic rearrangement of the double bond within the lactone ring of muconolactone to yield 3-oxoadipate enol lactone. Although both 5-CMLD and muconolactone isomerase share the ability to dechlorinate 5-chloromuconolactone, 5-CMLD shows a significant degree of specialization, having lost the capacity to convert its original substrate muconolactone. The active site of 5-CMLD was previously hypothesized to reside in a deep pocket at the interface of two different subunits, on the basis of a muconolactone isomerase structure analysis. In this study we also performed molecular docking calculations that confirmed these previous findings, and allowed us furthermore to determine the residues involved in the catalytic process. [Copyright &y& Elsevier]

This work investigates the ability of representatives of four actinobacterial genera ( Arthrobacter , Microbacterium , Rhodococcus , Gordonia ) to degrade benzoic acid and its hydroxylated or chlorinated analogs. Benzoate, 3-chlorobenzoate and 4-hydroxybenzoate were shown to support growth of most strains. We determined the maximal specific growth rates and doubling times for bacteria grown on these substrates. We examined the substrate specificities of actinobacterial benzoate dioxygenases towards 16 aromatic chemicals. The specificities varied among species, they were narrow for Rhodococcus opacus 1CP and Gordonia polyisoprenivorans 135 and broad for Rhodococcus ruber P25 and Microbacterium sp. B51. The genes encoding the benzoate dioxygenase α-subunit from R. opacus 1CP, R. opacus 6a, R. ruber P25, Rhodococcus wratislaviensis P1 and R. wratislaviensis G10 which were isolated at distant sites exhibited between 88 and 97% homology with the homologous gene of Rhodococcus jostii RHA1. This indicates a common origin of these genes. Notably, in some strains, catechol 1,2-dioxygenase and gentisate dioxygenase were induced simultaneously during growth on benzoate. [ABSTRACT FROM AUTHOR]

Abstract: The crystallographic structures of 4-chlorocatechol 1,2-dioxygenase (4-CCD) complexes with 3,5-dichlorocatechol, protocatechuate (3,4-dihydroxybenzoate), hydroxyquinol (benzen-1,2,4-triol) and pyrogallol (benzen-1,2,3-triol), which act as substrates or inhibitors of the enzyme, have been determined and analyzed. 4-CCD from the Gram-positive bacterium Rhodococcus opacus 1CP is a Fe(III) ion containing enzyme specialized in the aerobic biodegradation of chlorocatechols. The structures of the 4-CCD complexes show that the catechols bind the catalytic iron ion in a bidentate mode displacing Tyr169 and the benzoate ion (found in the native enzyme structure) from the metal coordination sphere, as found in other adducts of intradiol dioxygenases with substrates. The analysis of the present structures allowed to identify the residues selectively involved in recognition of the diverse substrates. Furthermore the structural comparison with the corresponding complexes of catechol 1,2-dioxygenase from the same Rhodococcus strain (Rho-1,2-CTD) highlights significant differences in the binding of the tested catechols to the active site of the enzyme, particularly in the orientation of the aromatic ring substituents. As an example the 3-substituted catechols are bound with the substituent oriented towards the external part of the 4-CCD active site cavity, whereas in the Rho-1,2-CTD complexes the 3-substituents were placed in the internal position. The present crystallographic study shed light on the mechanism that allows substrate recognition inside this class of high specific enzymes involved in the biodegradation of recalcitrant pollutants. [Copyright &y& Elsevier]

Abstract: The first crystallographic structures of a catechol 1,2-dioxygenase from a Gram-positive bacterium Rhodococcus opacus 1CP (Rho 1,2-CTD), a Fe(III) ion containing enzyme specialized in the aerobic biodegradation of catechols, and its adducts with catechol, 3-methylcatechol, 4-methylcatechol, pyrogallol (benzene-1,2,3-triol), 3-chlorocatechol, 4-chlorocatechol, 3,5-dichlorocatechol, 4,5-dichlorocatechol and protocatechuate (3,4-dihydroxybenzoate) have been determined and analyzed. This study represents the first extensive characterization of catechols adducts of 1,2-CTDs. The structural analyses reveal the diverse modes of binding to the active metal iron ion of the tested catechols thus allowing to identify the residues selectively involved in recognition of the diverse substrates by this class of enzymes. The comparison is further extended to the structural and functional characteristics of the other 1,2-CTDs isolated from Gram-positive and Gram-negative bacteria. Moreover the high structural homology of the present enzyme with the 3-chlorocatechol 1,2-dioxygenase from the same bacterium are discussed in terms of their different substrate specificity. The catalytic rates for Rho 1,2-CTD conversion of the tested compounds are also compared with the calculated energies of the highest occupied molecular orbital (E HOMO) of the substrates. A quantitative relationship (R =0.966) between the ln k cat and the calculated electronic parameter E HOMO was obtained for catechol, 3-methylcatechol, 4-methylcatechol, pyrogallol, 3-chlorocatechol, 4-chlorocatechol. This indicates that for these substrates the rate-limiting step of the reaction cycle is dependent on their nucleophilic reactivity. The discrepancies observed in the quantitative relationship for 3,5-dichlorocatechol, 4,5-dichlorocatechol and protocatechuate are ascribed to the sterical hindrances leading to the distorted binding of such catechols observed in the corresponding structures. [Copyright &y& Elsevier]

Abstract: The crystal structure of the 3-chlorocatechol 1,2-dioxygenase from the Gram-positive bacterium Rhodococcus opacus (erythropolis) 1CP, a Fe(III) ion-containing enzyme specialized in the aerobic biodegradation of 3-chloro- and methyl-substituted catechols, has been solved by molecular replacement techniques using the coordinates of 4-chlorocatechol 1,2-dioxygenase from the same organism (PDB code 1S9A) as a starting model and refined at 1.9 Å resolution (R free 21.9%; R-factor 17.4%). The analysis of the structure and of the kinetic parameters for a series of different substrates, and the comparison with the corresponding data for the 4-chlorocatechol 1,2-dioxygenase isolated from the same bacterial strain, provides evidence of which active site residues are responsible for the observed differences in substrate specificity. Among the amino acid residues expected to interact with substrates, only three are altered Val53(Ala53), Tyr78(Phe78) and Ala221(Cys224) (3-chlorocatechol 1,2-dioxygenase(4-chlorocatechol 1,2-dioxygenase)), clearly identifying the substitutions influencing substrate selectivity in these enzymes. The crystallographic asymmetric unit contains eight subunits (corresponding to four dimers) that show heterogeneity in the conformation of a co-crystallized molecule bound to the catalytic non-heme iron(III) ion resembling a benzohydroxamate moiety, probably a result of the breakdown of recently discovered siderophores synthesized by Gram-positive bacteria. Several different modes of binding benzohydroxamate into the active site induce distinct conformations of the interacting protein ligands Tyr167 and Arg188, illustrating the plasticity of the active site origin of the more promiscuous substrate preferences of the present enzyme. [Copyright &y& Elsevier]

The crystal structure of the 4-chlorocatechol 1,2-di- oxygenase from the Gram-positive bacterium Rhodococcus opacus (erythropolis) 1CP, a Fe(III) ion-containing enzyme involved in the aerobic biodegradation of chloroaromatic compounds, has been solved by multiple wavelength anomalous dispersion using the weak anomalous signal of the two catalytic irons (1 Fe/257 amino acids) and refined at a 2.5 Å resolution (Rfree 28.7%; R factor 21.4%). The analysis of the structure and its comparison with the structure of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus ADP1 (Ac 1,2-CTD) highlight significant differences between these enzymes. The general topology of the present enzyme comprises two catalytic domains (one for each subunit) related by a noncrystallographic 2-fold axis and separated by a common α-helical zipper motif consisting of five N-terminal helices from each subunit; furthermore the C-terminal tail is shortened significantly with respect to the known Ac 1,2-CTD. The presence of two phospholipids binding in a hydrophobic tunnel along the dimer axis is shown here to be a common feature for this class of enzyme. The active site cavity presents several dissimilarities with respect to the known catechol-cleaving enzyme. The catalytic nonheme iron(III) ion is bound to the side chains of Tyr-134, Tyr-169, His-194, and His-196, and a cocrystallized benzoate ion, bound to the metal center, reveals details on a novel mode of binding of bidentate inhibitors and a distinctive hydrogen bond network with the surrounding ligands. Among the amino acid residues expected to interact with substrates, several are different from the corresponding analogs of Ac 1,2-CTD: Asp-52, Ala-53, Gly-76, Phe-78, and Cys-224; in addition, regions of largely conserved amino acid residues in the catalytic cleft show different shapes [ABSTRACT FROM AUTHOR]

The goals of the present work were as follows: to obtain the dormant forms of R. opacus 1cp; to study the phenotypic variability during their germination; to compare phenotypic variants during the growth on selective and elective media; and to reveal changes in the ability of the strain to destruct xenobiotics that had not been degradable before dormancy. It was shown that Rhodococcus opacus 1cp (the strain degrading chlorinated phenols) became able to utilize a broader spectrum of xenobiotics after storage in the dormant state. Germination of the dormant forms of R. opacus 1cp on an agarized medium was followed by emergence and development of phenotypic variants that could grow on 4-chlorophenol and 2,4,6-trichlorophenol without adaptation. The cells of R. opacus 1cp phenotypic variants also utilized all of the tested chlorinated phenols: 2,3-, 2,5-, and 2,6-dichloro-, 2,3,4- and 2,4,5-trichloro-, pentachlorophenol, and 1,2,4,5-tetrachlorobenzene in concentrations up to 60 mg/L, though at the lower rates than 4-CP and 2,4,6-TCP. The improved degradation of chlorinated phenols by R. opacus strain 1cp exposed to the growth arrest conditions demonstrates the significance of dormancy for further manifestation of the adaptive potential of populations. A new principle of selection of variants with improved biodegradative properties was proposed. It embraces introduction of the dormancy stage into the cell life cycle with subsequent direct inoculation of morphologically different colonies into the media with different toxicants, including those previously not degraded by the strain.

The aim of the present work was to investigate the influence of alkylhydroxybenzenes (AHBs) and tyrosol, which belong to cell differentiation factors d(1) group of autoregulators on properties of biodegradation enzymes, catechol 1,2-dioxygenase (Cat 1,2-DO) and methylcatechol 1,2-dioxygenase (MCat 1,2-DO) of Rhodococcus opacus 6a. AHBs were found to have a greater effect on MCat 1,2-DO than on Cat 1,2-DO. It was expressed by more pronounced changes in the activity of MCat 1,2-DO with unsubstituted catechol at different AHB concentrations and by increasing thermostability of MCat 1,2-DO compared to Cat 1,2-DO under the protective action of AHBs. The compound C(7)-AHB shifted the maximum of dioxygenase activities towards higher temperatures and increased their operation optimum. AHBs changed the specificity constant of dioxygenases by decreasing/increasing the K(m)/V(max) value. For example, the increase in the V(max) value of 3,6-dichlorocatechol oxidation by Cat 1,2-DO in the presence of C(7)-AHB was 300-fold higher compared to the same reaction without AHB. The influence of cell differentiation factors on the properties of dimeric enzymes has been shown for the first time. It gives an idea of how the specificity of enzymes can be changed in vivo when strains contact new substrates. The work has shown the possibility of modification of the properties of dimeric enzymes towards the extension of enzyme activity with difficulty converted substrates or in more extreme conditions, which may be important for biotechnological processes.