A Recently Assembled Degradation Pathway for 2,3-Dichloronitrobenzene in Diaphorobacter sp. Strain JS3051.

Diaphorobacter sp. strain JS3051 utilizes 2,3-dichloronitrobenzene (23DCNB), a toxic anthropogenic compound, as the sole carbon, nitrogen, and energy source for growth, but the metabolic pathway and its origins are unknown. Here, we establish that a gene cluster (dcb), encoding a Nag-like dioxygenase, is responsible for the initial oxidation of the 23DCNB molecule. The 2,3-dichloronitrobenzene dioxygenase system (DcbAaAbAcAd) catalyzes conversion of 23DCNB to 3,4-dichlorocatechol (34DCC). Site-directed mutagenesis studies indicated that residue 204 of DcbAc is crucial for the substrate specificity of 23DCNB dioxygenase. The presence of glutamic acid at position 204 of 23DCNB dioxygenase is unique among Nag-like dioxygenases. Genetic, biochemical, and structural evidence indicate that the 23DCNB dioxygenase is more closely related to 2-nitrotoluene dioxygenase from Acidovorax sp. strain JS42 than to the 34DCNB dioxygenase from Diaphorobacter sp. strain JS3050, which was isolated from the same site as strain JS3051. A gene cluster (dcc) encoding the enzymes for 34DCC catabolism, homologous to a clc operon in Pseudomonas knackmussii strain B13, is also on the chromosome at a distance of 2.5 Mb from the dcb genes. Heterologously expressed DccA catalyzed ring cleavage of 34DCC with high affinity and catalytic efficiency. This work not only establishes the molecular mechanism for 23DCNB mineralization, but also enhances the understanding of the recent evolution of the catabolic pathways for nitroarenes. IMPORTANCE Because anthropogenic nitroaromatic compounds have entered the biosphere relatively recently, exploration of the recently evolved catabolic pathways can provide clues for adaptive evolutionary mechanisms in bacteria. The concept that nitroarene dioxygenases shared a common ancestor with naphthalene dioxygenase is well established. But their phylogeny and how they evolved in response to novel nitroaromatic compounds are largely unknown. Elucidation of the molecular basis for 23DCNB degradation revealed that the catabolic pathways of two DCNB isomers in different isolates from the same site were derived from different recent origins. Integrating structural models of catalytic subunits and enzymatic activities data provided new insight about how recently modified enzymes were selected depending on the structure of new substrates. This study enhances understanding and prediction of adaptive evolution of catabolic pathways in bacteria in response to new chemicals.

Switching the secondary and natural activity of Nitrilase from Acidovorax facilis 72 W for the efficient production of 2-picolinamide.

OBJECTIVES: Catalytic promiscuity, or the ability to catalyze a secondary reaction, provides new opportunities for industrial biocatalysis by expanding the range of biocatalytic reactions. Some nitrilases converting nitriles to amides, referred to as the secondary activity, show great potential for amides production. And our goal was exploiting the amide-forming potential of nitrilases. RESULTS: In this study, we characterized and altered the secondary activity of nitrilase from Acidovorax facilis 72 W (Nit72W) towards different substrates. We increased the secondary activity of Nit72W towards 2-cyanopyridine by 196-fold and created activity toward benzonitrile and p-nitrophenylacetonitrile by modifying the active pocket. Surprisingly, the best mutant, W188M, completely converted 250 mM 2-cyanopyridine to more than 98% 2-picolinamide in 12 h with a specific activity of 90 U/mg and showed potential for industrial applications. CONCLUSIONS: Nit72W was modified to increase its secondary activity for the amides production, especially 2-picolinamide.

Characterization of different biocatalyst formats for BVMO-catalyzed cyclohexanone oxidation.

Cyclohexanone monooxygenase (CHMO), a member of the Baeyer-Villiger monooxygenase family, is a versatile biocatalyst that efficiently catalyzes the conversion of cyclic ketones to lactones. In this study, an Acidovorax-derived CHMO gene was expressed in Pseudomonas taiwanensis VLB120. Upon purification, the enzyme was characterized in vitro and shown to feature a broad substrate spectrum and up to 100% conversion in 6 h. Furthermore, we determined and compared the cyclohexanone conversion kinetics for different CHMO-biocatalyst formats, that is, isolated enzyme, suspended whole cells, and biofilms, the latter two based on recombinant CHMO-containing P. taiwanensis VLB120. Biofilms showed less favorable values for KS (9.3-fold higher) and kcat (4.8-fold lower) compared with corresponding KM and kcat values of isolated CHMO, but a favorable KI for cyclohexanone (5.3-fold higher). The unfavorable KS and kcat values are related to mass transfer- and possibly heterogeneity issues and deserve further investigation and engineering, to exploit the high potential of biofilms regarding process stability. Suspended cells showed only 1.8-fold higher KS , but 1.3- and 4.2-fold higher kcat and KI values than isolated CHMO. This together with the efficient NADPH regeneration via glucose metabolism makes this format highly promising from a kinetics perspective.

A Nag-like dioxygenase initiates 3,4-dichloronitrobenzene degradation via 4,5-dichlorocatechol in Diaphorobacter sp. strain JS3050.

The chemical synthesis intermediate 3,4-dichloronitrobenzene (3,4-DCNB) is an environmental pollutant. Diaphorobacter sp. strain JS3050 utilizes 3,4-DCNB as a sole source of carbon, nitrogen and energy. However, the molecular determinants of its catabolism are poorly understood. Here, the complete genome of strain JS3050 was sequenced and key genes were expressed heterologously to establish the details of its degradation pathway. A chromosome-encoded three-component nitroarene dioxygenase (DcnAaAbAcAd) converted 3,4-DCNB stoichiometrically to 4,5-dichlorocatechol, which was transformed to 3,4-dichloromuconate by a plasmid-borne ring-cleavage chlorocatechol 1,2-dioxygenase (DcnC). On the chromosome, there are also genes encoding enzymes (DcnDEF) responsible for the subsequent transformation of 3,4-dichloromuconate to ß-ketoadipic acid. The fact that the genes responsible for the catabolic pathway are separately located on plasmid and chromosome indicates that recent assembly and ongoing evolution of the genes encoding the pathway is likely. The regiospecificity of 4,5-dichlorocatechol formation from 3,4-DCNB by DcnAaAbAcAd represents a sophisticated evolution of the nitroarene dioxygenase that avoids misrouting of toxic intermediates. The findings enhance the understanding of microbial catabolic diversity during adaptive evolution in response to xenobiotics released into the environment.

Whole-cell biocatalysis using the Acidovorax sp. CHX100 Δ6HX for the production of ω-hydroxycarboxylic acids from cycloalkanes.

Omega hydroxycarboxylic acids (ω-HAs) possess two functional groups, a hydroxyl group and a carboxyl group, and are essential precursors for the production of biodegradable polyester polymers. In this work, an Acidovorax mutant was investigated as a whole-cell biocatalyst for the conversion of cycloalkanes to their respective ω-hydroxycarboxylic acids. This Acidovorax sp. strain CHX100 originated from a wastewater treatment plant and uses cyclohexane as the sole source of carbon and energy with excellent growth rates (0.199 h-1). The metabolic efficiency of Acidovorax CHX100 is based on a highly efficient enzyme cascade used for the mineralization of cyclohexane. A deletion of 6-hydroxyhexanoate dehydrogenase in the native cycloalkane pathway resulted in the Acidovorax sp. strain CHX100 Δ6HX mutant, which accumulated short ω-hydroxycarboxylic acids (C5 to C10) from cycloalkanes. This mutant transformed cyclopentane and cyclohexane (5 mM) to 5-hydroxypentanoic acid and 6-hydroxyhexanoic acid, respectively, with a molar conversion above 98% in 6 h. An elementary environmental and economical assessment based on E-factor and biocatalyst yield suggests the use of inexpensive electron donor and carbon sources, with subsequent efforts to minimize waste generation. Such an early-stage analysis highlights the main bottlenecks that need to be solved in developing a sustainable bioprocess.

[Expression and characterization of a novel cytochrome P450 enzyme from Variovorax paradoxus S110].

Cytochrome P450 monooxygenases as powerful biocatalysts catalyze a wide range of chemical reactions to facilitate exogenous substances metabolism and biosynthesis of natural products. In order to explore new catalytic reactions and increase the number of P450 biocatalysts used in synthetic biology, a new self-sufficient cytochrome P450 monooxygenase (P450(VpMO)), belongs to CYP116B class, was mined from Variovorax paradoxus S110 genome and expressed in Escherichia coli. Based on characterization of the enzymatic properties, it shows that the optimal pH and temperature for P450(VpMO) reaction activity are 8.0 and 45 °C, respectively. P450(VpMO) is relatively stable at temperatures below 35 °C. The Km and kcat of P450(VpMO) toward 4-Methoxyacetophenone are 0.458 mmol/L and 2.438 min⁻¹, respectively. Importantly, P450(VpMO) was able to catalyze the demethylation reaction for a range of substrates containing methoxy group. Its demethylation reactivity is reasonably better than other P450s belongs to CYP116B class, particularly, for 4-methoxyacetophenone with a great conversion efficiency at 91%, showing that P450(VpMO) could be used as a great biocatalyst candidate for further analysis.

Functional and structural characterization of a novel L-fucose mutarotase involved in non-phosphorylative pathway of L-fucose metabolism.

Mutarotases catalyze the α-ß anomeric conversion of monosaccharide, and play a key role in utilizing sugar as enzymes involved in sugar metabolism have specificity for the α- or ß-anomer. In spite of the sequential similarity to l-rhamnose mutarotase protein superfamily (COG3254: RhaM), the ACAV_RS08160 gene in Acidovorax avenae ATCC 19860 (AaFucM) is located in a gene cluster related to non-phosphorylative l-fucose and l-galactose metabolism, and transcriptionally induced by these carbon sources; therefore, the physiological role remains unclear. Here, we report that AaFucM possesses mutarotation activity only toward l-fucose by saturation difference (SD) NMR experiments. Moreover, we determined the crystal structures of AaFucM in the apo form and in the l-fucose-bound form at resolutions of 2.21 and 1.75 Å, respectively. The overall structural folding was clearly similar to the RhaM members, differed from the known l-fucose mutarotase (COG4154: FucU), strongly indicating their convergent evolution. The structure-based mutational analyses suggest that Tyr18 is important for catalytic action, and that Gln87 and Trp99 are involved in the l-fucose-specific recognition.

Recombinant expression, characterization and application of maltotetraohydrolase from Pseudomonas saccharophila.

BACKGROUND: Maltotetraohydrolase, widely used in food and medical fields, possesses the ability to hydrolyze starch to produce maltooligosaccharides with maltotetraose as the main product. It also has the potential usage in delaying bread aging. RESULTS: Pseudomonas saccharophila maltotetraohydrolase was expressed in Bacillus subtilis WS11. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed obvious bands at 57 kDa (maltotetraohydrolase I) and 47 kDa (maltotetraohydrolase II). Both showed similar enzymatic properties, although the catalytic efficiency of maltotetraohydrolase I was 4.93 fold higher than that of maltotetraohydrolase II using soluble starch as substrate. In addition, the maltotetraohydrolase production was further scaled up in a 3-L fermentor, and the highest activity reached 1907 U mL-1 . Then, the recombinant maltotetraohydrolase was used to produce maltotetraose. The maltotetraose yields catalyzed by maltotetraohydrolase I and II reached 73.2% and 69.7%, respectively. Finally, when recombinant maltotetraohydrolase was used in bread-making, texture profile analysis of the bread indicated recombinant maltotetraohydrolase I exhibited a significant anti-aging effect. CONCLUSION: This is the first describing high-efficient expression of P. saccharophila maltotetraohydrolase in the food safety strain B. subtilis, and the yield represented the highest level ever reported. Excellent results were also obtained with respect to the preparation of maltotetraose and delaying bread aging using the recombinant maltotetraohydrolase. The present study will help lay the foundation for the industrial production and application of maltotetraohydrolase. © 2020 Society of Chemical Industry.

Production of a novel dimeric 4-deoxy-L-erythro-5-hexoseulose uronic acid by a PL-17 exolytic alginate lyase from Hydrogenophaga sp. UMI-18.

Hydrogenopahaga sp. strain UMI-18 is an alginolytic bacterium that can produce poly(3-hydroxybutylate) (PHB) using alginate as its sole carbon source. Genome analysis indicated that this strain harbors both PHB-synthesizing and alginate-assimilating gene clusters. In the present study, we cloned HyAly-I gene that encodes a PL-17 exolytic alginate lyase and investigated its enzymatic properties using recombinant HyAly-I (recHyAly-I) that was produced by Escherichia coli. The recHyAly-I preferably depolymerized poly(ß-D-mannuronate) block of alginate in an exolytic manner at an optimal temperature and a pH at 40 °C and pH 6.0, respectively. It released 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) from the non-reducing terminus of polymer and oligomer substrates. Interestingly, recHyAly-I was found to produce a novel unsaturated disaccharide, i.e., dimeric DEH (diDEH), along with monomeric DEH. Production of diDEH was prominent in the degradation of trisaccharides.

Structure of maltotetraose-forming amylase from Pseudomonas saccharophila STB07 provides insights into its product specificity.

The maltooligosaccharide-forming amylases (MFAses) degrade starch into maltooligosaccharides which potentially benefit human diet and grow popular in food processing, but little has been studied about their product specificity and structures. We focused on this topic and provide evidence through an X-ray crystal structure of the maltotetraose (G4)-forming amylase from Pseudomonas saccharophila STB07 (MFAps), as well as co-crystal structures of MFAps with G4 and with pseudo-maltoheptaose (pseudo-G7) determined at up to 1.1 Å resolution. G4 and pseudo-G7 occupy active cleft subsites -4 to -1 and -4 to +3 respectively. Binding induces conformational changes in the active sites except Asp193, working as the base catalyst. Comparison of the MFAps structure with those of other α-amylases revealed obvious differences in the loop structures providing dominant interactions between protein and substrate in the non-reducing side of the active sites cleft. These structures at the non-reducing end may govern the G4 specificity of MFAps and also be relevant to its exo-type action pattern.

H2 Metabolism revealed by metagenomic analysis of subglacial sediment from East Antarctica.

Subglacial ecosystems harbor diverse chemoautotrophic microbial communities in areas with limited organic carbon, and lithological H2 produced during glacial erosion has been considered an important energy source in these ecosystems. To verify the H2-utilizing potential there and to identify the related energy-converting metabolic mechanisms of these communities, we performed metagenomic analysis on subglacial sediment samples from East Antarctica with and without H2 supplementation. Genes coding for several [NiFe]-hydrogenases were identified in raw sediment and were enriched after H2 incubation. All genes in the dissimilatory nitrate reduction and denitrification pathways were detected in the subglacial community, and the genes coding for these pathways became enriched after H2 was supplied. Similarly, genes transcribing key enzymes in the Calvin cycle were detected in raw sediment and were also enriched. Moreover, key genes involved in H2 oxidization, nitrate reduction, oxidative phosphorylation, and the Calvin cycle were identified within one metagenome-assembled genome belonging to a Polaromonas sp. As suggested by our results, the microbial community in the subglacial environment we investigated consisted of chemoautotrophic populations supported by H2 oxidation. These results further confirm the importance of H2 in the cryosphere.

Production of Cyanocarboxylic Acid by Acidovorax facilis 72W Nitrilase Displayed on the Spore Surface of Bacillus subtilis.

Nitrilase is a valuable type of hydrolase that catalyzes nitriles into carboxylic acid and ammonia. Its applications, however, are severely restricted by the harsh conditions of industrial reaction processes. To solve this problem, a nitrilase from Acidovorax facilis 72W was inserted into an Escherichia coli-Bacillus subtilis shuttle vector for spore surface display. Western blot, enzyme activity measurements and flow cytometric analysis results all indicated a successful spore surface display of the CotB-nit fusion protein. In addition, the optimal catalytic pH value and temperature of the displayed nitrilase were determined to be 7.0 and 50°C, respectively. Moreover, results of reusability tests revealed that 64% of the initial activity of the displayed nitrilase was still retained at the 10th cycle. Furthermore, hydrolysis efficiency of upscale production of cyanocarboxylic acid was significantly higher in the displayed nitrilase-treated group than in the free group expressed by E. coli (pET-28a-nit). Generally, the display of A. facilis 72W nitrilase on the spore surface of Bacillus subtilis may be a useful method for immobilization of enzyme and consequent biocatalytic stabilization.

Identification and Characterization of a Dominant Sulfolane-Degrading Rhodoferax sp. via Stable Isotope Probing Combined with Metagenomics.

Sulfolane is an industrial solvent and emerging organic contaminant affecting groundwater around the world, but little is known about microbes capable of biodegrading sulfolane or the pathways involved. We combined DNA-based stable isotope probing (SIP) with genome-resolved metagenomics to identify microorganisms associated with sulfolane biodegradation in a contaminated subarctic aquifer. In addition to 16S rRNA gene amplicon sequencing, we performed shotgun metagenomics on the 13C-labeled DNA to obtain functional and taxonomic information about the active sulfolane-degrading community. We identified the primary sulfolane degrader, comprising ~85% of the labeled community in the amplicon sequencing dataset, as closely related to Rhodoferax ferrireducens strain T118. We obtained a 99.8%-complete metagenome-assembled genome for this strain, allowing us to identify putative pathways of sulfolane biodegradation. Although the 4S dibenzothiophene desulfurization pathway has been proposed as an analog for sulfolane biodegradation, we found only a subset of the required genes, suggesting a novel pathway specific to sulfolane. DszA, the enzyme likely responsible for opening the sulfolane ring structure, was encoded on both the chromosome and a plasmid. This study demonstrates the power of integrating DNA-SIP with metagenomics to characterize emerging organic contaminant degraders without culture bias and expands the known taxonomic distribution of sulfolane biodegradation.

Highly efficient conversion of 1-cyanocycloalkaneacetonitrile using a "super nitrilase mutant".

Nitrilase is the member of carbon-nitrogen hydrogen hydrolase superfamily, which has been widely used for the hydrolysis of nitriles into corresponding carboxylic acids. But most nitrilases are plagued by product inhibition in the industrial application. In this study, a "super nitrilase mutant" of nitrilase with high activity, thermostability and improved product tolerance from Acidovorax facilis ZJB09122 was characterized. Then, an efficient process was developed by employing the whole cell of recombinant E. coli for the conversion of high concentration of 1-cyanocyclohexylacetonitrile-to-1-cyanocyclohexaneacetic acid, an important intermediate of gabapentin. Under the optimized conditions, the higher substrate concentrations such as 1.3 M, 1.5 M and 1.8 M could be hydrolyzed by 13.58 g DCW/L with outstanding productivity (> 740 g/L/day). This study developed a highly efficient bioprocess for the preparation of 1-cyanocyclohexaneacetic acid which has the great potential for industrial application.

Functional analysis of active amino acid residues of the mercaptosuccinate dioxygenase of Variovorax paradoxus B4.

Thiol dioxygenases are non-heme mononuclear-iron proteins and belong to the cupin superfamily. In 2014, mercaptosuccinate dioxygenase (Msdo) of Variovorax paradoxus B4 was identified as another bacterial cysteine dioxygenase (Cdo) homolog catalyzing the conversion of mercaptosuccinate (MS) into succinate and sulfite. To gain further insights into potentially important amino acid residues for enzyme activity, seven enzyme variants were generated and analyzed. (i) Three variants comprised the substitution of one conserved histidine residue each by leucine, either supposed to be mandatory for coordination of the Fe(II) cofactor (H93 and H95) or to be important for substrate positioning within the active site (H163). The corresponding enzyme variants were completely inactive confirming their essential roles for enzyme activity. (ii) Mutation C100S resulted as well in an inactive enzyme demonstrating its importance for either stability or activity of the protein. (iii) For eukaryotic Cdo, a hydrogen bond network for substrate positioning was postulated, and the corresponding amino acids are basically present in Msdo. Albeit the MsdoQ64A mutation exhibited an increased Km of 0.29 mM when compared to the wildtype with 0.06 mM, it did not significantly affect the specific activity. (iv) The variant MsdoR66A showed only very low activity even when high amounts of enzyme were applied indicating that this residue might be important for catalysis. (v) No strong effect had the mutation Y165F for which a specific enzyme activity of 10.22 µmol min-1 mg-1 protein and a Km value of 0.06 mM with high similarity to those of the wildtype enzyme were obtained. This residue corresponds to Y157 of human Cdo, which is part of the catalytic triad and is supposed to be involved in substrate positioning. Apparently, another residue could fulfill this role in Msdo, since the loss of Y165 did not have a strong effect.

Gene probing reveals the widespread distribution, diversity and abundance of isoprene-degrading bacteria in the environment.

BACKGROUND: Approximately 500 Tg of isoprene are emitted to the atmosphere annually, an amount similar to that of methane, and despite its significant effects on the climate, very little is known about the biological degradation of isoprene in the environment. Isolation and characterisation of isoprene degraders at the molecular level has allowed the development of probes targeting isoA encoding the α-subunit of the isoprene monooxygenase. This enzyme belongs to the soluble diiron centre monooxygenase family and catalyses the first step in the isoprene degradation pathway. The use of probes targeting key metabolic genes is a successful approach in molecular ecology to study specific groups of bacteria in complex environments. Here, we developed and tested a novel isoA PCR primer set to study the distribution, abundance, and diversity of isoprene degraders in a wide range of environments. RESULTS: The new isoA probes specifically amplified isoA genes from taxonomically diverse isoprene-degrading bacteria including members of the genera Rhodococcus, Variovorax, and Sphingopyxis. There was no cross-reactivity with genes encoding related oxygenases from non-isoprene degraders. Sequencing of isoA amplicons from DNA extracted from environmental samples enriched with isoprene revealed that most environments tested harboured a considerable variety of isoA sequences, with poplar leaf enrichments containing more phylogenetically diverse isoA genes. Quantification by qPCR using these isoA probes revealed that isoprene degraders are widespread in the phyllosphere, terrestrial, freshwater and marine environments. Specifically, soils in the vicinity of high isoprene-emitting trees contained the highest number of isoprene-degrading bacteria. CONCLUSION: This study provides the molecular ecology tools to broaden our knowledge of the distribution, abundance and diversity of isoprene degraders in the environment, which is a fundamental step necessary to assess the impact that microbes have in mitigating the effects of this important climate-active gas.

The Plant Growth-Promoting Rhizobacterium Variovorax boronicumulans CGMCC 4969 Regulates the Level of Indole-3-Acetic Acid Synthesized from Indole-3-Acetonitrile.

Variovorax is a metabolically diverse genus of plant growth-promoting rhizobacteria (PGPR) that engages in mutually beneficial interactions between plants and microbes. Unlike most PGPR, Variovorax cannot synthesize the phytohormone indole-3-acetic acid (IAA) via tryptophan. However, we found that Variovorax boronicumulans strain CGMCC 4969 can produce IAA using indole-3-acetonitrile (IAN) as the precursor. Thus, in the present study, the IAA synthesis mechanism of V. boronicumulans CGMCC 4969 was investigated. V. boronicumulans CGMCC 4969 metabolized IAN to IAA through both a nitrilase-dependent pathway and a nitrile hydratase (NHase) and amidase-dependent pathway. Cobalt enhanced the metabolic flux via the NHase/amidase, by which IAN was rapidly converted to indole-3-acetamide (IAM) and in turn to IAA. IAN stimulated metabolic flux via the nitrilase, by which IAN was rapidly converted to IAA. Subsequently, the IAA was degraded. V. boronicumulans CGMCC 4969 can use IAN as the sole carbon and nitrogen source for growth. Genome sequencing confirmed the IAA synthesis pathways. Gene cloning and overexpression in Escherichia coli indicated that NitA has nitrilase activity and IamA has amidase activity to respectively transform IAN and IAM to IAA. Interestingly, NitA showed a close genetic relationship with the nitrilase of the phytopathogen Pseudomonas syringae Quantitative PCR analysis indicated that the NHase/amidase system is constitutively expressed, whereas the nitrilase is inducible. The present study helps our understanding of the versatile functions of Variovorax nitrile-converting enzymes that mediate IAA synthesis and the interactions between plants and these bacteria.IMPORTANCE We demonstrated that Variovorax boronicumulans CGMCC 4969 has two enzymatic systems-nitrilase and nitrile hydratase/amidase-that convert indole-3-acetonitrile (IAN) to the important plant hormone indole-3-acetic acid (IAA). The two IAA synthesis systems have very different regulatory mechanisms, affecting the IAA synthesis rate and duration. The nitrilase was induced by IAN, which was rapidly converted to IAA; subsequently, IAA was rapidly consumed for cell growth. The nitrile hydratase (NHase) and amidase system was constitutively expressed and slowly but continuously synthesized IAA. In addition to synthesizing IAA from IAN, CGMCC 4969 has a rapid IAA degradation system, which would be helpful for a host plant to eliminate redundant IAA. This study indicates that the plant growth-promoting rhizobacterium V. boronicumulans CGMCC 4969 has the potential to be used by host plants to regulate the IAA level.

Significant improvement of the nitrilase activity by semi-rational protein engineering and its application in the production of iminodiacetic acid.

Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacturing of chelating agents, glyphosate herbicides and surfactants. To improve activity and tolerance to the substrate for IDA production, Acidovorax facilis nitrilase was selected for further modification by the gene site saturation mutagenesis method. After screened by a two-step screening method, the best mutant (Mut-F168V/T201N/S192F/M191T/F192S) was selected. Compared to the wild-type nitrilase, Mut-F168V/T201N/S192F/M191T/F192S showed 136% improvement in specific activity. Co2+ stimulated nitrilase activity, whereas Cu2+, Zn2+ and Tween 80 showed a strong inhibitory effect. The Vmax and kcat of Mut-F168V/T201N/S192F/M191T/F192S were enhanced 1.23 and 1.23-fold, while the Km was decreased 1.53-fold. The yield of Mut-F168V/T201N/S192F/M191T/F192S with 453.2 mM of IDA reached 71.9% in 5 h when 630 mM iminodiacetonitrile was used as substrate. This study indicated that mutant nitrilase obtained in this study is promising in applications for the upscale production of IDAN.

Functional Role of Tyr12 in the Catalytic Activity of Novel Zeta-like Glutathione S-transferase from Acidovorax sp. KKS102.

Glutathione S-transferases (GSTs) are a family of enzymes that function in the detoxification of variety of electrophilic substrates. In the present work, we report a novel zeta-like GST (designated as KKSG9) from the biphenyl/polychlorobiphenyl degrading organism Acidovorax sp. KKS102. KKSG9 possessed low sequence similarity but similar biochemical properties to zeta class GSTs. Functional analysis showed that the enzyme exhibits wider substrate specificity compared to most zeta class GSTs by reacting with 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrobenzyl chloride (NBC), ethacrynic acid (EA), hydrogen peroxide, and cumene hydroperoxide. The enzyme also displayed dehalogenation function against dichloroacetate, permethrin, and dieldrin. The functional role of Tyr12 was also investigated by site-directed mutagenesis. The mutant (Y12C) displayed low catalytic activity and dehalogenation function against all the substrates when compared with the wild type. Kinetic analysis using NBC and GSH as substrates showed that the mutant (Y12C) displayed a higher affinity for NBC when compared with the wild type, however, no significant change in GSH affinity was observed. These findings suggest that the presence of tyrosine residue in the motif might represent an evolutionary trend toward improving the catalytic activity of the enzyme. The enzyme as well could be useful in the bioremediation of various types of organochlorine pollutants.

Active Site Flexibility as a Hallmark for Efficient PET Degradation by I. sakaiensis PETase.

Polyethylene terephthalate (PET) is one of the most-consumed synthetic polymers, with an annual production of 50 million tons. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation. Recently, fungal and bacterial thermophilic hydrolases were found to catalyze PET hydrolysis with optimal activities at high temperatures. Strikingly, an enzyme from Ideonella sakaiensis, termed PETase, was described to efficiently degrade PET at room temperature, but the molecular basis of its activity is not currently understood. Here, a crystal structure of PETase was determined at 2.02 Å resolution and employed in molecular dynamics simulations showing that the active site of PETase has higher flexibility at room temperature than its thermophilic counterparts. This flexibility is controlled by a novel disulfide bond in its active site, with its removal leading to destabilization of the catalytic triad and reduction of the hydrolase activity. Molecular docking of a model substrate predicts that PET binds to PETase in a unique and energetically favorable conformation facilitated by several residue substitutions within its active site when compared to other enzymes. These computational predictions are in excellent agreement with recent mutagenesis and PET film degradation analyses. Finally, we rationalize the increased catalytic activity of PETase at room temperature through molecular dynamics simulations of enzyme-ligand complexes for PETase and other thermophilic PET-degrading enzymes at 298, 323, and 353 K. Our results reveal that both the binding pose and residue substitutions within PETase favor proximity between the catalytic residues and the labile carbonyl of the substrate at room temperature, suggesting a more favorable hydrolytic reaction. These results are valuable for enabling detailed evolutionary analysis of PET-degrading enzymes and for rational design endeavors aiming at increasing the efficiency of PETase and similar enzymes toward plastic degradation.