Erratum to: Molecular dynamics simulation reveals DNA-specific recognition mechanism via c-Myb in pseudo-palindromic consensus of mim-1 promoter.

The original version of this article (Weng et al., 2023) unfortunately contained a mistake. In Acknowledgments, the number (No. 226-2022-00213) of the Fundamental Research Funds for the Central Universities is wrong. The correct number should be No. 2022FZZX01-33.

ZmNRT1.1B (ZmNPF6.6) determines nitrogen use efficiency via regulation of nitrate transport and signalling in maize.

Nitrate (NO3 - ) is crucial for optimal plant growth and development and often limits crop productivity under low availability. In comparison with model plant Arabidopsis, the molecular mechanisms underlying NO3 - acquisition and utilization remain largely unclear in maize. In particular, only a few genes have been exploited to improve nitrogen use efficiency (NUE). Here, we demonstrated that NO3 - -inducible ZmNRT1.1B (ZmNPF6.6) positively regulated NO3 - -dependent growth and NUE in maize. We showed that the tandem duplicated proteoform ZmNRT1.1C is irrelevant to maize seedling growth under NO3 - supply; however, the loss of function of ZmNRT1.1B significantly weakened plant growth under adequate NO3 - supply under both hydroponic and field conditions. The 15 N-labelled NO3 - absorption assay indicated that ZmNRT1.1B mediated the high-affinity NO3 - -transport and root-to-shoot NO3 - translocation. Transcriptome analysis further showed, upon NO3 - supply, ZmNRT1.1B promotes cytoplasmic-to-nuclear shuttling of ZmNLP3.1 (ZmNLP8), which co-regulates the expression of genes involved in NO3 - response, cytokinin biosynthesis and carbon metabolism. Remarkably, overexpression of ZmNRT1.1B in modern maize hybrids improved grain yield under N-limiting fields. Taken together, our study revealed a crucial role of ZmNRT1.1B in high-affinity NO3 - transport and signalling and offers valuable genetic resource for breeding N use efficient high-yield cultivars.

Molecular dynamics simulation reveals DNA-specific recognition mechanism via c-Myb in pseudo-palindromic consensus of mim-1 promoter. / é€šè¿‡åˆ†å­åŠ¨åŠ›å­¦æ¨¡æ‹Ÿæ–¹æ³•ç ”ç©¶c-Myb在mim-1启动子区域伪回文序列的结合特性揭示DNA特异性识别机制.

This study aims to gain insight into the DNA-specific recognition mechanism of c-Myb transcription factor during the regulation of cell early differentiation and proliferation. Therefore, we chose the chicken myeloid gene, mitochondrial import protein 1 (mim-1), as a target to study the binding specificity between potential dual-Myb-binding sites. The c-Myb-binding site in mim-1 is a pseudo-palindromic sequence AACGGTT, which contains two AACNG consensuses. Simulation studies in different biological scenarios revealed that c-Myb binding with mim-1 in the forward strand (complex F) ismore stable than that inthereverse strand (complex R). The principal component analysis (PCA) dynamics trajectory analyses suggested an opening motion of the recognition helices of R2 and R3 (R2R3), resulting in the dissociation of DNA from c-Myb in complex R at 330 K, triggered by the reduced electrostatic potential on the surface of R2R3. Furthermore, the DNA confirmation and hydrogen-bond interaction analyses indicated that the major groove width of DNA increased in complex R, which affected on the hydrogen-bond formation ability between R2R3 and DNA, and directly resulted in the dissociation of DNA from R2R3. The steered molecular dynamics (SMD) simulation studies also suggested that the electrostatic potential, major groove width, and hydrogen bonds made major contribution to the DNA|-specific recognition. In vitro trials confirmed the simulation results that c-Myb specifically bound to mim-1 in the forward strand. This study indicates that the three-dimensional (3D) structure features play an important role in the DNA-specific recognition mechanism by c-Myb besides the AACNG consensuses, which is beneficial to understanding the cell early differentiation and proliferation regulated by c-Myb, as well as the prediction of novel c-Myb-binding motifs in tumorigenesis.

Engineered chimeric insecticidal crystalline protein improves resistance to lepidopteran insects in rice (Oryza sativa L.) and maize (Zea mays L.).

The insecticidal crystalline proteins (Crys) are a family of insect endotoxin functioning in crop protection. As insects keep evolving into tolerance to the existing Crys, it is necessary to discover new Cry proteins to overcome potential threatens. Crys possess three functional domains at their N-termini, and the most active region throughout evolution was found at the domain-III. We swapped domain-IIIs from various Cry proteins and generated seven chimeric proteins. All recombinants were expressed in Escherichia coli and their toxicity was assessed by dietary exposure assays. Three of the seven Crys exhibited a high toxicity to Asian corn borer over the controls. One of them, Cry1Ab-Gc, a chimeric Cry1Ab being replaced with the domain-III of Cry1Gc, showed the highest toxicity to rice stem borer when it was over-expressed in Oryza sativa. Furthermore, it was also transformed into maize, backcrossed into commercial maize inbred lines and then produced hybrid to evaluate their commercial value. Transgenic maize performed significant resistance to the Asian corn borer without affecting the yield. We further showed that this new protein did not have adverse effects on the environment. Our results indicated that domain III swapped of Crys could be used as an efficient method for developing new engineered insecticidal protein.

Phylogenetic analyses of the 19 STR loci in X chromosome revealed discriminant forensic characteristics for Chinese Daur and Oroqen minorities.

In respect of forensic genetics, X-STRs are widely applied for deficiency paternity cases. Given the popularization of AGCU-X19 STR Kit in China, there has been investigation conducted into the genetic data and forensic parameters of 19 X-STR loci in many of the Chinese ethnic groups, which makes it possible to perform nationwide phylogenetic comparation. To evaluate the allele and haplotype diversity of 19 X-STR loci and to explore their forensic efficiency in the Daur and Oroqen minorities, unrelated healthy Daur (n = 86) and Oroqen (n = 165) individuals were recruited from Heilongjiang province, so as to reveal the phylogenetic relationship between the two minorities and other Chinese ethnic groups. Of the Daur and Oroqen minorities, 172 and 183 alleles at the 19 X-STR loci were observed, respectively. Haplotype diversity exceeded 0.9 among all the linkage clusters. High cumulative value was observed for the power of discrimination, the probability of exclusion, and the mean exclusion chance for deficiency cases (normal trios and duo cases). As revealed by this study, the panel of 19 X-STR loci is an effective supplementary tool for the kinship test of the studied nationalities.

Resequencing 250 Soybean Accessions: New Insights into Genes Associated with Agronomic Traits and Genetic Networks.

The limited knowledge of genomic diversity and functional genes associated with the traits of soybean varieties has resulted in slow progress in breeding. In this study, we sequenced the genomes of 250 soybean landraces and cultivars from China, America, and Europe, and investigated their population structure, genetic diversity and architecture, and the selective sweep regions of these accessions. Five novel agronomically important genes were identified, and the effects of functional mutations in respective genes were examined. The candidate genes GSTT1, GL3, and GSTL3 associated with the isoflavone content, CKX3 associated with yield traits, and CYP85A2 associated with both architecture and yield traits were found. The phenotype-gene network analysis revealed that hub nodes play a crucial role in complex phenotypic associations. This study describes novel agronomic trait-associated genes and a complex genetic network, providing a valuable resource for future soybean molecular breeding.

Potential interaction between autophagy and auxin during maize leaf senescence.

Leaf senescence is important for crop yield as delaying it can increase the average yield. In this study, population genetics and transcriptomic profiling were combined to dissect its genetic basis in maize. To do this, the progenies of an elite maize hybrid Jidan27 and its parental lines Si-287 (early senescence) and Si-144 (stay-green), as well as 173 maize inbred lines were used. We identified two novel loci and their candidate genes, Stg3 (ZmATG18b) and Stg7 (ZmGH3.8), which are predicted to be members of autophagy and auxin pathways, respectively. Genomic variations in the promoter regions of these two genes were detected, and four allelic combinations existed in the examined maize inbred lines. The Stg3Si-144/Stg7Si-144 allelic combination with lower ZmATG18b expression and higher ZmGH3.8 expression could distinctively delay leaf senescence, increase ear weight and the improved hybrid of NIL-Stg3Si-144/Stg7Si-144 × Si-144 significantly reduced ear weight loss under drought stress, while opposite effects were observed in the Stg3Si-287/Stg7Si-287 combination with a higher ZmATG18b expression and lower ZmGH3.8 expression. Thus, we identify a potential interaction between autophagy and auxin which could modulate the timing of maize leaf senescence.

QTL Mapping of Fusarium Ear Rot Resistance in Maize.

Ear rot is a globally prevalent class of disease in maize, of which Fusarium ear rot (FER), caused by the fungal pathogen Fusarium verticillioides, is the most commonly reported. In this study, three F2 populations, namely F2-C, F2-D, and F2-J, and their corresponding F2:3 families were produced by crossing three highly FER-resistant inbred lines, Cheng351, Dan598, and JiV203, with the same susceptible line, ZW18, for quantitative trait locus (QTL) mapping of FER resistance. The individual crop plants were inoculated with a spore suspension of the pathogen injected into the kernels of the maize ears. The broad-sense heritability (H2) for FER resistance was estimated to be as high as 0.76, 0.81, and 0.78 in F2-C, F2-D, and F2-J, respectively, indicating that genetic factors played a key role in the phenotypic variation. We detected a total of 20 FER-resistant QTLs in the three F2 populations, among which QTLs derived from the resistant parent Cheng351, Dan598, and JiV203 explained 62.89 to 82.25%, 43.19 to 61.51%, and 54.70 to 75.77% of the phenotypic variation, respectively. Among all FER-resistant QTLs detected, qRfer1, qRfer10, and qRfer17 accounted for the phenotypic variation as high as 26.58 to 43.36%, 11.76 to 18.02%, and 12.02 to 21.81%, respectively. Furthermore, QTLs mapped in different F2 populations showed some extent of overlaps indicating potential resistance hotspots. The FER-resistant QTLs detected in this study can be explored as useful candidates to improve FER resistance in maize by introducing these QTLs into susceptible maize inbred lines via molecular marker-assisted selection.

A Na2CO3-Responsive Chitinase Gene From Leymus chinensis Improve Pathogen Resistance and Saline-Alkali Stress Tolerance in Transgenic Tobacco and Maize.

Salinity and microbial pathogens are the major limiting factors for crop production. Although the manipulation of many genes could improve plant performance under either of these stresses, few genes have reported that could improve both pathogen resistance and saline-alkali stress tolerance. In this study, we identified a new chitinase gene CHITINASE 2 (LcCHI2) that encodes a class II chitinase from Leymus chinensis, which grows naturally on alkaline-sodic soil. Overexpression of LcCHI2 increased chitinase activity in transgenic plants. The transgenic tobacco and maize exhibited improved pathogen resistance and enhanced both neutral salt and alkaline salt stress tolerance. Overexpression of LcCHI2 reduced sodium (Na+) accumulation, malondialdehyde content and relative electrical conductivity in transgenic tobacco under salt stress. In addition, the transgenic tobacco showed diminished lesion against bacterial and fungal pathogen challenge, suggesting an improved disease resistance. Similar improved performance was also observed in LcCHI2-overexpressed maize under both pathogen and salt stresses. It is worth noting that this genetic manipulation does not impair the growth and yield of transgenic tobacco and maize under normal cultivation condition. Apparently, application of LcCHI2 provides a new train of thought for genetically engineering saline-alkali and pathogen resistant crops of both dicots and monocots.

The defective effect of starch branching enzyme IIb from weak to strong induces the formation of biphasic starch granules in amylose-extender maize endosperm.

KEYMESSAGE: Biphasic starch granules in maize ae mutant underwent the weak to strong SBEIIb-defective effect during endosperm development, leading to no birefringence in their exterior due to extended long branch-chains of amylopectin. Biphasic starch granules are usually detected regionally in cereal endosperm lacking starch branching enzyme (SBE). However, their molecular structure, formation mechanism, and regional distribution are unclear. In this research, biphasic starch granules were observed in the inner region of crown endosperm of maize ae mutant, and had poorly oriented structure with comb-like profiles in their exterior. The inner endosperm (IE) rich in biphasic starch granules and outer endosperm (OE) without biphasic starch granules were investigated. The starch had lower amylose content and higher proportion of long branch-chains of amylopectin in IE than in OE, and the exterior of biphasic starch granules had less amylose and more long branch-chains of amylopectin than the interior. Compared with OE, the expression pattern of starch synthesis related enzymes changed significantly in IE. The granule-bound starch synthase I activity within biphasic starch granules decreased slightly. The IE experienced more severe hypoxic stress than OE, and the up-regulated anaerobic respiration pathway indicated an increase in carbon consumption. The starch in IE underwent the SBEIIb-defective effect from weak to strong due to the lack of sufficient carbon inflow, leading to the formation of biphasic starch granules and their regional distribution in endosperm. The results provided information for understanding the biphasic starch granules.

Development of dwarfish and yield-effective GM maize through passivation of bioactive gibberellin.

During the Green Revolution in the 1960s, breeding dwarf cultivars turned out to be a landmark, leading to a significant increase in the global production of wheat and rice. The most direct and effective strategy for breeding dwarf crops, among others, would be to control endogenous gibberellin (GA) levels of the crops. GA 2-oxidases are a group of 2-oxoglutarate-dependent dioxygenases that catalyze the deactivation of bioactive GAs. The ArabidopsisAtGA2ox1 gene was transformed into maize with the aim of obtaining a height-reduced GM maize. The characterization of the GM maize revealed that the highest plant height reduction was accomplished by a 74% decline in GA1 level, and by approximately twofold increases in both chlorophyll content and root/shoot ratio over the wild-type (WT). Interestingly, the stem cells of the GM maize were condensed, and the typical vascular bundle structure was found to be deformed. Based on a 2-season field trial, the GM maize exhibited a higher harvest index (9-17%) and grain yield (10-14%) than the WT. The current results suggest that a modulation of the endogenous GA level would be a sensible approach for improving the crop architecture and grain yield in maize.

Sweet Sorghum Originated through Selection of Dry, a Plant-Specific NAC Transcription Factor Gene.

Sorghum (Sorghum bicolor) is the fifth most popular crop worldwide and a C4 model plant. Domesticated sorghum comes in many forms, including sweet cultivars with juicy stems and grain sorghum with dry, pithy stems at maturity. The Dry locus, which controls the pithy/juicy stem trait, was discovered over a century ago. Here, we found that Dry gene encodes a plant-specific NAC transcription factor. Dry was either deleted or acquired loss-of-function mutations in sweet sorghum, resulting in cell collapse and altered secondary cell wall composition in the stem. Twenty-three Dry ancestral haplotypes, all with dry, pithy stems, were found among wild sorghum and wild sorghum relatives. Two of the haplotypes were detected in domesticated landraces, with four additional dry haplotypes with juicy stems detected in improved lines. These results imply that selection for Dry gene mutations was a major step leading to the origin of sweet sorghum. The Dry gene is conserved in major cereals; fine-tuning its regulatory network could provide a molecular tool to control crop stem texture.

ZmCOL3, a CCT gene represses flowering in maize by interfering with the circadian clock and activating expression of ZmCCT.

Flowering time is a trait vital to the adaptation of flowering plants to different environments. Here, we report that CCT domain genes play an important role in flowering in maize (Zea mays L.). Among the 53 CCT family genes we identified in maize, 28 were located in flowering time quantitative trait locus regions and 15 were significantly associated with flowering time, based on candidate-gene association mapping analysis. Furthermore, a CCT gene named ZmCOL3 was shown to be a repressor of flowering. Overexpressing ZmCOL3 delayed flowering time by approximately 4 d, in either long-day or short-day conditions. The absence of one cytosine in the ZmCOL3 3'UTR and the presence of a 551 bp fragment in the promoter region are likely the causal polymorphisms contributing to the maize adaptation from tropical to temperate regions. We propose a modified model of the maize photoperiod pathway, wherein ZmCOL3 acts as an inhibitor of flowering either by transactivating transcription of ZmCCT, one of the key genes regulating maize flowering, or by interfering with the circadian clock.

Identification of Drought Tolerant Mechanisms in Maize Seedlings Based on Transcriptome Analysis of Recombination Inbred Lines.

Zea mays is an important crop that is sensitive to drought stress, but survival rates and growth status remain strong in some drought-tolerant lines under stress conditions. Under drought conditions, many biological processes, such as photosynthesis, carbohydrate metabolism and energy metabolism, are suppressed, while little is known about how the transcripts of genes respond to drought stress in the genome-wide rang in the seedling stage. In our study, the transcriptome profiles of two maize recombination inbred lines (drought-tolerant RIL70 and drought-sensitive RIL93) were analyzed at different drought stages to elucidate the dynamic mechanisms underlying drought tolerance in maize seedlings during drought conditions. Different numbers of differentially expressed genes presented in the different stages of drought stress in the two RILs, for the numbers of RIL93 vs. RIL70 were: 9 vs. 358, 477 vs. 103, and 5207 vs. 152 respectively in DT1, DT2, and DT5. Gene Ontology enrichment analysis revealed that in the initial drought-stressed stage, the primary differentially expressed genes involved in cell wall biosynthesis and transmembrane transport biological processes were overrepresented in RIL70 compared to RIL93. On the contrary, differentially expressed genes profiles presented at 2 and 5 day-treatments, the primary differentially expressed genes involved in response to stress, protein folding, oxidation-reduction, photosynthesis and carbohydrate metabolism, were overrepresented in RIL93 compared to RIL70. In addition, the transcription of genes encoding key members of the cell cycle and cell division processes were blocked, but ABA- and programmed cell death-related processes responded positively in RIL93. In contrast, the expression of cell cycle genes, ABA- and programmed cell death-related genes was relatively stable in RIL70. The results we obtained supported the working hypothesis that signaling events associated with turgor homeostasis, as established by cell wall biosynthesis regulation- and aquaporin-related genes, responded early in RIL70, which led to more efficient detoxification signaling (response to stress, protein folding, oxidation-reduction) during drought stress. This energy saving response at the early stages of drought should facilitate more cell activity under stress conditions and result in drought tolerance in RIL70.

Affinity chromatography revealed insights into unique functionality of two 14-3-3 protein species in developing maize kernels.

The 14-3-3 proteins are a group of regulatory proteins of divergent functions in plants. However, little is known about their roles in maize kernel development. Using publically available gene expression profiling data, we found that two 14-3-3 species genes, zmgf14-4 and zmgf14-6, exhibited prominent expression profiles over other 14-3-3 protein genes during maize kernel development. More than 5000 transcripts of these two genes were identified accounting for about 1/10 of the total transcripts of genes correlating to maize kernel development. We constructed a proteomics pipeline based on the affinity chromatography, in combination with 2-DE and LC-MS/MS technologies to identify the specific client proteins of the two proteins for their functional characterization. Consequently, we identified 77 specific client proteins from the developing kernels of the inbred maize B73. More than 60% of the client proteins were commonly affinity-identified by the two 14-3-3 species and are predicted to be implicated in the fundamental functions of metabolism, protein destination and storage. In addition, we found ZmGF14-4 specifically bound to the disease- or defense-relating proteins, whilst ZmGF14-6 tended to interact with the proteins involving metabolism and cell structure. Our findings provide primary insights into the functional roles of 14-3-3 proteins in maize kernel development. BIOLOGICAL SIGNIFICANCE: Maize kernel development is a complicated physiological process for its importance in both genetics and cereal breeding. 14-3-3 proteins form a multi-gene family participating in regulations of developmental processes in plants. However, the correlation between this protein family and maize kernel development has hardly been studied. We have for the first time found 12 14-3-3 protein genes from maize genome and studied in silico the gene transcription profiling of these genes. Comparative studies revealed that maize kernel development aroused a great number of gene expression, among which 14-3-3 protein genes took a significant proportion. We applied affinity chromatographic approach, in combination with 2-DE and LC-MS/MS, to explore the specific client proteins of two crucial 14-3-3 protein species that exhibit prominent gene expression over other members in the family during the kernel development. Assessments of the identified client proteins resulted in important information toward understanding the functional mechanism of 14-3-3 protein family in maize kernel development.

Genome-wide patterns of large-size presence/absence variants in sorghum.

The presence/absence variants (PAVs) are a major source of genome structural variation and have profound effects on phenotypic and genomic variation in animals and humans. However, little is understood about PAVs in plant genomes. Our previous resequencing effort on three sorghum (Sorghum bicolour L.) genomes, each 12× coverage, uncovered 5 364 PAVs. Here, we report a detailed characterization of 51 large-size (>30 kb) PAVs. These PAVs spanned a total size of 2.92 Mb of the sorghum genome containing 202 known and predicted genes, including 38 genes annotated to encode cell death and stress response genes. The PAVs varied considerably for repeat sequences and mobile elements with DNA transposons as the major components. The frequency and distribution of these PAVs differed substantially across 96 sorghum inbred lines, and the low- and high frequency PAVs differed in their gene categories. This report shed new light on the occurrence and diversity of PAVs in sorghum genomes. Our research exemplifies a new perspective to explore genome structural variation for genetic improvement in plant breeding.

Early transcriptomic adaptation to Na2CO3 stress altered the expression of a quarter of the total genes in the maize genome and exhibited shared and distinctive profiles with NaCl and high pH stresses.

Sodium carbonate (Na2CO3) presents a huge challenge to plants by the combined damaging effects of Na⁺, high pH, and CO3²â». Little is known about the cellular responses to Na2CO3 stress. In this study, the transcriptome of maize (Zea mays L. cv. B73) roots exposed to Na2CO3 stress for 5 h was compared with those of NaCl and NaOH stresses. The expression of 8,319 genes, representing over a quarter of the total number of genes in the maize genome, was altered by Na2CO3 stress, and the downregulated genes (5,232) outnumbered the upregulated genes (3,087). The effects of Na2CO3 differed from those of NaCl and NaOH, primarily by downregulating different categories of genes. Pathways commonly altered by Na2CO3, NaCl, and NaOH were enriched in phenylpropanoid biosynthesis, oxidation of unsaturated fatty acids, ATP-binding cassette (ABC) transporters, as well as the metabolism of secondary metabolites. Genes for brassinosteroid biosynthesis were specifically upregulated by Na2CO3, while genes involved in ascorbate and aldarate metabolism, protein processing in the endoplasmic reticulum and by N-glycosylation, fatty acid biosynthesis, and the circadian rhythm were downregulated. This work provides the first holistic picture of early transcriptomic adaptation to Na2CO3 stress, and highlights potential molecular pathways that could be manipulated to improve tolerance in maize.

Genome-scale identification of cell-wall related genes in Arabidopsis based on co-expression network analysis.

BACKGROUND: Identification of the novel genes relevant to plant cell-wall (PCW) synthesis represents a highly important and challenging problem. Although substantial efforts have been invested into studying this problem, the vast majority of the PCW related genes remain unknown. RESULTS: Here we present a computational study focused on identification of the novel PCW genes in Arabidopsis based on the co-expression analyses of transcriptomic data collected under 351 conditions, using a bi-clustering technique. Our analysis identified 217 highly co-expressed gene clusters (modules) under some experimental conditions, each containing at least one gene annotated as PCW related according to the Purdue Cell Wall Gene Families database. These co-expression modules cover 349 known/annotated PCW genes and 2,438 new candidates. For each candidate gene, we annotated the specific PCW synthesis stages in which it is involved and predicted the detailed function. In addition, for the co-expressed genes in each module, we predicted and analyzed their cis regulatory motifs in the promoters using our motif discovery pipeline, providing strong evidence that the genes in each co-expression module are transcriptionally co-regulated. From the all co-expression modules, we infer that 108 modules are related to four major PCW synthesis components, using three complementary methods. CONCLUSIONS: We believe our approach and data presented here will be useful for further identification and characterization of PCW genes. All the predicted PCW genes, co-expression modules, motifs and their annotations are available at a web-based database: http://csbl.bmb.uga.edu/publications/materials/shanwang/CWRPdb/index.html.

Molecular dynamics analysis reveals structural insights into mechanism of nicotine N-demethylation catalyzed by tobacco cytochrome P450 mono-oxygenase.

CYP82E4, a cytochrome P450 monooxygenase, has nicotine N-demethylase (NND) activity, which mediates the bioconversion of nicotine into nornicotine in senescing tobacco leaves. Nornicotine is a precursor of the carcinogen, tobacco-specific nitrosamine. CYP82E3 is an ortholog of CYP82E4 with 95% sequence identity, but it lacks NND activity. A recent site-directed mutagenesis study revealed that a single amino acid substitution, i.e., cysteine to tryptophan at the 330 position in the middle of protein, restores the NND activity of CYP82E3 entirely. However, the same amino acid change caused the loss of the NND activity of CYP82E4. To determine the mechanism of the functional turnover of the two molecules, four 3D structures, i.e., the two molecules and their corresponding cys-trp mutants were modeled. The resulting structures exhibited that the mutation site is far from the active site, which suggests that no direct interaction occurs between the two sites. Simulation studies in different biological scenarios revealed that the mutation introduces a conformation drift with the largest change at the F-G loop. The dynamics trajectories analysis using principal component analysis and covariance analysis suggests that the single amino acid change causes the opening and closing of the transfer channels of the substrates, products, and water by altering the motion of the F-G and B-C loops. The motion of helix I is also correlated with the motion of both the F-G loop and the B-C loop and; the single amino acid mutation resulted in the curvature of helix I. These results suggest that the single amino acid mutation outside the active site region may have indirectly mediated the flexibility of the F-G and B-C loops through helix I, causing a functional turnover of the P450 monooxygenase.

Cloning and characterisation of a novel 2,4-dichlorophenol hydroxylase from a metagenomic library derived from polychlorinated biphenyl-contaminated soil.

A novel 2,4-dichlorophenol hydroxylase (TfdB, EC 1.14.13.20) gene, designated as tfdB-JLU, was identified from a metagenome constructed from polychlorinated biphenyl-contaminated soil by functional screening and heterologously expressed in Escherichia coli. The deduced amino acid sequence of tfdB-JLU exhibited less than 48% homology with other known TfdBs. The enzyme exhibited a wider substrate spectrum than the previously reported TfdBs and higher relative activity towards ortho-substituted dichlorophenols, 2-chlorophenol, and 3-chlorophenol than towards 2,4-dichlorophenol, the preferred substrate of other known TfdBs. The enzyme had a K ( m ) of 5 µM for 2,4-dichlorophenol and 6 µM for NADPH. The optimal temperature and pH of the enzyme were 25°C and 7.5, respectively. Activity of the purified TfdB-JLU was slightly enhanced by Ca(2+), Mn(2+), Co(2+), and Fe(2+), and completely inhibited by Cu(2+), Hg(2+), and Zn(2+). This study is the first report to identify a novel TfdB from a metagenome.