Kinetic and dynamical properties of truncated hemoglobins of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

Due to the low temperature, the Antarctic marine environment is challenging for protein functioning. Cold-adapted organisms have evolved proteins endowed with higher flexibility and lower stability in comparison to their thermophilic homologs, resulting in enhanced reaction rates at low temperatures. The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) genome is one of the few examples of coexistence of multiple hemoglobin genes encoding, among others, two constitutively transcribed 2/2 hemoglobins (2/2Hbs), also named truncated Hbs (TrHbs), belonging to the Group II (or O), annotated as PSHAa0030 and PSHAa2217. In this work, we describe the ligand binding kinetics and their interrelationship with the dynamical properties of globin Ph-2/2HbO-2217 by combining experimental and computational approaches and implementing a new computational method to retrieve information from molecular dynamic trajectories. We show that our approach allows us to identify docking sites within the protein matrix that are potentially able to transiently accommodate ligands and migration pathways connecting them. Consistently with ligand rebinding studies, our modeling suggests that the distal heme pocket is connected to the solvent through a low energy barrier, while inner cavities play only a minor role in modulating rebinding kinetics.

Sphaerotilus natans hemoglobins have an NADH oxidation activity and promote the yield of limonene in an engineered E. coli strain.

Bacterial hemoglobins play important roles inside the cell. Phylogenetically, they belong to three different families: the single domain hemoglobin, flavohemoglobin and truncated hemoglobin. Vitreoscilla hemoglobin (VHb) is the first characterized bacterial hemoglobin, and belongs to the single domain hemoglobin family. Heterologous expression of VHb promotes the growth of host cells under microaerobic conditions, and enhances the yield of products during fermentation. Although VHb has been widely applied in the biotechnology field, other bacterial hemoglobins have not demonstrated similar applications. In this study, we identified four bacterial hemoglobins from the microaerobic growing bacterium Sphaerotilus natans, including one flavohemoglobins (FHB) and three truncated hemoglobins (THB1, THB2 and THB3). Absorption spectrum studies validate the existent of the Soret peak and Q-band characteristic to heme and suggest heme groups in FHB and THB1 are hexa- or penta-coordinated, respectively. Our studies demonstrate that FHB and all three truncated hemoglobins have NADH oxidation and radical production activities, which is surprising since truncated hemoglobins do not have a reductase domain that could bind NADH. However, the M. tuberculosis HbN does not show these activities, indicating they are not universal among truncated hemoglobins. Docking studies suggest the nicotinamide ring of NADH may bind to the distal heme pocket of THB1, suggesting the direct electron transfer from NADH to heme might be possible. Our truncated hemoglobins also show peroxidase activities that in THB2 and THB3 could be inhibited by FdR, indicating possible interactions between FdR and truncate hemoglobins. Expression of FHB and THB1 in E. coli could promote cell growth. THB1 also enhances the production of limonene in an engineered E. coli strain, while VHb does not have this effect, which suggests that studies on truncated hemoglobins may lead to the discovery of new and more powerful tools that could have profound impact on biotechnology.

Group II truncated haemoglobin YjbI prevents reactive oxygen species-induced protein aggregation in Bacillus subtilis.

Oxidative stress-mediated formation of protein hydroperoxides can induce irreversible fragmentation of the peptide backbone and accumulation of cross-linked protein aggregates, leading to cellular toxicity, dysfunction, and death. However, how bacteria protect themselves from damages caused by protein hydroperoxidation is unknown. Here, we show that YjbI, a group II truncated haemoglobin from Bacillus subtilis, prevents oxidative aggregation of cell-surface proteins by its protein hydroperoxide peroxidase-like activity, which removes hydroperoxide groups from oxidised proteins. Disruption of the yjbI gene in B. subtilis lowered biofilm water repellence, which associated with the cross-linked aggregation of the biofilm matrix protein TasA. YjbI was localised to the cell surface or the biofilm matrix, and the sensitivity of planktonically grown cells to generators of reactive oxygen species was significantly increased upon yjbI disruption, suggesting that YjbI pleiotropically protects labile cell-surface proteins from oxidative damage. YjbI removed hydroperoxide residues from the model oxidised protein substrate bovine serum albumin and biofilm component TasA, preventing oxidative aggregation in vitro. Furthermore, the replacement of Tyr63 near the haem of YjbI with phenylalanine resulted in the loss of its protein peroxidase-like activity, and the mutant gene failed to rescue biofilm water repellency and resistance to oxidative stress induced by hypochlorous acid in the yjbI-deficient strain. These findings provide new insights into the role of truncated haemoglobin and the importance of hydroperoxide removal from proteins in the survival of aerobic bacteria.

Transcript analysis and expression of the glbO gene, encoding truncated hemoglobin,O, of M. Smegmatis implicate its role under hypoxia and oxidative stress.

Although truncated hemoglobin O, (trHbO), is ubiquitous among mycobacteria, its physiological function is not very obvious and may be diverse. In an attempt to understand role of trHbO in cellular metabolism of a non-pathogenic mycobacterium, we analysed expression profile of the glbO gene, encoding trHbO, in M. smegmatis and studied implications of its overexpression on physiology of its host under different environmental conditions. Quantitative RT-PCR indicated that transcript level of the glbO gene remains low at a basal level under aerobic growth cycle of M. smegmatis but its level gets induced significantly during low oxygen, oxidative stress and macrophage infection. Overexpression of the glbO gene enhanced growth of M. smegmatis under hypoxia, promoted pellicle biofilm formation and provided resistance towards oxidative stress. Additionally, glbO gene overexpressing M. smegmatis exhibited enhanced cell survival over isogenic control cells and altered the level of pro- and anti- inflammatory cytokines during intracellular infection. These results suggested important role of trHbO, in supporting the cellular metabolism and survival of M, smegmatis both under low oxygen and oxidative stress.

Vitreoscilla hemoglobin enhances the catalytic performance of industrial oxidases in vitro.

Oxidases are a group of oxidoreductases and need molecular oxygen in the catalytic process. Vitreoscilla hemoglobin (VHb) can improve the growth and productivity of host cells under hypoxic conditions, rendering it attractive for industrial application. In this work, we demonstrated the addition of immobilized VHb increased the catalytic activity of immobilized D-amino acid oxidase of Trigonopsis variabilis by two-fold when catalyzing cephalosporin C under oxygen-limited conditions. A similar increase of activities was observed in glucose oxidase, alcohol oxidase, and p-hydroxymandelate synthase by adding free VHb or immobilized VHb under hypoxic conditions. When L-glutamate oxidase was used to catalyze L-glutamate to produce α-ketoglutarate, the yield increased from 80.6 to 96.9% by fusing VHb with L-glutamate oxidase. Results demonstrated that the addition of free VHb, immobilized VHb, or fused VHb could increase the catalytic efficiency of oxidases, which was considered by increasing the concentration of the microenvironmental oxygen. Thus, VHb may become a potential additive agent to promote the efficiency of oxidases on industrial scale . KEY POINTS: • First time confirmation of facilitation of VHb on several industrial oxidases in vitro • VHb functions under hypoxic conditions rather than oxygen-enriched conditions • VHb functions in vitro in the form of free, immobilized protein and fusion enzyme.

Overexpression of Shinorhizobium meliloti flavohemoglobin improves cell growth and fatty acid biosynthesis in oleaginous fungus Mucor circinelloides.

Oxygen availability is a limiting factor for lipid biosynthesis in eukaryotic microorganisms. Two bacterial hemoglobins from Vitreoscilla sp. (VHb) and Shinorhizobium meliloti (SHb), which deliver oxygen to the respiratory chain to produce more ATP, were introduced into Mucor circinelloides to alleviate oxygen limitation, thereby improving cell growth and fatty acid production. The VHb and SHb genes were integrated into the M. circinelloides MU402 genome by homologous recombination. VHb and SHb protein expression was verified by carbon monoxide difference spectrum analysis. The biomass was increased by ~ 50% in the strain expressing SHb compared with VHb. The total fatty acid (TFA) content of the strain expressing SHb reached 15.7% of the dry cell weight (~ 40% higher than that of the control strain) during flask cultivation. The biomass and TFA content were markedly increased (12.1 g/L and 21.1% dry cell weight, respectively) in strains expressing SHb than strains expressing VHb during fermenter cultivation. VHb and SHb expression also increased the proportion of polyunsaturated fatty acids. Overexpressed bacterial hemoglobins, especially SHb, increased cell growth and TFA content in M. circinelloides at low and high aeration, suggesting that SHb improves fatty acid production more effectively than VHb in oleaginous microorganisms.

Truncated (2/2) hemoglobin: Unconventional structures and functional roles in vivo and in human pathogenesis.

Truncated hemoglobins (trHbs) build a sub-class of the globin family, found in eubacteria, cyanobacteria, unicellular eukaryotes, and in higher plants; among these, selected human pathogens are found. The trHb fold is based on a 2/2 α-helical sandwich, consisting of a simplified and reduced-size version of the classical 3/3 α-helical sandwich of vertebrate and invertebrate globins. Phylogenetic analysis indicates that trHbs further branch into three groups: group I (or trHbN), group II (or trHbO), and group III (or trHbP), each group being characterized by specific structural features. Among these, a protein matrix tunnel, or a cavity system implicated in diatomic ligand diffusion through the protein matrix, is typical of group I and group II, respectively. In general, a highly intertwined network of hydrogen bonds stabilizes the heme bound ligand, despite variability of the heme distal residues in the different trHb groups. Notably, some organisms display genes from more than one trHb group, suggesting that trHbN, trHbO, and trHbP may support different functions in vivo, such as detoxification of reactive nitrogen and oxygen species, respiration, oxygen storage/sensoring, thus aiding survival of an invading microorganism. Here, structural features and proposed functions of trHbs from human pathogens are reviewed.

Coexpression of VHb and MceG genes in Mycobacterium sp. Strain LZ2 enhances androstenone production via immobilized repeated batch fermentation.

Androstenone production is limited by low-efficiency substrate transport and dissolved oxygen levels during fermentation. In this study, the coexpression of the optimized Vitreoscilla hemoglobin (VHb) and sterol transporter ATPase (MceG) genes in Mycobacterium sp. LZ2 (Msp) was investigated to alleviate dissolved oxygen and mass transfer limitations. Results revealed that Msp-vgb/mceG effectively improved the growth, production, and adaptation to dissolved oxygen compared with those of Msp. The increased catalase activity and reduced intracellular ROS levels enhanced cell viability and promoted transcription of genes critical for phytosterol metabolism. Bagasse as an immobilization carrier increased the productivity of Msp-vgb/mceG by 56%. Immobilized repeat batch fermentation reduced the biotransformation period from 60 days to 37 days and improved the productivity from 0.039 g/L/h to 0.069 g/L/h. To the best of our knowledge, this work is the first study on the immobilization of recombinant mycobacteria on bagasse for androstenone production.

Expression of the Vitreoscilla hemoglobin gene in Nannochloropsis oceanica regulates intracellular oxygen balance under high-light.

Nannochloropsis oceanica is widely used as a model photosynthetic chassis to produce fatty acids and carotenoid pigments. However, intense light typically causes excessive generation of reactive oxygen species (ROS) and photorespiration in microalgal cells, which results in decreased cell growth rate and unsaturated fatty acid content. In this study, the Vitreoscilla hemoglobin gene (vgb) was introduced into N. oceanica cells and expressed by using the light-harvesting complex promoter and its signal peptide. Compared with wild type (WT), the growth rate of transformants increased by 7.4%-18.5%, and the eicosapentaenoic acid content in an optimal transformant increased by 21.0%. Correspondingly, the intracellular ROS levels decreased by 56.9%-70.0%, and the catalase content in transformants was about 1.8 times that of WT. The photorespiration level of transformants was reduced by the measurement and calculation of the dissolved oxygen concentration under the condition of light-dark transition. The expression level of the key genes related to the photorespiration pathway in transformants was more than 80% lower than that in WT. These results indicated that Vitreoscilla hemoglobin could improve microalgal growth by reducing ROS damage and modulating photorespiration under stress conditions.

Distal lysine (de)coordination in the algal hemoglobin THB1: A combined computer simulation and experimental study.

THB1 is a monomeric truncated hemoglobin from the green alga Chlamydomonas reinhardtii. In the absence of exogenous ligands and at neutral pH, the heme group of THB1 is coordinated by two protein residues, Lys53 and His77. THB1 is thought to function as a nitric oxide dioxygenase, and the distal binding of O2 requires the cleavage of the Fe-Lys53 bond accompanied by protonation and expulsion of the lysine from the heme cavity into the solvent. Nuclear magnetic resonance spectroscopy and crystallographic data have provided dynamic and structural insights of the process, but the details of the mechanism have not been fully elucidated. We applied a combination of computer simulations and site-directed mutagenesis experiments to shed light on this issue. Molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics restrained optimizations were performed to explore the nature of the transition between the decoordinated and lysine-bound states of the ferrous heme in THB1. Lys49 and Arg52, which form ionic interactions with the heme propionates in the X-ray structure of lysine-bound THB1, were observed to assist in maintaining Lys53 inside the protein cavity and play a key role in the transition. Lys49Ala, Arg52Ala and Lys49Ala/Arg52Ala THB1 variants were prepared, and the consequences of the replacements on the Lys (de)coordination equilibrium were characterized experimentally for comparison with computational prediction. The results reinforced the dynamic role of protein-propionate interactions and strongly suggested that cleavage of the Fe-Lys53 bond and ensuing conformational rearrangement is facilitated by protonation of the amino group inside the distal cavity.

Multiple putative methemoglobin reductases in C. reinhardtii may support enzymatic functions for its multiple hemoglobins.

The ubiquitous nature of hemoglobins, their presence in multiple forms and low cellular expression in organisms suggests alternative physiological functions of hemoglobins in addition to oxygen transport and storage. Previous research has proposed enzymatic function of hemoglobins such as nitric oxide dioxygenase, nitrite reductase and hydroxylamine reductase. In all these enzymatic functions, active ferrous form of hemoglobin is converted to ferric form and reconversion of ferric to ferrous through reduction partners is under active investigation. The model alga C. reinhardtii contains multiple globins and is thus expected to have multiple putative methemoglobin reductases to augment the physiological functions of the novel hemoglobins. In this regard, three putative methemoglobin reductases and three algal hemoglobins were characterized. Our results signify that the identified putative methemoglobin reductases can reduce algal methemoglobins in a nonspecific manner under in vitro conditions. Enzyme kinetics of two putative methemoglobin reductases with methemoglobins as substrates and in silico analysis support interaction between the hemoglobins and the two reduction partners as also observed in vitro. Our investigation on algal methemoglobin reductases underpins the valuable chemistry of nitric oxide with the newly discovered hemoglobins to ensure their physiological relevance, with multiple hemoglobins probably necessitating the presence of multiple reductases.

Combining co-culturing of Paenibacillus strains and Vitreoscilla hemoglobin expression as a strategy to improve biodesulfurization.

Enhancement of the desulfurization activities of Paenibacillus strains 32O-W and 32O-Y were investigated using dibenzothiophene (DBT) and DBT sulfone (DBTS) as sources of sulphur in growth experiments. Strains 32O-W, 32O-Y and their co-culture (32O-W plus 32O-Y), and Vitreoscilla hemoglobin (VHb) expressing recombinant strain 32O-Yvgb and its co-culture with strain 32O-W were grown at varying concentrations (0·1-2 mmol l-1 ) of DBT or DBTS for 96 h, and desulfurization measured by production of 2-hydroxybiphenyl (2-HBP) and disappearance of DBT or DBTS. Of the four cultures grown with DBT as sulphur source, the best growth occurred for the 32O-Yvgb plus 32O-W co-culture at 0·1 and 0·5 mmol l-1 DBT. Although the presence of vgb provided no consistent advantage regarding growth on DBTS, strain 32O-W, as predicted by previous work, was shown to contain a partial 4S desulfurization pathway allowing it to metabolize this 4S pathway intermediate.

Enhancement of glucaric acid production in Saccharomyces cerevisiae by expressing Vitreoscilla hemoglobin.

OBJECTIVE: To enhance the glucaric acid (GA) production in Saccharomyces cerevisiae, the Vitreoscilla hemoglobin was employed to reinforce cellular oxygen supplement. Additionally, the pH-free fermentation strategy was engaged to lower the cost brought by base feeding during the acid-accumulated and long-period glucaric acid production. RESULTS: Recombinant yeast Bga-4 was constructed harboring Vitreoscilla hemoglobin on the basis of previous Bga-3. Higher glucose uptake rate, growth rate, and ethanol reuse rate were achieved in Bga-4 in shake-flask fermentation than those in Bga-3. Furthermore, the fed-batch fermentation in a 5-L bioreactor was performed without pH control, resulting in a final glucaric acid titer of 6.38 g/L. CONCLUSIONS: Both the GA titer and biomass were enhanced along with the efficiency of ethanol re-utilization in the presence of VHb. Moreover, the absence of base feeding for long-period fermentation reduced production cost, which is meaningful for industrial applications.

Physiological Response of Escherichia coli W3110 and BL21 to the Aerobic Expression of Vitreoscilla Hemoglobin.

The aerobic growth and metabolic performance of Escherichia coli strains BL21 and W3110 were studied when the Vitreoscilla hemoglobin (VHb) was constitutively expressed in the chromosome. When VHb was expressed, acetate production decreased in both strains and was nearly eliminated in BL21. Transcriptional levels of the glyoxylate shunt genes decreased in both strains when VHb was expressed. However, higher transcription of the α-ketoglutarate dehydrogenase genes were observed for W3110, while for BL21 transcription levels decreased. VHb expression reduced the transcription of the cytochrome bo3 genes only in BL21. These results are useful for better selecting a production host.

Truncated Hemoglobin O Carries an Autokinase Activity and Facilitates Adaptation of Mycobacterium tuberculosis Under Hypoxia.

Aims: Although the human pathogen, Mycobacterium tuberculosis (Mtb), is strictly aerobic and requires efficient supply of oxygen, it can survive long stretches of severe hypoxia. The mechanism responsible for this metabolic flexibility is unknown. We have investigated a novel mechanism by which hemoglobin O (HbO), operates and supports its host under oxygen stress. Results: We discovered that the HbO exists in a phospho-bound state in Mtb and remains associated with the cell membrane under hypoxia. Deoxy-HbO carries an autokinase activity that disrupts its dimeric assembly into monomer and facilitates its association with the cell membrane, supporting survival and adaptation of Mtb under low oxygen conditions. Consistent with these observations, deletion of the glbO gene in Mycobacterium bovis bacillus Calmette-Guerin, which is identical to the glbO gene of Mtb, attenuated its survival under hypoxia and complementation of the glbO gene of Mtb rescued this inhibition, but phosphorylation-deficient mutant did not. These results demonstrated that autokinase activity of the HbO modulates its physiological function and plays a vital role in supporting the survival of its host under hypoxia. Innovation and Conclusion: Our study demonstrates that the redox-dependent autokinase activity regulates oligomeric state and membrane association of HbO that generates a reservoir of oxygen in the proximity of respiratory membranes to sustain viability of Mtb under hypoxia. These results thus provide a novel insight into the physiological function of the HbO and demonstrate its pivotal role in supporting the survival and adaptation of Mtb under hypoxia.

Truncated Hemoglobins 1 and 2 Are Implicated in the Modulation of Phosphorus Deficiency-Induced Nitric Oxide Levels in Chlamydomonas.

Truncated hemoglobins (trHbs) form a widely distributed family of proteins found in archaea, bacteria, and eukaryotes. Accumulating evidence suggests that trHbs may be implicated in functions other than oxygen delivery, but these roles are largely unknown. Characterization of the conditions that affect trHb expression and investigation of their regulatory mechanisms will provide a framework for elucidating the functions of these globins. Here, the transcription of Chlamydomonas trHb genes (THB1-12) under conditions of phosphorus (P) deprivation was analyzed. Three THB genes, THB1, THB2, and THB12 were expressed at the highest level. For the first time, we demonstrate the synthesis of nitric oxide (NO) under P-limiting conditions and the production of NO by cells via a nitrate reductase-independent pathway. To clarify the functions of THB1 and THB2, we generated and analyzed strains in which these THBs were strongly under-expressed by using an artificial microRNA approach. Similar to THB1 knockdown, the depletion of THB2 led to a decrease in cell size and chlorophyll levels. We provide evidence that the knockdown of THB1 or THB2 enhanced NO production under P deprivation. Overall, these results demonstrate that THB1 and THB2 are likely to contribute, at least in part, to acclimation responses in P-deprived Chlamydomonas.

Enhanced production of individual ganoderic acids by integrating Vitreoscilla haemoglobin expression and calcium ion induction in liquid static cultures of Ganoderma lingzhi.

Ganoderic acids produced by Ganoderma exhibit anticancer and antimetastatic activities. A novel approach by combining Vitreoscilla haemoglobin (VHb) expression and calcium ion induction was developed to enhance ganoderic acid (GA) production in liquid static cultures of G. lingzhi. The maximum contents of GA-O, GA-S and GA-Me were 1451.33 ± 67.50, 1431.23 ± 79.74 and 1283.81 ± 85.13 µg per 100 mg cell weight, respectively under the integrated approach, which are the highest contents as ever reported in Ganoderma. The contents of squalene and lanosterol were increased by 2.0- and 3.0-fold in this case compared with those in the control. The transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, farnesyl-diphosphate synthase, squalene synthase and cytochrome P450 CYP5150L8 were upregulated by 2.56-, 3.31-, 2.59- and 6.12-fold respectively. Additionally, the expression of VHb improved the ratio of type I to type II GA in liquid static cultivation of G. lingzhi. The transcription levels of cyp512a2, cyp512v2 and cyp512a13, candidate cytochrome P450 genes involved in oxidative modification of the lanostane skeleton in GA biosynthesis, were also increased by 2.28-, 2.65- and 3.54-fold in the VHb-expressing strain respectively. Our results illustrated that the approach described here efficiently improved GA production in G. lingzhi fermentation.

Myxococcus xanthus truncated globin HbO: in silico analysis and functional characterization.

Truncated globins are 20-40 amino acids shorter than full length globins. Till date, globins have been characterized predominantly from bacteria involved in pathogenicity, nitrogen fixation and photosynthesis, where they are implicated in bacterial virulence within the host, protection of nitrogenase from oxygen inactivation and prevention of oxidative damage to the photosynthetic machinery respectively. Myxococcus xanthus, the model myxobacterium, is an obligate aerobe with a multicellular stage in its life cycle where cells encounter oxygen limitation. This work was undertaken to investigate the potential role of the truncated globin in M. xanthus. To examine the role of globins in this unique group of bacteria, the gene coding for a putative truncated globin (HbO) was identified in the genome of M. xanthus DK 1622. The sequence analysis by bioinformatics approaches revealed that HbO from M. xanthus (Mx-HbO) likely adopts a 2-on-2 alpha helical fold of the truncated globins. The gene coding for Mx-HbO was cloned and its expression in E. coli imparted reddish tinge to the cells. The spectral analysis confirmed it to be a functional globin. The expression of Mx-HbO in the heterologous host improved its growth, resulting in the attainment of higher cell density in culture. The transcript of Mx-hbO was induced threefold in the host cells when grown under low aeration condition as compared to the cells grown under high aeration condition. In M. xanthus, an obligate aerobe, where cell growth accompanies swarming, there is a higher density of cells in the middle of the swarm. Our results suggest that Mx-HbO is a functional globin and could facilitate the growth of cells facing oxygen deprivation, the condition prevailing in the middle of the swarm.

Targeted expression of Vitreoscilla hemoglobin improves the production of tropane alkaloids in Hyoscyamus niger hairy roots.

Under hypoxic conditions, the expression of Vitreoscilla hemoglobin (VHb) in plants is proposed to increase the productivity of certain oxygen-requiring metabolic pathways by promoting the delivery of oxygen. Tropane alkaloids (TAs) are a class of important plant secondary metabolites with significant medicinal value; the final step in their biosynthesis requires oxygen. Whether heterologous expression of VHb, especially in different subcellular compartments, can accelerate the accumulation of TAs is not known. Herein, the effect of heterologous expression of VHb in different subcellular locations on the TA profile of H. niger hairy roots was investigated. The targeted expression of VHb in the plastids (using pVHb-RecA construct), led to the accumulation of 197.68 µg/g hyoscyamine in the transgenic H. niger hairy roots, which was 1.25-fold of the content present in the lines in which VHb expression was not targeted, and 3.66-fold of that present in the wild type (WT) lines. The content of scopolamine was increased by 2.20- and 4.70-fold in the pVHb-RecA transgenic lines compared to that in the VHb transgenic and WT lines. Our results demonstrate that VHb could stimulate the accumulation of TAs in the transgenic H. niger hairy roots. Quantitative RT-PCR analysis revealed that the expression of key genes involved in TA biosynthesis increased significantly in the VHb transgenic lines. We present the first description of a highly efficient strategy to increase TA content in H. niger. Moreover, our results also shed light on how the production of desired metabolites can be efficiently enhanced by using more accurate and appropriate genetic engineering strategies.

Replacement of the Distal Histidine Reveals a Noncanonical Heme Binding Site in a 2-on-2 Hemoglobin.

Heme ligation in hemoglobin is typically assumed by the "proximal" histidine. Hydrophobic contacts, ionic interactions, and the ligation bond secure the heme between two α-helices denoted E and F. Across the hemoglobin superfamily, several proteins also use a "distal" histidine, making the native state a bis-histidine complex. The group 1 truncated hemoglobin from Synechocystis sp. PCC 6803, GlbN, is one such bis-histidine protein. Ferric GlbN, in which the distal histidine (His46 or E10) has been replaced with a leucine, though expected to bind a water molecule and yield a high-spin iron complex at neutral pH, has low-spin spectral properties. Here, we applied nuclear magnetic resonance and electronic absorption spectroscopic methods to GlbN modified with heme and amino acid replacements to identify the distal ligand in H46L GlbN. We found that His117, a residue located in the C-terminal portion of the protein and on the proximal side of the heme, is responsible for the formation of an alternative bis-histidine complex. Simultaneous coordination by His70 and His117 situates the heme in a binding site different from the canonical site. This new holoprotein form is achieved with only local conformational changes. Heme affinity in the alternative site is weaker than in the normal site, likely because of strained coordination and a reduced number of specific heme-protein interactions. The observation of an unconventional heme binding site has important implications for the interpretation of mutagenesis results and globin homology modeling.