Histidinol-phosphate Aminotransferase Research Articles

Rv2231c, a unique histidinol phosphate aminotransferase from Mycobacterium tuberculosis, supports virulence by inhibiting host-directed defense

Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de novo synthesis and uptake of various amino acids from host cells for metabolic demands. Pyridoxal phosphate-dependent histidinol phosphate aminotransferase-HspAT enzyme is critically required for histidine biosynthesis. HspAT is involved in metabolic synthesis of histidine, phenylalanine, tyrosine, tryptophan, and novobiocin. We showed that M. tuberculosis Rv2231c is a conserved enzyme with HspAT activity. Rv2231c is a monomeric globular protein that contains α-helices and β-sheets. It is a secretory and cell wall-localized protein that regulates critical pathogenic attributes. Rv2231c enhances the survival and virulence of recombinant M. smegmatis in infected RAW264.7 macrophage cells. Rv2231c is recognized by the TLR4 innate immune receptor and modulates the host immune response by suppressing the secretion of the antibacterial pro-inflammatory cytokines TNF, IL-12, and IL-6. It also inhibits the expression of co-stimulatory molecules CD80 and CD86 along with antigen presenting molecule MHC-I on macrophage and suppresses reactive nitrogen species formation, thereby promoting M2 macrophage polarization. Recombinant M. smegmatis expressing Rv2231c inhibited apoptosis in macrophages, promoting efficient bacterial survival and proliferation, thereby increasing virulence. Our results indicate that Rv2231c is a moonlighting protein that regulates multiple functions of M. tuberculosis pathophysiology to increase its virulence. These mechanistic insights can be used to better understand the pathogenesis of M. tuberculosis and to design strategies for tuberculosis mitigation.

Multifactorial interaction of selenium, iron, xylose, and glycine on cordycepin metabolism in Cordyceps militaris.

Cordycepin, a nucleoside analog, is the main antioxidative and antimicrobial substance in Cordyceps militaris. Toimprove the metabolism of cordycepin, carbon sources, nitrogen sources, trace elements, and precursors were studied by single factor, Plackett-Burman, and central composite designs in C. militaris mycelial fermentation. Under the regulation of the multifactorial interactions of selenite, ferrous chloride, xylose, and glycine, cordycepin production was increased by 5.2-fold compared with the control. The gene expression of hexokinase, ATP phosphoribosyltransferase, adenylosuccinate synthetase, and cns1-3 in the glycolysis, pentose phosphate, and adenosine synthesis pathways were increased by 3.2-7.5 times due to multifactorial interactions, while the gene expression of histidine biosynthesis trifunctional protein and histidinol-phosphate aminotransferase in histidine synthesis pathway were decreased by 23.4%-56.2%. Increasing with cordycepin production, glucose uptake was accelerated, mycelia growth was inhibited, and the cell wall was damaged. Selenomethionine (SeMet), selenocysteine (SeCys), and selenium nanoparticles (SeNPs) were the major Se species in C. militaris mycelia. This study provides a new insight for promoting cordycepin production by regulating glycolysis, pentose phosphate, and histidine metabolism. KEY POINTS: • Cordycepin production in the CCDmax group was 5.2-fold than that of the control. •Glucose uptake of the CCDmax group was accelerated and cell wall was damaged. •The metabolic flux was concentrated to the cordycepin synthesis pathway.

Insights into the substrate specificity, structure, and dynamics of plant histidinol-phosphate aminotransferase (HISN6)

Histidinol-phosphate aminotransferase is the sixth protein (hence HISN6) in the histidine biosynthetic pathway in plants. HISN6 is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the reversible conversion of imidazole acetol phosphate into L-histidinol phosphate (HOLP). Here, we show that plant HISN6 enzymes are closely related to the orthologs from Chloroflexota. The studied example, HISN6 from Medicago truncatula (MtHISN6), exhibits a surprisingly high affinity for HOLP, which is much higher than reported for bacterial homologs. Moreover, unlike the latter, MtHISN6 does not transaminate phenylalanine. High-resolution crystal structures of MtHISN6 in the open and closed states, as well as the complex with HOLP and the apo structure without PLP, bring new insights into the enzyme dynamics, pointing at a particular role of a string-like fragment that oscillates near the active site and participates in the HOLP binding. When MtHISN6 is compared to bacterial orthologs with known structures, significant differences arise in or near the string region. The high affinity of MtHISN6 appears linked to the particularly tight active site cavity. Finally, a virtual screening against a library of over 1.3 mln compounds revealed three sites in the MtHISN6 structure with the potential to bind small molecules. Such compounds could be developed into herbicides inhibiting plant HISN6 enzymes absent in animals, which makes them a potential target for weed control agents.

Comparative proteome and transcriptome analyses of the response of postharvest pears to Penicillium expansum infection

Penicillium expansum causes blue mold disease in pears during storage and transportation, causing huge economic losses. However, researches on the defense mechanisms of pears against P. expansum are still in the early stages. Therefore, many unknown disease-resistant factors have yet to be excavated, severely limiting the breakthrough progress of pear blue mold control strategies. The current study used transcriptome and proteome approaches to evaluate pear defense mechanisms following infection with P. expansum. 96% of pear proteins were associated with the transcriptome data. Gene Ontology (GO) enrichment analysis determined that the remaining associated genes/proteins were significantly enriched in 80 GO terms. The significantly enriched GO terms included oxyacid metabolism, aldehyde-lyase activity, transition metal ion binding, terpenoid metabolic process, response to stimulus, etc. An enrichment analysis conducted by the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that 122 metabolic pathways were enriched, of which 11 were closely related to other metabolic pathways. In these 11 metabolic pathways, the genes/proteins (such as peroxidase, histidinol-phosphate aminotransferase (hisC), prephenate dehydratase (ADT), etc.) related to the biosynthesis of plant secondary metabolites, and the genes/proteins (such as ethylene-responsive transcription factor 1 (ERF), BR-signaling kinase (BSK), brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1), etc.) related to plant hormones were significantly enriched. Overall, this study provides a deep understanding of pear’s defense response, which could be used in future research to develop strategies to control fungal diseases.

Repurposed HisC Aminotransferases Complete the Biosynthesis of Some Methanobactins.

Methanobactins (Mbns) are ribosomally produced, post-translationally modified bacterial natural products with a high affinity for copper. MbnN, a pyridoxal 5'-phosphate-dependent aminotransferase, performs a transamination reaction that is the last step in the biosynthesis of Mbns produced by several Methylosinus species. Our bioinformatic analyses indicate that MbnNs likely derive from histidinol-phosphate aminotransferases (HisCs), which play a key role in histidine biosynthesis. A comparison of the HisC active site with the predicted MbnN structure suggests that MbnN's active site is altered to accommodate the larger and more hydrophobic substrates necessary for Mbn biosynthesis. Moreover, we have confirmed that MbnN is capable of catalyzing the final transamination step in Mbn biosynthesis in vitro and in vivo. We also demonstrate that without this final modification, Mbn exhibits significantly decreased stability under physiological conditions. An examination of other Mbns and Mbn operons suggests that N-terminal protection of this family of natural products is of critical importance and that several different means of N-terminal stabilization have evolved independently in Mbn subfamilies.

Crystal structures of Mycobacterium tuberculosis HspAT and ArAT reveal structural basis of their distinct substrate specificities.

Aminotransferases of subfamily Iβ, which include histidinol phosphate aminotransferases (HspATs) and aromatic amino acid aminotransferases (ArATs), are structurally similar but possess distinct substrate specificities. This study, encompassing structural and biochemical characterisation of HspAT and ArAT from Mycobacterium tuberculosis demonstrates that the residues lining the substrate binding pocket and N-terminal lid are the primary determinants of their substrate specificities. In mHspAT, hydrophilic residues in the substrate binding pocket and N-terminal lid allow the entry and binding of its preferential substrate, Hsp. On the other hand, the hydrophobic nature of both the substrate binding pocket and the N-terminal lid of mArAT is responsible for the discrimination of a polar substrate such as Hsp, while facilitating the binding of Phe and other aromatic residues such as Tyr and Trp. In addition, the present study delineates the ligand induced conformational rearrangements, providing insights into the plasticity of aminotransferases. Furthermore, the study also demonstrates that the adventitiously bound ligand 2-(N-morpholino)ethanesulfonic acid (MES) is indeed a specific inhibitor of HspAT. These results suggest that previously untapped morpholine-ring scaffold compounds could be explored for the design of new anti-TB agents.

Sample preparation, crystallization and structure solution of HisC from Mycobacterium tuberculosis.

Histidinolphosphate aminotransferase (HisC; Rv1600) from Mycobacterium tuberculosis was overexpressed in M. smegmatis and purified to homogeneity using nickel-nitrilotriacetic acid metal-affinity and gel-filtration chromatography. Diffraction-quality crystals suitable for X-ray analysis were grown by the hanging-drop vapour-diffusion technique using 30% polyethylene glycol monomethyl ether 2000 as the precipitant. The crystals belonged to the hexagonal space group P3221, with an unusual high solvent content of 74.5%. X-ray diffraction data were recorded to 3.08 Å resolution from a single crystal using in-house Cu Kα radiation. The structure of HisC was solved by the molecular-replacement method using its Corynebacterium glutamicum counterpart as a search model. HisC is a dimer in the crystal as well as in solution.

Molecular cloning, overexpression, purification, crystallization and preliminary X-ray diffraction studies of histidinol phosphate aminotransferase (HisC2) from Mycobacterium tuberculosis.

HisC2 from Mycobacterium tuberculosis was overexpressed in M. smegmatis and purified to homogeneity using nickel-nitrilotriacetic acid metal-affinity and gel-filtration chromatography. Diffraction-quality crystals were grown using the hanging-drop vapour-diffusion technique from a condition consisting of 7 mg ml(-1) HisC2 (in 20 mM Tris pH 8.8, 50 mM NaCl and 5% glycerol), 1 M succinic acid pH 7.0, 0.1 M HEPES pH 7.0 and 1%(w/v) polyethylene glycol monomethyl ether 2000. The crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 255.98, b=77.09, c = 117.97 Å. X-ray diffraction data were recorded to 2.45 Å resolution from a single crystal using the in-house X-ray facility.

Adaptation by stochastic switching of a monostable genetic circuit in Escherichia coli

Stochastic switching is considered as a cost-saving strategy for adaptation to environmental challenges. We show here that stochastic switching of a monostable circuit can mediate the adaptation of the engineered OSU12-hisC Escherichia coli strain to histidine starvation. In this strain, the hisC gene was deleted from the His operon and placed under the control of a monostable foreign promoter. In response to histidine depletion, the OSU12-hisC population shifted to a higher HisC expression level, which is beneficial under starving conditions but is not favoured by the monostable circuit. The population shift was accompanied by growth recovery and was reversible upon histidine addition. A weak directionality in stochastic switching of hisC was observed in growing microcolonies under histidine-free conditions. Directionality and fate decision were in part dependent on the initial cellular status. Finally, microarray analysis indicated that OSU12-hisC reorganized its transcriptome to reach the appropriate physiological state upon starvation. These findings suggest that bacteria do not necessarily need to evolve signalling mechanisms to control gene expression appropriately, even for essential genes.

Protein interaction network analysis—Approach for potential drug target identification in Mycobacterium tuberculosis

In host–parasite diseases like tuberculosis, non-homologous proteins (enzymes) as drug target are first preference. Most potent drug target can be identified among large number of non-homologous protein through protein interaction network analysis. In this study, the entire promising dimension has been explored for identification of potential drug target. A comparative metabolic pathway analysis of the host Homo sapiens and the pathogen M. tuberculosis H37Rv has been performed with three level of analysis. In first level, the unique metabolic pathways of M. tuberculosis have been identified through its comparative study with H. sapiens and identification of non-homologous proteins has been done through BLAST similarity search. In second level, choke-point analysis has been performed with identified non-homologous proteins of metabolic pathways. In third level, two type of analysis have been performed through protein interaction network. First analysis has been done to find out the most potential metabolic functional associations among all identified choke point proteins whereas second analysis has been performed to find out the functional association of high metabolic interacting proteins to pathogenesis causing proteins. Most interactive metabolic proteins which have highest number of functional association with pathogenesis causing proteins have been considered as potential drug target. A list of 18 potential drug targets has been proposed which are various stages of progress at the TBSGC and proposed drug targets are also studied for other pathogenic strains. As a case study, we have built a homology model of identified drug targets histidinol-phosphate aminotransferase (HisC1) using MODELLER software and various information have been generated through molecular dynamics which will be useful in wetlab structure determination. The generated model could be further explored for insilico docking studies with suitable inhibitors.

Insights into the structural basis of substrate recognition by histidinol-phosphate aminotransferase fromCorynebacterium glutamicum

Histidinol-phosphate aminotransferase (HisC) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the reversible transamination reaction between histidinol phosphate (His-P) and 2-oxoglutarate (O-Glu). The crystal structures of apo histidinol-phosphate aminotransferase from Corynebacterium glutamicum, of the internal PLP aldimine adduct and of a pyridoxamine 5-phosphate-enzyme complex were determined at resolutions of 2.2, 2.1 and 1.8 A, respectively. Residues important for substrate specificity were identified by modelling His-P into the active site and comparison with crystal structures of HisC from Thermotoga maritima and Escherichia coli. Four of the residues lining the substrate-binding pocket were studied by site-directed mutagenesis. Kinetic analysis of the Tyr21Phe mutant suggested that the hydrogen bond between the side chain of this residue and the phosphate group of His-P is important for recognition of the natural substrate and discrimination against other potential amino donors such as phenylalanine and leucine. The mutagenesis studies further indicated that residue Asn99 does not contribute to the specific recognition of the amino-acid donor, but may be involved in binding of the phosphate group of pyridoxal 5'-phosphate. The conserved residues Tyr123 and Tyr257 interact with the substrate through van der Waals interactions and their potential for hydrogen-bonding interactions is not utilized in substrate recognition, as the corresponding phenylalanine mutants show only a moderate effect on the catalytic efficiency kcat/Km.

The hpa1 Mutant of Arabidopsis Reveals a Crucial Role of Histidine Homeostasis in Root Meristem Maintenance

Histidine (His) is an essential ingredient for protein synthesis and is required by all living organisms. In higher plants, although there is considerable evidence that His is essential for plant growth and survival, there is very little information as to whether it plays any specific role in plant development. Here, we present evidence for such a role of this amino acid in root development in Arabidopsis (Arabidopsis thaliana) from the characterization of a novel Arabidopsis mutant, hpa1, which has a very short root system and carries a mutation in one of the two Arabidopsis histidinol-phosphate aminotransferase (HPA) genes, AtHPA1. We have established that AtHPA1 encodes a functional HPA and that its complete knockout is embryo lethal. Biochemical analysis shows that the mutation in hpa1 only resulted in a 30% reduction in free His content and had no significant impact on the total His content. It did not cause any known symptoms of His starvation. However, the mutant displayed a specific developmental defect in root meristem maintenance and was unable to sustain primary root growth 2 d after germination. We have demonstrated that the root meristem failure in the mutant is tightly linked to the reduction in free His content and could be rescued by either exogenous His supplementation or AtHPA1 overexpression. Our results therefore reveal an important role of His homeostasis in plant development.

Structural Studies of the Catalytic Reaction Pathway of a Hyperthermophilic Histidinol-phosphate Aminotransferase

In histidine biosynthesis, histidinol-phosphate aminotransferase catalyzes the transfer of the amino group from glutamate to imidazole acetol-phosphate producing 2-oxoglutarate and histidinol phosphate. In some organisms such as the hyperthermophile Thermotoga maritima, specific tyrosine and aromatic amino acid transaminases have not been identified to date, suggesting an additional role for histidinol-phosphate aminotransferase in other transamination reactions generating aromatic amino acids. To gain insight into the specific function of this transaminase, we have determined its crystal structure in the absence of any ligand except phosphate, in the presence of covalently bound pyridoxal 5'-phosphate, of the coenzyme histidinol phosphate adduct, and of pyridoxamine 5'-phosphate. The enzyme accepts histidinol phosphate, tyrosine, tryptophan, and phenylalanine, but not histidine, as substrates. The structures provide a model of how these different substrates could be accommodated by histidinol-phosphate aminotransferase. Some of the structural features of the enzyme are more preserved between the T. maritima enzyme and a related threonine-phosphate decarboxylase from S. typhimurium than with histidinol-phosphate aminotransferases from different organisms.

Crystal structures of branched-chain amino acid aminotransferase complexed with glutamate and glutarate: true reaction intermediate and double substrate recognition of the enzyme.

Branched-chain amino acid aminotransferase (BCAT), which has pyridoxal 5'-phosphate as a cofactor, is a key enzyme in the biosynthetic pathway of hydrophobic amino acids (leucine, isoleucine, and valine). The enzyme reversibly catalyzes the transfer of the amino group of a hydrophobic amino acid to 2-oxoglutarate to form a 2-oxo acid and glutamate. Therefore, the active site of BCAT should have a mechanism to enable recognition of an acidic amino acid as well as a hydrophobic amino acid (double substrate recognition). The three-dimensional structures of Escherichia coli BCAT (eBCAT) in complex with the acidic substrate (glutamate) and the acidic substrate analogue (glutarate) have been determined by X-ray diffraction at 1.82 and 2.15 A resolution, respectively. The enzyme is a homo hexamer, with the polypeptide chain of the subunit folded into small and large domains, and an interdomain loop. The eBCAT in complex with the natural substrate, glutamate, was assigned as a ketimine as the most probable form based upon absorption spectra of the crystal complex and the shape of the residual electron density corresponding to the cofactor-glutamate bond structure. Upon binding of an acidic substrate, the interdomain loop approaches the substrate to shield it from the solvent region, as observed in the complex with a hydrophobic substrate. Both the acidic and the hydrophobic side chains of the substrates are bound to almost the same position in the pocket of the enzyme and are identical in structure. The inner side of the pocket is mostly hydrophobic to accommodate the hydrophobic side chain but has four sites to coordinate with the gamma-carboxylate of glutamate. The mechanism for the double substrate recognition observed in eBCAT is in contrast to those in aromatic amino acid and histidinol-phosphate aminotransferases. In an aromatic amino acid aminotransferase, the acidic side chain is located at the same position as that for the aromatic side chain because of large-scale rearrangements of the hydrogen bond network. In the histidinol-phosphate aminotransferase, the acidic and basic side chains are located at different sites and interact with different residues of the disordered loop.

Characterization of histidinol phosphate aminotransferase from Escherichia coli

Histidinol phosphate aminotransferase (HPAT) is a pyridoxal 5′-phosphate (PLP)-dependent aminotransferase classified into Subgroup I aminotransferase, in which aspartate aminotransferase (AspAT) is the prototype. In order to expand our knowledge on the reaction mechanism of Subgroup I aminotransferases, HPAT is an enzyme suitable for detailed mechanistic studies because of having low sequence identity with AspAT and a unique substrate recognition mode. Here we investigated the spectroscopic properties of HPAT and the effect of the C4–C4′ strain of the PLP–Lys 214 Schiff base on regulating the Schiff base p K a in HPAT. Similar to AspAT, the PLP-form HPAT showed pH-dependent absorption spectral change with maxima at 340 nm at high pH and 420 nm at low pH, having a low p K a of 6.6. The p K a value of the methylamine-reconstituted K214A mutant enzyme was increased from 6.6 to 10.6. Mutation of Asn 157 to Ala increased the p K a to 9.2. Replacement of Arg 335 by Leu increased the p K a to 8.6. On the other hand, the p K a value of the N157A/R335L double mutant enzyme was 10.6. These data indicate that the strain of the Schiff base is the principal factor to decrease the p K a in HPAT and is crucial for the subsequent increase in the Schiff base p K a during catalysis, although the electrostatic effect of the arginine residue that binds the negatively charged group of the substrate is larger in HPAT than that in AspAT. Our findings also support the idea that the strain mechanism is common to Subgroup I aminotransferases.

Structural studies of the L-threonine-O-3-phosphate decarboxylase (CobD) enzyme from Salmonella enterica: the apo, substrate, and product-aldimine complexes.

The evolution of biosynthetic pathways is difficult to reconstruct in hindsight; however, the structures of the enzymes that are involved may provide insight into their development. One enzyme in the cobalamin biosynthetic pathway that appears to have evolved from a protein with different function is L-threonine-O-3-phosphate decarboxylase (CobD) from Salmonella enterica, which is structurally similar to histidinol phosphate aminotransferase [Cheong, C. G., Bauer, C. B., Brushaber, K. R., Escalante-Semerena, J. C., and Rayment, I. (2002) Biochemistry 41, 4798-4808]. This enzyme is responsible for synthesizing (R)-1-amino-2-propanol phosphate which is the precursor for the linkage between the nucleotide loop and the corrin ring in cobalamin. To understand the relationship between this decarboxylase and the aspartate aminotransferase family to which it belongs, the structures of CobD in its apo state, the apo state complexed with the substrate, and its product external aldimine complex have been determined at 1.46, 1.8, and 1.8 A resolution, respectively. These structures show that the enzyme steers the breakdown of the external aldimine toward decarboxylation instead of amino transfer by positioning the carboxylate moiety of the substrate out of the plane of the pyridoxal ring and by placing the alpha-hydrogen out of reach of the catalytic base provided by the lysine that forms the internal aldimine. It would appear that CobD evolved from a primordial PLP-dependent aminotransferase, where the selection was based on similarities between the stereochemical properties of the substrates rather than preservation of the fate of the external aldimine. These structures provide a sequence signature for distinguishing between L-threonine-O-3-phosphate decarboxylase and histidinol phosphate aminotransferases, many of which appear to have been misannotated.

Three-Dimensional Structure of the l-Threonine-O-3-phosphate Decarboxylase (CobD) Enzyme from Salmonella enterica,

The three-dimensional structure of the pyridoxal 5'-phosphate (PLP)-dependent L-threonine-O-3-phosphate decarboxylase (CobD) from Salmonella enterica is described here. This enzyme is responsible for synthesizing (R)-1-amino-2-propanol phosphate which is the precursor for the linkage between the nucleotide loop and the corrin ring in cobalamin. The molecule is a molecular dimer where each subunit consists of a large and small domain. Overall the protein is very similar to the members of the family of aspartate aminotransferases. Indeed, the arrangement of the ligands surrounding the cofactor and putative substrate binding site are remarkably close to that observed in histidinol phosphate aminotransferase, which suggests that this latter enzyme might have been its progenitor. The only significant differences in structure occur at the N-terminus, which is approximately 12 residues shorter in CobD and does not form the same type of interdomain interaction common to other aminotransferases. CobD is unusual since within the aspartate aminotransferase subfamily of PLP-dependent enzymes the chemical transformations are substantially conserved, where the only exceptions are 1-aminocyclopropane-1-carboxylate synthase and CobD. Although there are a large number of PLP-dependent amino acid decarboxylases, these are generally larger and structurally distinct from the members of the aspartate aminotransferase subfamily of enzymes. The structure of CobD suggests that the chemical fate of the external aldimine can be redirected by modifications at the N-terminus of the protein. This study provides insight into the evolutionary history of the cobalamin biosynthetic pathway and raises the question of why most PLP-dependent decarboxylases are considerably larger enzymes.

Crystal structure of histidinol phosphate aminotransferase (HisC) from Escherichia coli, and its covalent complex with pyridoxal-5′-phosphate and l-histidinol phosphate

The biosynthesis of histidine is a central metabolic process in organisms ranging from bacteria to yeast and plants. The seventh step in the synthesis of histidine within eubacteria is carried out by a pyridoxal-5′-phosphate (PLP)-dependent l-histidinol phosphate aminotransferase (HisC, EC 2.6.1.9). Here, we report the crystal structure of l-histidinol phosphate aminotransferase from Escherichia coli, as a complex with pyridoxamine-5′-phosphate (PMP) at 1.5 Å resolution, as the internal aldimine with PLP, and in a covalent, tetrahedral complex consisting of PLP and l-histidinol phosphate attached to Lys214, both at 2.2 Å resolution. This covalent complex resembles, in structural terms, the gem-diamine intermediate that is formed transiently during conversion of the internal to external aldimine. HisC is a dimeric enzyme with a mass of approximately 80 kDa. Like most PLP-dependent enzymes, each HisC monomer consists of two domains, a larger PLP-binding domain having an α/β/α topology, and a smaller domain. An N-terminal arm contributes to the dimerization of the two monomers. The PLP-binding domain of HisC shows weak sequence similarity, but significant structural similarity with the PLP-binding domains of a number of PLP-dependent enzymes. Residues that interact with the PLP cofactor, including Tyr55, Asn157, Asp184, Tyr187, Ser213, Lys214 and Arg222, are conserved in the family of aspartate, tyrosine and histidinol phosphate aminotransferases. The imidazole ring of l-histidinol phosphate is bound, in part, through a hydrogen bond with Tyr110, a residue that is substituted by Phe in the broad substrate specific HisC enzymes from Zymomonas mobilis and Bacillus subtilis. Comparison of the structures of the HisC internal aldimine, the PMP complex and the HisC l-histidinol phosphate complex reveal minimal changes in protein or ligand structure. Proton transfer, required for conversion of the gem-diamine to the external aldimine, does not appear to be limited by the distance between substrate and lysine amino groups. We propose that the tetrahedral complex has resulted from non-productive binding of l-histidinol phosphate soaked into the HisC crystals, resulting in its inability to be converted to the external aldimine at the HisC active site.

Cloning, sequencing and function ofsanB, a gene related to nikkomycin biosynthesis ofStreptomyces ansochromogenes

A 6.0 kb DNA fragment related to nikkomycin biosynthesis was cloned from nikkomycin-producingStreptomyces ansochromogenes 7100. Sequence analysis showed that the 1.9 kbTth111 I fragment, a part of the 6.0 kb DNA fragment, contains one complete ORF designatedsanB (GenBank accession No. AF224501), which is composed of 1740 bp encoding a protein consisting of 580 amino acid residues. Its start codon is GTG at 100 bp position and stop codon is TGA at 1840-bp position. Database searching indicated that the deduced protein ofsanB is homologous to the histidinol-phosphate aminotransferase inStreptomyces coelicolor with 31% identities and 47% positives. Gene disruption was performed to study the function ofsanB. It was found that disruptants ofsanB lost the ability to synthesize nikkomycin, which reveals thatsanB is a novel gene essential for nikkomycin biosynthesis.

Suppression of an ethanol-sensitive mutation of Acetobacter pasteurianus by overexpression of the his1 gene encoding histidinol phosphate aminotransferase

An ethanol-sensitive mutant strain of Acetobacter pasteurianus was isolated by chemical mutagenesis with N-methyl- N′-nitro- N-nitrosoguanidine. The his1 gene of A. pasteurianus, which codes for histidinol phosphate aminotransferase (HPAT), was cloned on a multicopy plasmid pMV24 because of its ability to complement the ethanol-sensitive mutation. The mutant was found still to possess almost the same HPAT activity as the parental strain, which was increased to five times that of the basal activity by the introduction of a multicopy plasmid containing the his1 gene into the mutant. It was thus concluded that an increase in the HPAT activity led to the suppression of the ethanol-sensitive mutation.