Crystal structure of MJ1247 protein from M. jannaschii at 2.0 A resolution infers a molecular function of 3-hexulose-6-phosphate isomerase

The crystal structure of the hypothetical protein MJ1247 from Methanococccus jannaschii at 2 A resolution, a detailed sequence analysis, and biochemical assays infer its molecular function to be 3-hexulose-6-phosphate isomerase (PHI). In the dissimilatory ribulose monophosphate (RuMP) cycle, ribulose-5-phosphate is coupled to formaldehyde by the 3-hexulose-6-phosphate synthase (HPS), yielding hexulose-6-phosphate, which is then isomerized to fructose-6-phosphate by the enzyme 3-hexulose-6-phosphate isomerase. MJ1247 is an alpha/beta structure consisting of a five-stranded parallel beta sheet flanked on both sides by alpha helices, forming a three-layered alpha-beta-alpha sandwich. The fold represents the nucleotide binding motif of a flavodoxin type. MJ1247 is a tetramer in the crystal and in solution and each monomer has a folding similar to the isomerase domain of glucosamine-6-phosphate synthase (GlmS).     

Cloning and nucleotide sequences of the genes encoding triose phosphate isomerase, phosphoglycerate mutase, and enolase from Bacillus subtilis.

The Bacillus subtilis genes tpi, pgm, and eno, encoding triose phosphate isomerase, phosphoglycerate mutase (PGM), and enolase, respectively, have been cloned and sequenced. These genes are the last three in a large putative operon coding for glycolytic enzymes; the operon includes pgk (coding for phosphoglycerate kinase) followed by tpi, pgm, and eno. The triose phosphate isomerase and enolase from B. subtilis are extremely similar to those from all other species, both eukaryotic and prokaryotic. However, B. subtilis PGM bears no resemblance to mammalian, fungal, or gram-negative bacterial PGMs, which are dependent on 2,3-diphosphoglycerate (DPG) for activity. Instead, B. subtilis PGM, which is DPG independent, is very similar to a DPG-independent PGM from a plant species but differs from the latter in the absolute requirement of B. subtilis PGM for Mn2+. The cloned pgm gene has been used to direct up to 25-fold overexpression of PGM in Escherichia coli; this should facilitate purification of large amounts of this novel Mn(2+)-dependent enzyme. Inactivation of pgm plus eno in B. subtilis resulted in extremely slow growth either on plates or in liquid, but growth of these mutants was enhanced by supplementation of media with malate. However, these mutants were asporogenous with or without malate supplementation.     

Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis.

The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and pH optima) identical to those of the native enzyme purified from C. thermosulfurogenes. Simple heat treatment of crude extracts from E. coli and B. subtilis cells carrying the recombinant plasmid at 85 degrees C for 15 min generated 80% pure glucose isomerase. The maximum conversion yield of glucose (35%, wt/wt) to fructose with the thermostable glucose isomerase (10.8 U/g of dry substrate) was 52% at pH 7.0 and 70 degrees C.     

Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis

The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and pH optima) identical to those of the native enzyme purified from C. thermosulfurogenes. Simple heat treatment of crude extracts from E. coli and B. subtilis cells carrying the recombinant plasmid at 85 degrees C for 15 min generated 80% pure glucose isomerase. The maximum conversion yield of glucose (35%, wt/wt) to fructose with the thermostable glucose isomerase (10.8 U/g of dry substrate) was 52% at pH 7.0 and 70 degrees C.     

Nucleotide sequence of the Bacillus subtilis xylose isomerase gene: extensive homology between the Bacillus and Escherichia coli enzyme.

The xylose isomerase gene from Bacillus subtilis was cloned from a genomic BamH1 library by complementation of an isomerase defective Escherichia coli strain as previously described. The ATG initiation codon is preceded by a Shine-Dalgarno sequence and two hexamers being characteristic for the promoter region of Bacillus genes. The structural gene consists of 1320 base pairs, thus coding for a polypeptide chain of 440 amino acids with a molecular weight of 49 680. The polypeptide primary structure shows over 50% homology to that of the E. coli xylose isomerase.     

Cloning and expression of the Escherichia coli D-xylose isomerase gene in Bacillus subtilis

A DNA fragment containing the Escherichia coli D-xylose isomerase gene and D-xylulokinase gene had been isolated from an E. coli genomic bank constructed by Clarke and Carbon. The D-xylose isomerase gene coding for the synthesis of an important industrial enzyme, xylose isomerase, was subcloned into a Bacillus-E. coli bifunctional plasmid. It was found that the intact E. coli gene was not expressed in B. subtilis, a host traditionally used to produce industrial enzymes. An attempt was then made to express the E. coli gene in B. subtilis by fusion of the E. coli xylose isomerase structural gene downstream to the promoter of the penicillinase gene isolated from Bacillus licheniformis. Two such fused genes were constructed and they were found able to be expressed in both B. subtilis and E. coli.     

Isolation and characterization of two tryptophan biosynthetic enzymes, indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, from Bacillus subtilis.

Two of the enzymes responsible for tryptophan biosynthesis in Bacillus subtilis have been extensively purified. These proteins are indole-3-glycerol phosphate synthase and N-(5'-phosphoribosyl) anthranilate isomerase. By comparison to the non-differentiating enteric bacteria in which these two enzymes are fused into a single polypeptide, the isolation of the indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase from B. subtilis has demonstrated that the two proteins are separate species in this organism. The two enzymes were clearly separable by anion-exchange chromatography without any significant loss of activity. Molecular weights were determined for both enzymes by gel filtration and sodium dodecyl sulfate-slab gel electrophoresis, and indicated that the indoleglycerol phosphate synthase is the slightly larger of the two proteins. The minimum molecular weight for indoleglycerol phosphate synthase was 23,500, and that for phosphoribosyl anthranilate isomerase was 21,800. Both enzymes have been examined as to conditions necessary to achieve maximal activity of their individual functions and to maintain that activity.     

The structure of rhamnose isomerase from Escherichia coli and its relation with xylose isomerase illustrates a change between inter and intra-subunit complementation during evolution

Using a new expression construct, rhamnose isomerase from Escherichia coli was purified and crystallized. The crystal structure was solved by multiple isomorphous replacement and refined to a crystallographic residual of 17.4 % at 1.6 A resolution. Rhamnose isomerase is a tight tetramer of four (beta/alpha)(8)-barrels. A comparison with other known structures reveals that rhamnose isomerase is most similar to xylose isomerase. Alignment of the sequences of the two enzymes based on their structures reveals a hitherto undetected sequence identity of 13 %, suggesting that the two enzymes evolved from a common precursor. The structure and arrangement of the (beta/alpha)(8)-barrels of rhamnose isomerase are very similar to xylose isomerase. Each enzyme does, however, have additional alpha-helical domains, which are involved in tetramer association, and largely differ in structure. The structures of complexes of rhamnose isomerase with the inhibitor l-rhamnitol and the natural substrate l-rhamnose were determined and suggest that an extended loop, which is disordered in the native enzyme, becomes ordered on substrate binding, and may exclude bulk solvent during catalysis. Unlike xylose isomerase, this loop does not extend across a subunit interface but contributes to the active site of its own subunit. It illustrates how an interconversion between inter and intra-subunit complementation can occur during evolution. In the crystal structure (although not necessarily in vivo) rhamnose isomerase appears to bind Zn(2+) at a "structural" site. In the presence of substrate the enzyme also binds Mn(2+) at a nearby "catalytic" site. An array of hydrophobic residues, not present in xylose isomerase, is likely to be responsible for the recognition of l-rhamnose as a substrate. The available structural data suggest that a metal-mediated hydride-shift mechanism, which is generally favored for xylose isomerase, is also feasible for rhamnose isomerase.     

A hyperthermostable D-ribose-5-phosphate isomerase from Pyrococcus horikoshii characterization and three-dimensional structure

A gene homologous to D-ribose-5-phosphate isomerase (EC 5.3.1.6) was found in the genome of Pyrococcus horikoshii. D-ribose-5-phosphate isomerase (PRI) is of particular metabolic importance since it catalyzes the interconversion between the ribose and ribulose forms involved in the pentose phosphate cycle and in the process of photosynthesis. The gene consisting of 687 bp was overexpressed in Escherichia coli, and the resulting enzyme showed activity at high temperatures with an optimum over 90 degrees C. The crystal structures of the enzyme, free and in complex with D-4-phosphoerythronic acid inhibitor, were determined. PRI is a tetramer in the crystal and in solution, and each monomer has a new fold consisting of two alpha/beta domains. The 3D structures and the characterization of different mutants indicate a direct or indirect catalytic role for the residues E107, D85, and K98.     

Cloning metabolic pathway genes by complementation in Escherichia coli. Isolation and expression of Plasmodium falciparum glucose phosphate isomerase

Genetic complementation of an Escherichia coli double mutant was used to isolate and express the gene coding for Plasmodium falciparum glucose phosphate isomerase. The gene contains a 1773-base pair open reading frame, has no introns, and maps to P. falciparum chromosome 14. 34% of the deduced amino acid sequence is identical to human glucose phosphate isomerase, with highest similarity in regions of the proposed active sites. The putative initiation site of translation was determined by deletional and oligonucleotide mediated, site-specific mutageneses. Our data suggest that key metabolic enzymes of Plasmodia can be cloned and expressed in E. coli without prior knowledge of the primary amino acid or nucleic acid structure.     

Bacillus subtilis yckG and yckF encode two key enzymes of the ribulose monophosphate pathway used by methylotrophs, and yckH is required for their expression

The ribulose monophosphate (RuMP) pathway is one of the metabolic pathways for the synthesis of compounds containing carbon-carbon bonds from one-carbon units and is found in many methane- and methanol-utilizing bacteria, which are known as methylotrophs. The characteristic enzymes of this pathway are 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), neither of which was thought to exist outside methylotrophs. However, the presumed yckG gene product (YckG) of Bacillus subtilis shows a primary structure similar to that of methylotroph HPS (F. Kunst et al., Nature 390:249-256, 1997). We have also investigated the sequence similarity between the yckF gene product (YckF) and methylotroph PHI (Y. Sakai, R. Mitsui, Y. Katayama, H. Yanase, and N. Kato, FEMS Microbiol. Lett. 176:125-130, 1999) and found that the yckG and yckF genes of B. subtilis express enzymatic activities of HPS and PHI, respectively. Both of these activities were concomitantly induced in B. subtilis by formaldehyde, with induction showing dependence on the yckH gene, but were not induced by methanol, formate, or methylamine. Disruption of either gene caused moderate sensitivity to formaldehyde, suggesting that these enzymes may act as a detoxification system for formaldehyde in B. subtilis. In conclusion, we found an active yckG (for HPS)-yckF (for PHI) gene structure (now named hxlA-hxlB) in a nonmethylotroph, B. subtilis, which inherently preserves the RuMP pathway.     

Enzymatic characterization of 5-methylthioribose 1-phosphate isomerase from Bacillus subtilis

The product of the mtnA gene of Bacillus subtilis catalyzes the isomerization of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P). The catalysis of MtnA is a novel isomerization of an aldose phosphate harboring a phosphate group on the hemiacetal group. This enzyme is distributed widely among bacteria through higher eukaryotes. The isomerase reaction analyzed using the recombinant B. subtilis enzyme showed a Michaelis constant for MTR-1-P of 138 microM, and showed that the maximum velocity of the reaction was 20.4 micromol min(-1) (mg protein)(-1). The optimum reaction temperature and reaction pH were 35 degrees C and 8.1. The activation energy of the reaction was calculated to be 68.7 kJ mol(-1). The enzyme, with a molecular mass of 76 kDa, was composed of two subunits. The equilibrium constant in the reversible isomerase reaction [MTRu-1-P]/[MTR-1-P] was 6. We discuss the possible reaction mechanism.     

Structural analysis of N-acetylglucosamine-6-phosphate deacetylase apoenzyme from Escherichia coli

We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn2+ in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that catalyzes the conversion of the GlcNAc6P into glucosamine 6-phosphate (GlcN6P). The crystal structure was phased by the single isomorphous replacement with anomalous scattering (SIRAS) method using low-resolution (2.9 A) iodine anomalous scattering and it was refined against a native dataset up to 2.0 A resolution. The structure is similar to two other NAGPases whose structures are known from Thermotoga maritima (TmNAGPase) and Bacillus subtilis (BsNAGPase); however, it shows a phosphate ion bound at the metal-binding site. Compared to these previous structures, the apoenzyme shows extensive conformational changes in two loops adjacent to the active site. The E. coli enzyme is a tetramer and its dimer-dimer interface was analyzed. The tetrameric structure was confirmed in solution by small-angle X-ray scattering data. Although no metal ions were detected in the present structure, experiments of photon-induced X-ray emission (PIXE) spectra and of inductively coupled plasma emission spectroscopy (ICP-AES) with enzyme that was neither exposed to chelating agents nor metal ions during purification, revealed the presence of 1.4 atoms of Zn per polypeptide chain. Enzyme inactivation by metal-sequestering agents and subsequent reactivation by the addition of several divalent cations, demonstrate the role of metal ions in EcNAGPase structure and catalysis.     

Sequence of the Ampullariella sp. strain 3876 gene coding for xylose isomerase.

The nucleotide sequence of the gene coding for xylose isomerase from Ampullariella sp. strain 3876, a gram-positive bacterium, has been determined. A clone of a fragment of strain 3876 DNA coding for a xylose isomerase activity was identified by its ability to complement a xylose isomerase-defective Escherichia coli strain. One such complementation positive fragment, 2,922 nucleotides in length, was sequenced in its entirety. There are two open reading frames 1,182 and 1,242 nucleotides in length, on opposite strands of this fragment, each of which could code for a protein the expected size of xylose isomerase. The 1,182-nucleotide open reading frame was identified as the coding sequence for the protein from the sequence analysis of the amino-terminal region and selected internal peptides. The gene initiates with GTG and has a high guanine and cytosine content (70%) and an exceptionally strong preference (97%) for guanine or cytosine in the third position of the codons. The gene codes for a 43,210-dalton polypeptide composed of 393 amino acids. The xylose isomerase from Ampullariella sp. strain 3876 is similar in size to other bacterial xylose isomerases and has limited amino acid sequence homology to the available sequences from E. coli, Bacillus subtilis, and Streptomyces violaceus-ruber. In all cases yet studied, the bacterial gene for xylulose kinase is downstream from the gene for xylose isomerase. We present evidence suggesting that in Ampullariella sp. strain 3876 these genes are similarly arranged.     

Characterization of mutants with single and multiple defects in the tryptophan biosynthetic pathway in Bacillus subtilis

Sixty-five tryptophan auxotrophs which map in a cluster on the genome of Bacillus subtilis were characterized on the basis of (i) growth response, (ii) accumulation of intermediate compounds, and (iii) determination of enzymatic defects. They could be placed into six phenotypic classes. Certain of the mutants exhibited pleiotropic effects on more than one enzymatic activity in a manner different from those effects reported for the tryptophan pathway in other organisms. Invariably, mutations in the second gene, that coding for phosphoribosyl transferase activity, were found to lack the indoleglycerol phosphate synthase activity specified by the third gene in the cluster; however, this polarity did not extend to genes more distal in the cluster. Furthermore, mutations in the gene which codes for phosphoribosyl-anthranilate isomerase not only led to a loss of this enzyme but also to a loss of phosphoribosyl transferase and indoleglycerol phosphate synthase. In contrast, mutations in either of the loci coding for these latter functions had no apparent effect on isomerase activity. No polarity of the conventional type was found, e.g., none of the mutations in any gene led to polarized effects on the levels of the enzymes specified by the other genes of the cluster. These observations indicated a possible in vivo aggregation involving the transferase, isomerase, and synthase enzymes, with the isomerase acting as the "key" enzyme in the aggregate.     

A genetic screen for the identification of thiamin metabolic genes

A genetic screen was developed for the identification of genes related to thiamin biosynthesis and degradation. Genes conferring resistance to bacimethrin or 4-amino-2-trifluoromethyl-5-hydroxymethylpyrimidine were selected from Escherichia coli and Bacillus subtilis genomic libraries. Hits from the selection included the known thiamin biosynthetic genes thiC, thiE, and dxs as well as five genes of previously unknown function (E. coli yjjX, yajO, ymfB, and cof and B. subtilis yveN). The gene products YmfB and Cof catalyze the hydrolysis of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate to 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate. YmfB also converts thiamin pyrophosphate into thiamin phosphate.     

Effect of a new non-cleavable substrate analog on wild-type and serine mutants in the signature sequence of adenylosuccinate lyase of Bacillus subtilis and Homo sapiens

Adenylosuccinate lyase (ASL) catalyzes two beta-elimination reactions in purine biosynthesis, leading to the question of whether the two substrates occupy the same or different active sites. Kinetic studies of Bacillus subtilis and human ASL with a new substrate analog, adenosine phosphonobutyric acid, 2'(3'), 5'-diphosphate (APBADP), show that it acts as a competitive inhibitor with respect to either substrate (K(I) approximately 0.1 microM), indicating that the two substrates occupy the same active site. Binding studies show that both the B. subtilis and human ASLs bind up to 4 mol of APBADP per mole of enzyme tetramer and that both enzymes exhibit cooperativity: negative for B. subtilis ASL and positive for human ASL. Mutant B. subtilis ASLs, with replacements for residues previously identified as critical for catalysis, bind the substrate analog similarly to wild-type ASL. Two serines in a flexible loop of ASL have been proposed to play roles in catalysis because they are close to the substrate in the crystal structure of Escherichia coli ASL. We have now mutated the corresponding serines to alanines in B. subtilis and human ASL to evaluate their involvement in enzyme function. Kinetic data reveal that human Ser(289) and B. subtilis Ser(262) and Ser(263) are essential for catalysis, while the ability of these Ser mutants to bind APBADP suggests that they do not contribute to substrate affinity. Although these serines are not visible in the crystal structure of human adenylosuccinate lyase complexed with substrate or products (PDB #2VD6), they may be interacting with the active sites.     

Production of D-arabitol by a metabolic engineered strain of Bacillus subtilis

A novel method for D-arabitol production with a metabolically engineered Bacillus subtilis strain is described. A known transketolase-deficient and D-ribose-producing mutant of B. subtilis (ATCC 31094) was further modified by disruption of its rpi (D-ribose phosphate isomerase) gene to create a D-ribulose- and D-xylulose-producing B. subtilis strain. Expression of the D-arabitol phosphate dehydrogenase gene of Enterococcus avium in the D-ribulose- and D-xylulose-producing strain resulted in a strain of B. subtilis capable of converting D-glucose to D-arabitol with a high yield (38%) and little by-product formation.     

Expression of genes for guanyl-specific ribonucleases in Bacillus intermedius and Bacillus pumilus is regulated by the PhoP-PhoR two-component signal transduction system of PHO regulon of Bacillus subtilis]

Promoters of the genes of guanyl-specific ribonucleases of Bacillus intermedius (binase) and Bacillus pumilus (RNase Bp) were found to contain sequences homologous to those recognizable by the regulatory protein PhoP in the promoters of the PHO regulon of B. subtilis, as well as regions partially homologous to the binding sites of another regulatory protein, PhoB, in the promoters of the PHO regulon of Escherichia coli. The role of the two-component regulatory systems PhoP-PhoR and PhoB-PhoR in the regulation of expression of the genes of binase and RNase Bp in recombinant strains of B. subtilis and E. coli was studied by using mutant strains. It was established that the expression of these genes in recombinant B. subtilis cells is stringently controlled by the PhoP-PhoR two-component regulatory system, whereas the expression of these genes in E. coli cells is not controlled by the regulatory proteins PhoB or PhoR. Presumably, regulatory systems of the response to phosphate starvation, analogous to the PHO regulon of B. subtilis, also function in other representatives of the genus Bacillus.