Role of glutamate 243 in the active site of 2-deoxy-scyllo-inosose synthase from Bacillus circulans

2-Deoxy-scyllo-inosose (DOI) synthase is involved in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics and catalyzes the carbocyclic formation from d-glucose-6-phosphate (G-6-P) into DOI. The reaction mechanism is proposed to be similar to that of dehydroquinate (DHQ) synthase in the shikimate pathway, and includes oxidation of C-4, beta-elimination of phosphate, reduction of C-4, ring opening, and intramolecular aldol cyclization. To investigate the reaction mechanism of DOI synthase, site-directed mutational analysis of three presumable catalytically important amino acids of DOI synthase derived from the butirosin producer Bacillus circulans (BtrC) was carried out. Steady state and pre-steady state kinetic analysis suggested that E243 of BtrC is catalytically involved in the phosphate elimination step. Further analysis of the mutant E243Q of BtrC using substrate analogue, glucose-6-phosphonate, clearly confirmed that E243 was responsible to abstract a proton at C-5 in G-6-P and set off phosphate elimination. This glutamate residue is completely conserved in all DOI synthases identified so far and the corresponding amino acid of DHQ synthase is completely conserved as asparagine. Therefore, this characteristic glutamate residue of DOI synthase is a key determinant to distinguish the reaction mechanism between DOI synthase and DHQ synthase as well as primary sequence.     

Stereospecificity of hydride transfer in NAD+-catalyzed 2-deoxy-scyllo-inosose synthase, the key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics

The key enzyme in the biosynthesis of clinically important aminocyclitol antibiotics is 2-deoxy-scyllo-inosose synthase (DOIS), which converts ubiquitous d-glucose 6-phosphate (G-6-P) into the specific carbocycle, 2-deoxy-scyllo-inosose with an aid of NAD(+)-NADH recycling. The NAD(+)-dependent first step of the DOIS reaction was examined in detail by the use of 6-phosphonate and 6-homophosphonate analogs of G-6-P. Both analogs showed competitive inhibition against the DOIS reaction with K(i) values of 1.3 and 2.8 mM, respectively, due to their inability for the subsequent phosphate elimination. Based on the direct spectrophotometric observation of NADH formed by the hydride transfer from 6-phosphonate to NAD(+), the stereospecificity of the hydride transfer in the DOIS reaction was analyzed with 6-[4-(2)H]phosphonate and was found to be pro-R specific.     

Susceptibility to butirosin and neomycin B in Bacillus circulans, the butirosin-producing organism.

Butirosin, an aminoglycoside antibiotic, is produced by Bacillus circulans B-3312. Experiments using recombined ribosomal and supernatant fractions from this strain and from B. megaterium KM have shown that the ribosome of both are sensitive to butirosin. The aminoglycoside 3'-phosphotransferase present in B. circulans modifies butirosin and neomycin in vitro but confers resistance only to the former in vivo. The phosphotransferase does not modifya detectable amount of extracellular butirosin while mediating resistance to the antibiotic. In vitro, however, the enzyme appears to protect against inhibition by butirosin by inactivating the bulk of the antibiotic in the system. An extrachromosomal element of unknown function has been detected in B. circulans.     

Structure of 2-deoxy-scyllo-inosose synthase, a key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics, in complex with a mechanism-based inhibitor and NAD+

A key enzyme in the biosynthesis of clinically important aminoglycoside antibiotics is 2-deoxy-scyllo-inosose synthase (DOIS), which catalyzes carbocycle formation from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose through a multistep reaction. This reaction mechanism is similar to the catalysis by dehydroquinate synthase (DHQS) of the cyclization of 3-deoxy-D-arabino-heputulosonate-7-phosphate to dehydroquinate in the shikimate pathway, but significant dissimilarity between these enzymes is also known, particularly in the stereochemistry of the phosphate elimination reaction and the cyclization. Here, the crystal structures of DOIS from Bacillus circulans and its complex with the substrate analog inhibitor carbaglucose-6-phosphate, NAD+, and Co2+ have been determined to provide structural insights into the reaction mechanism. The complex structure shows that an active site exists between the N-terminal and C-terminal domains and that the inhibitor coordinates a cobalt ion in this site. Two subunits exist as a dimer in the asymmetric unit. The two active sites of the dimer were observed to be different. One contains a dephosphorylated compound derived from the inhibitor and the other includes the inhibitor without change. The present study suggested that phosphate elimination proceeds through syn-elimination assisted by Glu 243 and the aldol condensation proceeds via a boat conformation. Also discussed are significant similarities and dissimilarities between DOIS and DHQS, particularly in terms of the structure at the active site and the reaction mechanism.     

Unique O-ribosylation in the biosynthesis of butirosin

Using a comparative genetics approach, one or more of the BtrA, BtrL, BtrP, and BtrV proteins encoded in the butirosin biosynthetic gene cluster (btr) from Bacillus circulans SANK72073 were identified as being responsible for an O-ribosylation process leading to the formation of ribostamycin, a key intermediate in this, and related antibiotic biosynthetic pathways. Functional analysis of the recombinantly expressed proteins revealed that both BtrL and BtrP were responsible for the ribosylation of neamine, using 5-phosphoribosyl-1-diphosphate (PRPP) as the ribosyl donor. Further detailed analysis indicated that this process occurs via two discrete steps: with BtrL first catalyzing the phosphoribosylaion of neamine to form 5'-phosphoribostamycin, followed by a BtrP-catalyzed dephosphorylation to generate ribostamycin. To the best of our knowledge, this is the first time that the functional characterization of a glycosyltransferase from an aminoglycoside biosynthetic gene cluster has been reported.     

Regulation of Amylase Synthesis in Bacillus circulans F-2

In the usual batch cultivation, Bacillus circulans F-2 produced amylase only when granular carbon sources such as raw starch or crosslinked starches (CLP) were added. In the dialysis cultivation, where CLP and partially purified amylase were incubated inside the dialysis tubing, the bacterium inoculated outside of the tubing grew and produced the amylase. Amylase production of this bacterium was further investigated in feeding cultivation, in which maltose was fed to the cultivation medium at various rates. The bacterial growth increased with the increase of the feeding rate of maltose, but maximum amylase production was observed at a feeding rate of 45 mg/hr/1. No amylase was produced on the media containing monosaccharides, sucrose, lactose, or isomaltose in the feeding cultivation although bacterial growth was observed. The amylase of this bacterium was found to be inducible. Replacement of 20% of the maltose with glucose resulted in a great decrease (70%) in the amylase production. This shows that the amylase synthesis of B. circulans F-2 is severely repressed by glucose.     

Identification of the 38-kDa subunit of rabbit skeletal muscle glycogen synthase as glycogenin

Glycogen synthase from rabbit skeletal muscle has been shown to be a complex of two types of subunit which have apparent molecular masses of 86 kDa and 38 kDa and are present in a 1:1 molar ratio. The 38-kDa component was separated from the 86-kDa catalytic subunit by gel filtration in the presence of 2 M LiBr, and a number of chymotryptic peptides were sequenced. This demonstrated that the 38-kDa subunit was glycogenin, the protein that is bound covalently to glycogen and believed to be the 'primer' involved in the initiation of de novo glycogen synthesis.     

Significance of the epsilon subunit in the thiol modulation of chloroplast ATP synthase

To understand the regulatory function of the gamma and epsilon subunits of chloroplast ATP synthase in the membrane integrated complex, we constructed a chimeric FoF1 complex of thermophilic bacteria. When a part of the chloroplast F1 gamma subunit was introduced into the bacterial FoF1 complex, the inverted membrane vesicles with this chimeric FoF1 did not exhibit the redox sensitive ATP hydrolysis activity, which is a common property of the chloroplast ATP synthase. However, when the whole part or the C-terminal alpha-helices region of the epsilon subunit was substituted with the corresponding region from CF1-epsilon together with the mutation of gamma, the redox regulation property emerged. In contrast, ATP synthesis activity did not become redox sensitive even if both the regulatory region of CF1-gamma and the entire epsilon subunit from CF1 were introduced. These results provide important features for the regulation of FoF1 by these subunits: (1) the interaction between gamma and epsilon is important for the redox regulation of FoF1 complex by the gamma subunit, and (2) a certain structural matching between these regulatory subunits and the catalytic core of the enzyme must be required to confer the complete redox regulation mechanism to the bacterial FoF1. In addition, a structural requirement for the redox regulation of ATP hydrolysis activity might be different from that for the ATP synthesis activity.     

Congenital deficiency of a 20-kDa subunit of mitochondrial complex I in fibroblasts.

The first component of the mitochondrial electron-transport chain is especially complex, consisting of 19 nuclear and seven mitochondrion-encoded subunits. Accordingly, a wide range of clinical manifestations are produced by the various mutations occurring in human populations. In this study, we analyze the subunit structure of complex I in fibroblasts from two patients who have distinct clinical phenotypes associated with complex I deficiency. The first patient died in the second week of life from overwhelming lactic acidosis. Severe complex I deficiency was evident in her fibroblasts, since alanine oxidation was markedly reduced whereas succinate oxidation was normal. Absence of a 20-kDa subunit was demonstrable when newly synthesized proteins were immunoprecipitated from pulse-labeled fibroblasts by anti-complex I antibody. Disordered assembly or decreased stability of the complex was suggested by deficiency of multiple subunits on Western immunoblots. The second patient exhibited a milder clinical phenotype, characterized by moderate lactic acidosis and developmental delay in childhood and by onset of seizures at 8 years of age. Oxidation studies demonstrated expression of the complex I deficiency in fibroblasts, but no subunit abnormalities were detected by immunoprecipitation or Western immunoblotting. This report demonstrates the utility of cultured fibroblasts in studying mutations affecting synthesis and assembly of complex I.     

Heavy riboflavin synthase of Bacillus subtilis. Primary structure of the beta subunit

Heavy riboflavin synthase is a 1,000,000-Da protein catalyzing the last two reactions of riboflavin biosynthesis. The enzyme complex consists of 60 beta subunits (Mr = 16,200) and approximately three alpha subunits (Mr = 23,000). beta subunits were isolated and cleaved with cyanogen bromide. Fragments were isolated and further digested with trypsin and staphylococcal protease. Peptides were isolated by high performance liquid chromatography. Sequences were determined by automated liquid-phase Edman degradation. The complete sequence of the beta subunit (154 amino acids) was established by direct sequencing of the NH2 terminus, sequencing of overlapping peptides, and carboxypeptidase degradation of the COOH terminus. The sequence shows no detectable homologies to other proteins. A computer prediction of secondary structure elements indicates 34% alpha helix and 30% beta sheet.     

Refolding the unfoldable: A systematic approach for renaturation of Bacillus circulans xylanase

Xylanases are important polysaccharide‐cleaving catalysts for the pulp and paper, animal feeds and biofuels industries. They have also proved to be valuable model systems for understanding enzymatic catalysis, with one of the best studied being the GH11 xylanase from Bacillus circulans (Bcx). However, proteins from this class are very recalcitrant to refolding in vitro. This both limits their high level expression in heterologous hosts, and prevents experimental approaches, such as peptide ligation or chemical modifications, to probe and engineer their stability and function. To solve this problem, a systematic screening approach was employed to identify suitable buffer conditions for renaturing Bcx in vitro. The fractional factorial screen employed identified starting conditions for refolding, which were then refined and developed into a generic protocol for renaturing preparative amounts of active Bcx in a 50–60% yield from inclusion bodies. The method is robust and proved equally proficient at refolding circularly permuted versions that carry cysteine mutations. This general approach should be applicable to related GH11 xylanases, as well as proteins adopting a similar β‐jellyroll fold, that are otherwise recalcitrant to refolding in vitro.     

The biosynthesis of the ubiquinol-cytochrome c reductase complex in yeast. DNA sequence analysis of the nuclear gene coding for the 14-kDa subunit

The nuclear gene coding for the imported 14-kDa subunit of the ubiquinol-cytochrome c reductase of yeast mitochondria has been sequenced in an attempt to define regulatory and protein topogenic elements. The gene has a length of 381 base pairs and is potentially capable of encoding a polypeptide of 14561 Da. It is transcribed into a single low-abundance RNA of 680 nucleotides whose 5' and 3' termini map, respectively, 30-35 nucleotides upstream and 180-190 nucleotides downstream of the initiator and termination codons. Consistent with the estimated low level of the mRNA, codon usage in the gene is not strongly biased and other features, characteristic of highly expressed genes in yeast, are absent. The 14-kDa protein is predicted to be a predominantly hydrophilic protein, with only a single, short hydrophobic stretch located between positions 19-38. Comparison with other imported mitochondrial proteins so far sequenced has failed to reveal unifying features that might serve as targeting elements. Steady-state levels of the 14-kDa and 11-kDa subunits are reduced in mit- mutants which synthesize truncated forms of apocytochrome b and in these, newly synthesized subunits exhibit a specifically increased turnover rate. We suggest that association of these two subunits with the complex may be mediated or enhanced by interaction with other subunits, in particular cytochrome b.     

Significance of mutations on the structural perturbation of thymidylate synthase: implications for their involvement in subunit exchange

Wild-type thymidylate synthase (WT-TS) from Escherichia coli and several of its mutants showed varying degrees of susceptibility to trypsin. While WT-TS was resistant to trypsin as were the mutants C146S, K48E, and R126K, others such as Y94A, Y94F, C146W, and R126E were digested but at different rates from one another. The peptides released from the mutants were identified by mass spectrometry and Edman sequence analysis. The known crystal structures for WT-TS, Y94F, and R126E, surprisingly, showed no structural differences that could explain the difference in their susceptibility to trypsin. One explanation is that the mutations could perturb the dynamic equilibrium of the dimeric state of the mutants as to increase their dissociation to monomers, which being less structured than the dimer, would be hydrolyzed more readily by trypsin. Earlier studies appear to support this proposal since conditions that promote subunit dissociation in solutions of R126E with other inactive mutants, such as dilution, low concentrations of urea, and elevated pH, greatly enhance the rate of restoration of TS activity. Analytic ultracentrifuge studies with various TSs in urea, or at pH 9.0, or that have been highly diluted are, for the most part, in agreement with this thesis, since these conditions are associated with an increase in dissociation to monomers, particularly with the mutant TSs. However, these studies do not rule out the possibility that conformation differences among the various TS dimers are responsible for the differences in susceptibility to trypsin, particularly at high concentrations of protein where the WT-TS and mutants are mainly dimers.     

Cloning and expression of raw-starch-digesting alpha-amylase gene from Bacillus circulans F-2 in Escherichia coli

The raw potato-starch-digesting alpha-amylase gene of Bacillus circulans F-2 was cloned for the first time in Escherichia coli C600, using plasmid pYEJ001. The recombinant plasmid, named pYKA3, has a 5.4 kb insert from a chromosome of the donor bacterium. Subcloning of this amylase gene gave plasmid pHA300 which carried 3.15 kb of the inserted DNA. The transformed bacterium, E. coli C600 (pYKA3), produced the amylase in the periplasmic space, whereas it is secreted outside the cell in the donor bacterium. The cloned raw-starch-digesting alpha-amylase has a molecular weight of 93,000 on SDS-PAGE, and its action pattern was absolutely the same as that of the potent raw-starch-digestible amylase produced by B. circulans F-2. The periplasmic amylase produced by the transformed E. coli (pHA300) could digest raw starch granules such as potato, corn and barley raw starch granules, indicating that the raw-starch-digesting amylase is active in E. coli. Furthermore, this amylase crossreacted with the rabbit antiserum raised against the raw potato-digesting alpha-amylase of B. circulans F-2. From these results it was concluded that the cloned amylase is the same amylase protein as B. circulans F-2 amylase, which has a potent raw-starch digestibility. Thus, this paper is to our knowledge the first describing the molecular cloning of raw-starch-digesting alpha-amylase from Bacillus species and its successful expression in E. coli.     

A unique sequence motif in the 54-kDa subunit of the chloroplast signal recognition particle mediates binding to the 43-kDa subunit

Chloroplasts contain a novel type of signal recognition particle (cpSRP) that consists of two proteins, cpSRP54 and cpSRP43. cpSRP is involved in the post-translational targeting of the nuclear encoded light-harvesting chlorophyll-binding proteins (LHCPs) to the thylakoid membrane by forming a soluble cpSRP.LHCP transit complex in the stroma. Despite high sequence homology between chloroplast and cytosolic SRP54 proteins, the 54-kDa subunit of cpSRP is unique in its ability to bind cpSRP43. In this report, we identified a 10-amino acid long segment of cpSRP54 that forms the cpSRP43-binding site. This segment is located at position 530-539 close to the C terminus of cpSRP54. In addition, we demonstrate that arginine at position 537 is essential for binding cpSRP43 and that mutation of arginine 536 drastically reduced cpSRP43 binding. Mutations within the cpSRP43-binding site of cpSRP54 that reduced or completely abolished cpSRP complex formation also did inhibit transit complex formation and integration of LHCP into the thylakoid membrane, reflecting the importance of these residues for LHCP targeting. Alignment studies revealed that the cpSRP43-binding site is conserved in chloroplast SRP54 proteins and is not present in any SRP54 subunit of cytosolic SRPs.     

Thiamin biosynthesis in Bacillus subtilis: structure of the thiazole synthase/sulfur carrier protein complex

Thiazole synthase is the key enzyme involved in the formation of the thiazole moiety of thiamin pyrophosphate. We have determined the structure of this enzyme in complex with ThiS, the sulfur carrier protein, at 3.15 A resolution. Thiazole synthase is a tetramer with 222 symmetry. The monomer is a (betaalpha)(8) barrel with similarities to the aldolase class 1 and flavin mononucleotide dependent oxidoreductase and phosphate binding superfamilies. The sulfur carrier protein (ThiS) is a compact protein with a fold similar to that of ubiquitin. The structure allowed us to model the substrate, deoxy-D-xylulose 5-phosphate (DXP), in the active site. This model identified Glu98 and Asp182 as new active site residues likely to be involved in the catalysis of thiazole formation. The function of these residues was probed by mutagenesis experiments, which confirmed that both residues are essential for thiazole formation and identified Asp182 as the base involved in the deprotonation at C3 of the thiazole synthase DXP imine. Comparison of the ThiS binding surface to the surface of ubiquitin identified a conserved hydrophobic patch of unknown function on ubiquitin that may be involved in complex formation between ubiquitin and one of its binding partners.