alpha-helix E of Spo0A is required for sigma(A)- but not for sigma(H)-dependent promoter activation in Bacillus subtilis

At the onset of endospore formation in Bacillus subtilis, the DNA binding protein Spo0A activates transcription from two types of promoters. The first type includes the spoIIG and spoIIE promoters, which are used by sigma(A)-RNA polymerase, whereas the second type includes the spoIIA promoter, which is used by RNA polymerase containing the secondary sigma factor sigma(H). Previous genetic analyses have identified specific amino acids in alpha-helix E of Spo0A that are important for activation of Spo0A-dependent, sigma(A)-dependent promoters. However, these amino acids are not required for activation of the sigma(H)-dependent spoIIA promoter. We now report the effects of additional single-amino-acid substitutions and the effects of deletions in alpha-helix E. The effects of alanine substitutions revealed one new position (239) in Spo0A that appears to be specifically required for activation of the sigma(A)-dependent promoters. Based on the effects of a deletion mutation, we suggest that alpha-helix E in Spo0A is not directly involved in interaction with sigma(H)-RNA polymerase

A new crystal form of penicillin acylase from Escherichia coli

A new crystal form of penicillin acylase (penicillin amidohydrolase, E.C. 3.5.1.11) from Escherichia coli W (ATCC 11105) is reported. The crystals were grown using a combination of hanging-drop and streak-seeding methods. The crystals are in the monoclinic space group P2(1) with cell dimensions a = 51.52, b = 131.95, c = 64.43 Angstrom, beta = 106.12 degrees. There is one heterodimer in the asymmetric unit (Vm = 2.45 Angstrom(3) Da(-1)) and the solvent content is 49%. Preliminary data have been collected to d(min) = 2.7 Angstrom using a MAR Research image plate and a rotating-anode X-ray source. Subsequent experiments show diffraction beyond 1.3 Angstrom at a synchrotron radiation source

Structure of d(TGCGCA)(2) and a comparison with other DNA Hexamers

The X-ray crystal structure of d(TGCGCA)(2) has been determined at 120 K to a resolution of 1.3 Angstrom. Hexamer duplexes, in the Z-DNA conformation, pack in an arrangement similar to the 'pure spermine form' [Egli et al. (1991). Biochemistry, 30, 11388-11402] but with significantly different cell dimensions. The phosphate backbone exists in two equally populated discrete conformations at one nucleotide step, around phosphate 11. The structure contains two ordered cobalt hexammine molecules which have roles in stabilization of both the Z-DNA conformation of the duplex and in crystal packing. A comparison of d(TGCGCA)(2) with other Z-DNA hexamer structures available in the Nucleic Acid Database illustrates the elusive nature of crystal packing. A review of the interactions with the metal cations Na+, Mg2+ and Co3+ reveals a relatively small proportion of phosphate binding and that close contacts between metal ions are common. A prediction of the water structure is compared with the observed pattern in the reported structure

The three-dimensional structure of the N-acetylglucosamine-6-phosphate deacetylase, NagA, from Bacillus subtilis

The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides. It is classified into carbohydrate esterase family CE-9 (see afmb.cnrs-mrs.fr/CAZY/). Here we report the cloning, expression, and three-dimensional structure (Protein Data Bank code 1un7) determination by x-ray crystallography of the Bacillus subtilis NagA at a resolution of 2.0 Å. The structure presents two domains, a ({beta}/{alpha})8 barrel enclosing the active center and a small {beta} barrel domain. The structure is dimeric, and the substrate phosphate coordination at the active center is provided by an Arg/His pair contributed from the second molecule of the dimer. Both the overall structure and the active center bear a striking similarity to the urease superfamily with two metals involved in substrate binding and catalysis. PIXE (Proton-Induced x-ray Emission) data show that iron is the predominant metal in the purified protein. We propose a catalytic mechanism involving proton donation to the leaving group by aspartate, nucleophilic attack by an Fe-bridged hydroxide, and stabilization of the carbonyl oxygen by one of the two Fe atoms of the pair. We believe that this is the first sugar deacetylase to utilize this fold and catalytic mechanism

Crystal structures of penicillin acylase enzyme-substrate complexes: Structural insights into the catalytic mechanism

The crystal structure of penicillin G acylase from Escherichia coli has been determined to a resolution of 1.3 Å from a crystal form grown in the presence of ethylene glycol. To study aspects of the substrate specificity and catalytic mechanism of this key biotechnological enzyme, mutants were made to generate inactive protein useful for producing enzyme-substrate complexes. Owing to the intimate association of enzyme activity and precursor processing in this protein family (the Ntn hydrolases), most attempts to alter active-site residues lead to processing defects. Mutation of the invariant residue Arg B263 results in the accumulation of a protein precursor form. However, the mutation of Asn B241, a residue implicated in stabilisation of the tetrahedral intermediate during catalysis, inactivates the enzyme but does not prevent autocatalytic processing or the ability to bind substrates. The crystal structure of the Asn B241 Ala oxyanion hole mutant enzyme has been determined in its native form and in complex with penicillin G and penicillin G sulphoxide. We show that Asn B241 has an important role in maintaining the active site geometry and in productive substrate binding, hence the structure of the mutant protein is a poor model for the Michaelis complex. For this reason, we subsequently solved the structure of the wild-type protein in complex with the slowly processed substrate penicillin G sulphoxide. Analysis of this structure suggests that the reaction mechanism proceeds via direct nucleophilic attack of Ser B1 on the scissile amide and not as previously proposed via a tightly H-bonded water molecule acting as a “virtual” base

The three-dimensional structure of the N-acetylglucosamine-6-phosphate deacetylase, NagA, from Bacillus subtilis

The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides. It is classified into carbohydrate esterase family CE-9 (see afmb.cnrs-mrs.fr/CAZY/). Here we report the cloning, expression, and three-dimensional structure (Protein Data Bank code 1un7) determination by x-ray crystallography of the Bacillus subtilis NagA at a resolution of 2.0 Å. The structure presents two domains, a ({beta}/{alpha})8 barrel enclosing the active center and a small {beta} barrel domain. The structure is dimeric, and the substrate phosphate coordination at the active center is provided by an Arg/His pair contributed from the second molecule of the dimer. Both the overall structure and the active center bear a striking similarity to the urease superfamily with two metals involved in substrate binding and catalysis. PIXE (Proton-Induced x-ray Emission) data show that iron is the predominant metal in the purified protein. We propose a catalytic mechanism involving proton donation to the leaving group by aspartate, nucleophilic attack by an Fe-bridged hydroxide, and stabilization of the carbonyl oxygen by one of the two Fe atoms of the pair. We believe that this is the first sugar deacetylase to utilize this fold and catalytic mechanism

Structural and biochemical characterization of a mitochondrial peroxiredoxin from <i>Plasmodium falciparum</i>

&lt;i&gt;Plasmodium falciparum&lt;/i&gt; possesses a single mitochondrion with a functional electron transport chain. During respiration, reactive oxygen species are generated that need to be removed to protect the organelle from oxidative damage. In the absence of catalase and glutathione peroxidase, the parasites rely primarily on peroxiredoxin-linked systems for protection. We have analysed the biochemical and structural features of the mitochondrial peroxiredoxin and thioredoxin of &lt;i&gt;P. falciparum&lt;/i&gt;. The mitochondrial localization of both proteins was confirmed by expressing green fluorescent protein fusions in parasite erythrocytic stages. Recombinant protein was kinetically characterized using the cytosolic and the mitochondrial thioredoxin (PfTrx1 and PfTrx2 respectively). The peroxiredoxin clearly preferred PfTrx2 to PfTrx1 as a reducing partner, reflected by the KM values of 11.6 μM and 130.4 μM respectively. Substitution of the two dyads asparagine-62/tyrosine-63 and phenylalanine-139/alanine-140 residues by aspartate-phenylalaine and valine-serine, respectively, reduced the &lt;i&gt;K&lt;/i&gt;&lt;sub&gt;M&lt;/sub&gt; for Trx1 but had no effect on the &lt;i&gt;K&lt;/i&gt;&lt;sub&gt;M&lt;/sub&gt; of Trx2 suggesting some role for these residues in the discrimination between the two substrates. Solution studies suggest that the protein exists primarily in a homodecameric form. The crystal structure of the mitochondrial peroxiredoxin reveals a fold typical of the 2-Cys class peroxiredoxins and a dimeric form with an intermolecular disulphide bridge between Cys67 and Cys187. These results show that the mitochondrial peroxiredoxin of &lt;i&gt;P. falciparum&lt;/i&gt; occurs in both dimeric and decameric forms when purified under non-reducing conditions

Multifunctional xylooligosaccharide/cephalosporin C deacetylase revealed by the hexameric structure of the Bacillus subtilis enzyme at 1.9 Å resolution

Esterases and deacetylases active on carbohydrate ligands have been classified into 14 families based upon amino acid sequence similarities. Enzymes from carbohydrate esterase family seven (CE-7) are unusual in that they display activity towards both acetylated xylooligosaccharides and the antibiotic, cephalosporin C. The 1.9 Å structure of the multifunctional CE-7 esterase (hereinafter CAH) from Bacillus subtilis 168 reveals a classical α/β hydrolase fold encased within a 32 hexamer. This is the first example of a hexameric α/β hydrolase and is further evidence of the versatility of this particular fold, which is used in a wide variety of biological contexts. A narrow entrance tunnel leads to the centre of the molecule, where the six active-centre catalytic triads point towards the tunnel interior and thus are sequestered away from cytoplasmic contents. By analogy to self-compartmentalising proteases, the tunnel entrance may function to hinder access of large substrates to the poly-specific active centre. This would explain the observation that the enzyme is active on a variety of small, acetylated molecules. The structure of an active site mutant in complex with the reaction product, acetate, reveals details of the putative oxyanion binding site, and suggests that substrates bind predominantly through non-specific contacts with protein hydrophobic residues. Protein residues involved in catalysis are tethered by interactions with protein excursions from the canonical α/β hydrolase fold. These excursions also mediate quaternary structure maintenance, so it would appear that catalytic competence is only achieved on protein multimerisation. We suggest that the acetyl xylan esterase (EC 3.1.1.72) and cephalosporin C deacetylase (EC 3.1.1.41) enzymes of the CE-7 family represent a single class of proteins with a multifunctional deacetylase activity against a range of small substrates