Michael D. Scawen

Cadmium-Resistant Pseudomonas putida Synthesizes Novel Cadmium Proteins

Three cysteine-rich proteins of molecular weight 4000 to 7000, containing 4 to 7 gram atoms of cadmium, zinc, and copper per mole were isolated from Pseudomonas putida growing in 3 mM cadmium. The three proteins were induced during different phases of growth, and the smallest (molecular weight 3600; 3 gram atoms of cadmium) was released into the medium when the cells lysed. The results of amino acid analyses and of ultraviolet, circular dichroism, electron paramagnetic resonance, and cadmium-113 nuclear magnetic resonance spectroscopy suggest a novel cadmium(II)-zinc(II)-copper(I) cluster structure for the major protein.

Molecular evolution of bacterial cell-surface proteins

The cell-surface proteins of the infective bacteria Streptococcus and Staphylococcus are probably involved in the process of infection. These proteins share many features including secretion signal peptides, cell-wall spanning regions, membrane anchor domains and repeated domains of various functions. These common features may have evolved by gene duplication and swapping of gene fragments.

Cadmium Resistance in Pseudomonas putida: Growth and Uptake of Cadmium

Summary: A strain of Pseudomonas putida resistant to low concentrations of cadmium (0.25 mM-Cd2+) adapted to growth in the presence of 3 mM-Cd2+ in a chemically defined medium. This increased resistance was rapidly lost if Cd2+ was omitted from the growth medium. P. putida concentrated 109Cd2+ actively. Mechanisms appeared to exist for expelling cadmium from cells during the lag and exponential phases and for continued growth in the presence of high intracellular Cd2+ concentrations. Cd2+-resistant cells adapted so as to control both the extent and rate of Cd2+ uptake when compared to control cells.

Nucleotide sequence of the Erwinia chrysanthemi NCPPB 1066 l-asparaginase gene

The complete nucleotide sequence of the Erwinia chrysanthemi NCPPB 1066 gene coding for the chemotherapeutic enzyme L-asparaginase has been determined. The structural gene consists of an open reading frame commencing with an ATG start codon of 1044 bp followed by a TGA stop codon. Confirmation of the nucleotide sequence was obtained by comparing the predicted amino acid (aa) sequence with that derived by N-terminal aa sequencing of the purified protein. The gene has been shown to code for a 21-aa signal peptide at its N terminus which closely resembles the signal peptides of other secreted proteins. In common with highly expressed Escherichia coli genes, little use is made of modulator codons. The predicted aa sequence of the enzyme exhibits 46% identity with the determined primary sequence of the E. coli L-asparaginase, although the predicted secondary structure of both proteins indicates more extensive homology. Downstream of the TGA stop codon is a G + C-rich region of dyad symmetry (delta G = -25.4 kcal) characteristic of E. coli Rho-independent transcription terminators. Upstream of the structural gene there are no sequences which bear a strong resemblance to the consensus -35 and -10 regions of E. coli promoters. A sequence is present (CTGGCTCTCCTCTTGAT), however, which exhibits strong homology to the nif promoter consensus sequence (CTGGCACN5TTGCA). Upstream of this region is a sequence which strongly resembles the consensus sequence for promoter regions which are subject to catabolite repression.

Cadmium-binding proteins in Pseudomonas putida: pseudothioneins.

Pseudomonas putida adapted to growth in 3 mM cadmium. The resistance mechanism involved complexation of cadmium in polyphosphate granules, changes in the structure of the cell membrane and induction of three cysteine-rich, low molecular weight proteins (3500-7000) containing 4 to 7 g-atoms per mole of cadmium, zinc, and copper. Each protein was produced during a different phase of growth, and the smallest protein (3500) was released into the environment when the cells lysed at the end of the exponential phase. The metal binding sites of the major protein were further characterized using a range of physical methods, including 113Cd NMR. The properties of the bacterial pseudothioneins are compared to those of metallothioneins. ImagesFIGURE 2.

The importance of arginine 102 for the substrate specificity of Escherichia coli malate dehydrogenase

The malate dehydrogenase from Escherichia coli has been specifically altered at a single amino acid residue by using site-directed mutagenesis. The conserved Arg residue at amino acid position 102 in the putative substrate binding site was replaced with a Gln residue. The result was the loss of the high degree of specificity for oxaloacetate. The difference in relative binding energy for oxaloacetate amounted to about 7 kcal/mol and a difference in specificity between oxaloacetate and pyruvate of 8 orders of magnitude between the wild-type and mutant enzymes. These differences may be explained by the large hydration potential of Arg and the formation of a salt bridge with a carboxylate group of oxaloacetate.

Isolation and characterisation of the glycerol dehydrogenase from Bacillus stearothermophilus.

A protocol for the rapid purification of the glycerol dehydrogenase (glycerol: NAD+ 2-oxidoreductase, EC 1.1.1.6) from the thermophile Bacillus stearothermophilus has been developed using a combination of chromatographic techniques including affinity chromatography on a Sepharose-immobilised triazine dye (Procion red, HE3B, ICI). Substrate specificity has been examined and Km values determined. The protein has been shown to have an oligomeric Mr of approx. 180,000 and consists of four identical subunits of Mr 42,000. Exposure to chelating agents (e.g., EDTA) leads to total loss of activity; the EDTA-inactivated enzyme can be reactivated by Zn2+ and requires 1 mol equivalent of zinc per subunit for full catalytic activity. Other divalent cations such as Cd2+ and Co2+ will reactivate the apo-enzyme but yields an enzyme of lower specific activity. The enzyme binds 1 equivalent of NADH per subunit and during catalysis transfers the 4-pro-R hydride from the nicotinamide ring of the reduced-coenzyme to the substrate. Glycerol increases the dissociation constant for the interaction between NADH and Zn-metallo-glycerol dehydrogenase (ZnGDH) but has no effect on the equilibrium between NADH and metal-depleted enzyme.

A study of the interactions between an IgG-binding domain based on the B domain of staphylococcal protein a and rabbit IgG

The nonantigenic interaction between a recombinant immunoglobulin G (IgG)-binding protein based on the B domain of Protein A fromStaphylococcus aureus (termed SpA1) and the Fc fragment of rabbit IgG has been investigated. The contribution to binding of four putative hydrogen bond contacts between SpA1 and IgG-Fc were examined by the individual substitution of the residues in SpA1 involved in these interactions by others unable to form hydrogen bonds. It was found that the most important of the hydrogen bonds involved Tyr 18 which, when replaced by Phe, resulted in a twofold decrease in IgG-binding affinity. The residues of SpA1 proposed to make close, mainly hydrophobic, contacts with Fc were replaced by residues with potential electrostatic charge to establish the importance of the hydrophobic interaction in the complex. The IgG-binding affinities of the mutant proteins were compared to the wild-type protein by a competitive enzyme-linked immunosorbant assay. The replacement of individual hydrophobic residues by His generated a number of novel IgG-binding proteins with reduced binding affinity at pH 5.0 but which maintained strong binding affinities at pH 8.0. The elution profile of human IgG1-Fc (Fc fragment of human IgG1) from a column made from an immobilized two-domain mutant protein shows that the complex dissociates at a higher pH relative to that of the non-mutated protein thus offering favorable elution characteristics.

The complete amino acid sequence of a hirudin variant from the leech Hirudinaria manillensis

Unlike the European leechHirudo medicinalis, the Asian jawed leechHirudinaria manillensis is specialized for feeding on mammalian blood. In the salivary glands of both these leeches, there is a potent inhibitor of thrombin, called hirudin, which acts as an anticoagulant. We have reported previously the isolation and purification of a variant of hirudin, called bufrudin, from the head portions ofHirudinaria. In the present study, the complete amino acid sequence of bufrudin was determined by automated Edman degradation of peptide fragments generated after cleavage of protein with trypsin or thermolysin. Comparison of the primary structure of bufrudin, with hirudin HV1, show about 70% sequence identity with deletion of two amino acids, but the key amino acids at the C-terminus, involved in the inhibition of thrombin, are conserved. However, similar sequence comparison of bufrudin with hirullin P18, a hirudin variant isolated from the same leech species but from whole leech, instead of heads, reveals even less sequence identity of about 60%. From the amino acid sequence, it is suggested that the conformation of the C-terminal portion of bufrudin may be significantly different from hirullin P18, but similar to hirudin HV1, upon its interaction with thrombin. These results indicate that, as withHirudo leech, various isoforms of hirudin also exist inHirudinaria leech, with a significant change occurring in the structure of the molecule during the evolution of leeches.

Fermentation and Downstream Processing of Bacillus

The genus Bacillus comprises a heterogeneous group of chemoorganotrophic, aerobic, rod-shaped microorganisms. These include both mesophilic and thermophilic species as well as acidophiles and alkalophiles. One of their main characteristics is their ability to produce heat-resistant endospores.