Characterization of cycP gene expression in Achromobacter xylosoxidans NCIMB 11015 and high-level heterologous synthesis of cytochrome c' in Escherichia coli.

The cycP gene encoding a periplasmic cytochrome c' from the denitrifying beta-proteobacterium Achromobacter xylosoxidans was characterized. The genes flanking cycP encode components of a mobile genetic element characteristic of the beta-proteobacteria, suggesting that cycP has inserted within a transposon or insertion element. The gene therefore does not form part of a denitrification operon or gene cluster. The level of expression of the cycP gene and the level of synthesis of its corresponding gene product were found to increase by maximally 3-fold anaerobically. Expression of cycP appears to occur mainly by non-specific read-through transcription from portions of the insertion element. Conditions were developed for high-level overproduction of cytochrome c' in Escherichia coli, which resulted in signal peptide cleavage concomitant with secretion of the protein into the periplasm. Using a single-step purification, 20-30 mg of pure protein were isolated from a 1-litre culture. Based on UV-visible spectrophotometry the dimeric protein was shown to have a full complement of haem and to be indistinguishable from the native protein purified from A. xylosoxidans. This system provides an excellent platform to facilitate biochemical and structural dissection of the mechanism underlying the novel specificity of NO binding to the proximal face of the haem.

Coordinate synthesis of azurin I and copper nitrite reductase in Alcaligenes xylosoxidans during denitrification.

The denitrifying bacterium Alcaligenes xylosoxidans synthesises two azurins (Az), which are termed Az I and Az 2. Both function as effective electron donors to copper nitrite reductase (NiR) in vitro. As a first step towards identifying the physiological relevance of these electron transfer proteins in the denitrification process, the gene (azuA) encoding Az I was characterised and its expression with respect to denitrification determined. We show that the azuA gene from A. xylosoxidans is monocistronic and its expression is increased when cells are grown under denitrifying conditions in the presence of nitrate or nitrite. The expression pattern of azuA was similar, though not identical, to that of the monocistronic nirK gene, which encodes copper NiR, and is in accord with both gene products being synthesised when the bacterium denitrifies. Recombinant Az I was exported to the periplasm of the heterologous host Escherichia coli, was synthesised at very high levels (80 mg purified protein per litre) and was fully loaded with copper. Electron donation from reduced recombinant Az to NiR was indistinguishable from the activity determined with the native protein. Taken together, these findings indicate that in A. xylosoxidans azuA expression is coordinated with denitrification and recombinant Az I is processed and matured in the periplasm of E. coli in the same way it is in A. xylosoxidans.

Initiation of plasminogen activation on the surface of monocytes expressing the type II transmembrane serine protease matriptase.

uPA (urokinase-type plasminogen activator) activates plasminogen with high efficiency when bound to its cellular receptor uPAR, but only after a prolonged lag phase during which generated plasmin activates pro-uPA. How the activity of this proteolytic system might be rapidly initiated is unknown. We have now found that 2 monocytic cell lines display distinct patterns of plasminogen activation. U937 cells, but not THP-1 cells, displayed the expected lag phase, suggesting a constitutive initiation mechanism on the latter. This was shown to be due to the plasmin-independent activation of uPAR-bound pro-uPA by a cell surface-associated protease and to correlate with the expression of matriptase, a type II transmembrane serine protease that was highly expressed in THP-1 cells but undetectable in U937 cells. Kinetic analysis demonstrated that matriptase is a relatively poor activator of pro-uPA in solution, approximately 100-fold less efficient than plasmin (k(cat)/K(m) 1.16 x 10(5) M(-1)s(-1) cf 1.21 x 10(7) M(-1)s(-1)). However, down-regulation of matriptase expression in THP-1 cells by siRNA reduced the activation of cell-associated pro-uPA and the subsequent rapid initiation of plasminogen activation by 76% to 93%. Matriptase was also found to be expressed by peripheral blood monocytes and may therefore be a specific mechanism for the rapid initiation and regulation of plasminogen activation by these cells.

Heterologous metalloprotein biosynthesis in Escherichia coli: conditions for the overproduction of functional copper-containing nitrite reductase and azurin from Alcaligenes xylosoxidans.

This paper reports on the optimization of conditions for the overproduction and isolation of two recombinant copper metalloproteins, originally encoded on the chromosome of the dentrifying soil bacterium Alcaligenes xylosoxidans, in the heterologous host Escherichia coli. The trimeric enzyme nitrite reductase (NiR) contains both type-1 and type-2 Cu centres, whilst its putative redox partner, azurin I, is monomeric and has only a type-1 Cu centre. Both proteins were processed and exported to the periplasm of E. coli, which is consistent with their periplasmic location in their native host A. xylosoxidans. NiR could be readily purified from the periplasmic fraction of E. coli but the enzyme as isolated possessed only type-1 Cu centres. The type-2 Cu centre could be fully reconstituted by incubation of the periplasmic fraction with copper sulfate prior to enzyme purification. Azurin I could only be isolated with a fully occupied type-1 centre when isolated from the crude cell extract but not after isolation from the periplasmic fraction, suggesting loss of the copper due to proteolysis. Based on a number of criteria, including spectroscopic, mass spectrometric, biochemical and structural analyses, both recombinant proteins were found to be indistinguishable from their native counterparts isolated from A. xylosoxidans. The findings of this work have important implications for the overproduction of recombinant metalloproteins in heterologous hosts.

Insights into redox partner interactions and substrate binding in nitrite reductase from Alcaligenes xylosoxidans: crystal structures of the Trp138His and His313Gln mutants.

Dissimilatory nitrite reductase catalyses the reduction of nitrite to nitric oxide within the key biological process of denitrification. We present biochemical and structural results on two key mutants, one postulated to be important for the interaction with the partner protein and the other for substrate entry. Trp138, adjacent to one of the type-1 Cu ligands, is one of the residues surrounding a small depression speculated to be important in complex formation with the physiological redox partners, azurin I and II. Our data reveal that the Trp138His mutant is fully active using methyl viologen as an artificial electron donor, but there is a large decrease in activity using azurin I. These observations together with its crystal structure at a high resolution of 1.6 A confirm the importance of Trp138 in electron transfer and thus in productive interaction with azurin. A "hydrophobic pocket" on the protein surface has been identified as the channel through which nitrite may be guided to the catalytic type-2 Cu site. Glu133 and His313 at the opening of the pocket are conserved among most blue and green copper nitrite reductases (CuNiRs). The failure to soak the substrate into our high-resolution crystal form of native and mutant CuNiRs has been linked to the observation of an extraneous poly(ethylene glycol) (PEG) molecule interacting with His313. We present the crystal structure of His313Gln and the substrate-bound mutant at high resolutions of 1.65 and 1.72 A, respectively. The observation of the substrate-bound structure for the His313Gln mutant and inhibitory studies with PEG establishes the role of the hydrophobic pocket as the port of substrate entry.

The iron-sulfur cluster in the L-serine dehydratase TdcG from Escherichia coli is required for enzyme activity.

The anaerobically inducible L-serine dehydratase, TdcG, from Escherichia coli was characterized. Based on UV-visible spectroscopy, iron and labile sulfide analyses, the homodimeric enzyme is proposed to have two oxygen-labile [4Fe-4S]2+ clusters. Anaerobically isolated dimeric TdcG had a kcat of 544 s(-1) and an apparent KM for L-serine of 4.8 mM. L-threonine did not act as a substrate for the enzyme. Exposure of the active enzyme to air resulted in disappearance of the broad absorption band at 400-420 nm, indicating a loss of the [4Fe-4S]2+ cluster. A concomitant loss of dehydratase activity was demonstrated, indicating that integrity of the [4Fe-4S]2+ cluster is essential for enzyme activity.