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At least 865 records · Page 48Linked to original sources
PubMed1996

Alcaligenes xylosoxidans subsp xylosoxidans in children with chronic otorrhea.

Because other known pathogens are frequently isolated with AXX, its clinical significance may be overlooked. AXX should not be considered a colonizer or contaminant, particularly in the presence of clinical signs and symptoms of infection. For a pediatric patient with AXX in the ear fluid, the choice of an antibiotic regimen should be based on in vitro activity of both AXX and other concurrent pathogens. Although the sensitivity of AXX may be highly variable, a number of antibiotics appear to be clinically useful.

Alcaligenes↗
PubMed1994

Overexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus: evidence for a required posttranslational modification for catalytic activity.

Polyhydroxyalkanoate (PHA) synthase has been expressed in Escherichia coli by reengineering the 5'-end of the wild-type (wt) gene and subsequent transformation of this gene into protease-deficient E. coli UT5600 (ompT-). Induction with IPTG results in soluble PHA synthase, which is approximately 5% of the total protein. The soluble synthase has been purified to > 90% homogeneity using FPLC chromatography on hydroxylapatite and Q-Sepharose and has a specific activity of 5 mumol min-1 mg-1. The molecular weight of the PHA product is approximately 10(6) Da based on PlGel chromatography and calibration using polystyrene molecular weight markers. The synthase in the absence of substrate appears to exist in both monomeric and dimeric forms. Incubation of the synthase with an excess of substrate converts it into a form that is now extractable into CHCl3 and sediments on sucrose density ultracentrifugation with PHA. Studies in which the ratio of substrate, 3-D-hydroxybutyrylCoA, to synthase is varied suggest that during polymerization the elongation process occurs at a rate much faster than during the initiation process. A mechanistic model has been proposed for the polymerization process [Griebel, R., Smith, Z., & Merrick, J. (1968) Biochemistry 7, 3676-3681] in which two cysteines are required for catalysis. This model is based on the well-characterized enzymes involved in fatty acid biosynthesis. To test this model, several site-directed mutants of synthase, selected based on sequence conservation among synthases, have been prepared. The C459S mutant has activity approximately 90% that of the wt protein, while the C319S and C319A synthases possess < 0.01% the activity of the wt protein. CD and antibody studies suggest that the mutant proteins are properly folded. The detection of only a single essential cysteine by mutagenesis and the requirement for posttranslational modification by phosphopantetheine to provide a second thiol in many enzymes utilizing coenzyme A thiol ester substrates made us consider the possibility that posttranslational modification was required for synthase activity as well. This hypothesis was confirmed when the plasmid containing PHA synthase (pKAS4) was transformed into E. coli SJ16, requiring beta-alanine for growth. Growth of SJ16/pKAS4 on [3H]-beta-alanine followed by Coomassie staining of the protein and autoradiography revealed that PHA synthase is overexpressed and that beta-alanine is incorporated into the protein. These results suggest PHA synthase is posttranslationally modified by phosphopantetheine.(ABSTRACT TRUNCATED AT 400 WORDS)

Acyltransferases↗
PubMed2004

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.

Alcaligenes↗
PubMed2006

Crystal structure of an electron transfer complex between aromatic amine dehydrogenase and azurin from Alcaligenes faecalis.

The crystal structure of an electron transfer complex of aromatic amine dehydrogenase (AADH) and azurin is presented. Electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of AADH to the type I copper of the cupredoxin azurin. This structure is compared with the complex of the TTQ-containing methylamine dehydrogenase (MADH) and the cupredoxin amicyanin. Despite significant similarities between the two quinoproteins and the two cupredoxins, each is specific for its respective partner and the ionic strength dependence and magnitude of the binding constant for each complex are quite different. The AADH-azurin interface is largely hydrophobic, covering approximately 500 A(2) of surface on each molecule, with one direct hydrogen bond linking them. The closest distance from TTQ to copper is 12.6 A compared with a distance of 9.3 A in the MADH-amicyanin complex. When the MADH-amicyanin complex is aligned with the AADH-azurin complex, the amicyanin lies on top of the azurin but is oriented quite differently. Although the copper atoms differ in position by approximately 4.7 A, the amicyanin bound to MADH appears to be rotated approximately 90 degrees from its aligned position with azurin. Comparison of the structures of the two complexes identifies features of the interface that dictate the specificity of the protein-protein interaction and determine the rate of interprotein electron transfer.

Alcaligenes faecalis↗