Alkaline Proteinase Inhibitor of Pseudomonas aeruginosa

Abstract

The apr locus of Pseudomonas aeruginosa encodes alkaline proteinase (APR), a member of the metzincin metalloendopeptidase superfamily, and an 11.4-kDa alkaline proteinase inhibitor (APRin). We describe here the expression in Escherichia coli and characterization of full-length and N-terminally truncated APRin proteins. Fluorescence and circular dichroism spectra indicated that the recombinant proteins were folded into native-like structures. Analytical ultracentrifugation showed that APRin was monomeric and formed a Pseudomonas aeruginosa is a ubiquitous microorganism that is usually benign with respect to healthy individuals but pathogenic in a number of clinical settings, including surgical patients, burn victims, and persons with cystic fibrosis (1). Eye infection by P. aeruginosa is relatively common, particularly in wearers of contact lenses (2); if not promptly treated, these infections can result in corneal ulceration and visual impairment. Among the virulence factors of P. aeruginosa are two secreted metalloproteinases (3), elastase (pseudolysin, PL) 1 and alkaline proteinase (aeruginolysin, APR). Substrates for PL include extracellular matrix structural proteins, immunoglobulins, complement components, and ␣ 1 -proteinase inhibitor (3). APR degrades fibrinogen, fibrin (4), laminin (5), and ␥-interferon (6); it also is capable of activating Hageman factor (7) and matrix metalloproteinases (MMPs) (8).APR and PL were initially characterized by Morihora (9, 10). Subsequent structural comparison revealed that PL is related to thermolysin of Bacillus thermolyticus, and APR is homologous to the 50-kDa metalloproteinases secreted by Serratia marcescens and Erwinia chrysanthemi (11,12). These enzymes constitute the serralysin branch of the metzincin superfamily of metalloendopeptidases (13). X-ray crystallography of APR (14) and SMP (15), the Serratia homolog of APR, shows that both consist of an N-terminal catalytic domain of about 230 residues and a C-terminal calcium binding domain of approximately 240 residues that appears to be required for proteinase secretion (16). The catalytic domain contains sequences characteristic of the metzincin superfamily of metalloendopeptidases, namely a zinc-binding motif (HEXXHXXGXXH) and a conserved Met located in a turn near the base of the metal binding pocket. Other metzincins include astacins, MMPs, reprolysins, and the adamalysins (12).Serralysin secretors are also capable of producing specific inhibitory proteins of about 125 residues that are transported to the periplasm where the signal peptide is removed (17, 18). The three known members of this inhibitor family exhibit approximately 20% sequence identity and an additional 20% sequence similarity, including a conserved disulfide bond (19). The mature inhibitors from E. chrysanthemi and P. aeruginosa have N-terminal Ser, whereas the N terminus of the S. marcescens inhibitor is Gly; in addition, 8 of 15 N-terminal residues are identical among the three inhibitors.X-ray crystallography of a complex between SMP and the

Alkaline Proteinase Inhibitor of Pseudomonas aeruginosa

“…Determination of Kinetic and Equilibrium Constants-Inhibition of APR by APRin in this assay system has been previously shown (15) to be compatible with the reaction steps defined in Scheme 1.…”

“…The dissociation constant K s for the ES complex and k cat for ES were set to values determined previously (15). Values for rate constants for enzyme-inhibitor association and dissociation (k on and k off ) and for the rate constant for spontaneous hydrolysis of the substrate (k h ) were estimated by global fitting of a series of progress curves obtained at different E:I ratios to the kinetic equations of Scheme 1 using the computer program Dynafit (15,19).…”

“…Alkaline Proteinase-Crystalline APR was purchased from Nagase Biochemicals Ltd., Tokyo, Japan, and purified as described (15). APR concentration was estimated by UV absorption using E 1 cm 1% ϭ 16.0 at 280 nm, in conjunction with a molecular weight of 50,000 (2).…”

“…The procedures of Feltzer et al (15) for culture, protein induction, osmotic shock, and inhibitor purification were followed except that a broad-spectrum proteinase inhibitor mixture designed for bacterial cell extracts (Sigma) was included in the crude osmotic lysate. The inhibitor proteins were purified by ion exchange and gel filtration chromatography and subsequently characterized by SDS-PAGE, electrospray mass spectrometry, fluorescence emission, and circular dichroism spectroscopy.…”

“…Each mutant gave a single band on SDS-PAGE and the expected molecular mass by mass spectrometry. For all inhibitors except those with an added Trp residue, protein concentrations were estimated from the absorbance at 280 nm using ⑀ ϭ 17.7 mM Ϫ1 cm Ϫ1 previously established for the wt protein (15). An ⑀ 280 of 25.3 mM Ϫ1 cm-1 was estimated for the S2W and I4W mutants using the method of Gill and von Hipple (16).…”