The metzincin superfamily of proteases includes the matrixins, astacins, reprolysins, snapalysins, leishmanolysins and serralysins 12. These zinc-dependent endoproteases may act on a large number of substrates, or they may be highly specific, targeting only one or a few proteins or oligopeptides. There are over 700 different enzymes that have been classified as metzincins and they are produced by organisms ranging from bacteria to plants and animals 3. As diverse as the members of this superfamily are, surprisingly they all share a structurally similar catalytic site which includes a common zinc binding motif and an absolutely conserved methionine-containing 1,4-tight β turn, termed the "Met-turn" in their active sites 4.

Many of the metzincin proteases are of medical significance. The matrixins, or matrix metalloproteinases (MMPs) include the collagenases, gelatinases and stromelysins 56. Members of this subfamily not only degrade extracellular matrix for tissue remodeling and maturation, but they have also been found to release and activate several growth factors and modulate chemotactic signals 5. In disease processes, however, they have been implicated in promoting tumour invasion and metastasis by degradation of matrix barriers, exacerbating periodontal disease, and destroying aggrecan and collagen in rheumatoid arthritis. For this reason, much interest has arisen in mechanistic and inhibitor studies of MMPs 789. The astacins comprise a subfamily of enzymes involved in digestion, developmental regulation, and peptide processing. The reprolysins or adamalysins are mainly responsible for the hemorrhagic effects and tissue necrosis typical of snake bites 2. These effects are caused mainly by destruction of the extra-cellular matrix surrounding capillaries in conjunction with the inhibition of platelet aggregation by disintegrins. The N-terminal catalytic domains of members of this family are usually zinc and calcium dependent, while the C-terminal portion may be disintegrin, cysteine-rich, or lectin type domains with hemorrhagic or cell adhesion properties. Mammalian adamalysins play important roles in reproduction, myogenesis and cytodifferentiation as well as in disorders such as asthma, cardiac hypertrophy, and endotoxic shock 10111213. The snapalysins are secreted proteases from several Streptomyces organisms and the membrane-bound leishmanolysins originate from a number of protozoan parasites. The serralysins, of which the alkaline protease from P. aeruginosa (AprA) is a member, are secreted by several pathogenic, gram-negative bacterial species 14. They are, on average, 50 kDa proteins consisting of 2 domains; the C-terminal domain binds up to eight calcium ions in a mainly structural role, but also contains the secretion signal. The catalytic site is located in a cleft on the N-terminal domain (Figure 1)15. The physiologic substrate(s) of these enzymes are not known with certainty, but are thought to be proteins of the extracellular matrix and possibly immune response elements such as interferon gamma and complement proteins 1617.

To add a further dimension to the techniques applied to the analysis of protein structure and function, the incorporation of nonnatural amino acids into proteins is currently of great interest. One aspect of this approach is the incorporation of amino acids containing fluorine which would be applicable to not only probing subtle steric interactions in proteins (Van der Waals' radius of F is 1.47 Å vs. 1.20 Å for H), but also in applying ¹⁹F spectroscopy to this area 1819. In order to probe the methionine position in this class of proteins (specifically AprA), the fluorinated analogue, S-difluoromethyl-L-homocysteine (L-difluoromethionine, (DFM)), was utilized to replace the methionine in the Met-turn and to determine its effects on catalytic activity and thermal stability.

AprA is a member of the metzincin superfamily of metalloproteases and is one of several proteases secreted from Pseudomonas aeruginosa into tissue of infected patients 163031 (Figure 1). It appears that a combination of AprA, elastase (LasB) and LasA protease and protease IV work in concert to produce tissue invasion 3233. AprA is biosynthesized in P. aeruginosa as a 479 residue polypeptide which is secreted via a C-terminal signal sequence directly into the extra cellular medium where a 9 amino acid N-terminal inhibitory propeptide is autocatalytically removed 26. A critical aspect to the structure-function of AprA and the metzincins in general is the active-site arrangement of the metal ligands and the still incompletely understood methionine-containing 1,4-tight β turn at the base of the active site present in all members of the metzincin superfamily. Although much is known concerning the cellular biochemistry of a number of metzincins, detailed knowledge of the function of the components of the active site is still lacking. For example, methionine residues in proteins are less frequent in occurrence than many other residues (third least abundant after tryptophan and cysteine 34) and it can frequently be replaced with other hydrophobic amino acids such as leucine 3536. However the surprising persistent presence of methionine in over 700 members of this class of protease has been noted343738. Attempts to define the contribution that this seemingly quintessential amino acid plays in this class of enzyme have indicated that, depending upon the specific superfamily member studied, some flexibility in replacement may be possible, at least in vitro. For example, in the case of MMP-2 (gelatinase A), enzymes with replacements of the active site Met with either Leu or Ser have only minor perturbations in catalytic activity 39. In the case of protease C from Erwinia chrysanthemi, replacement of the active site methionine (Met226) by leucine, alanine or isoleucine resulted in enzyme mutants sufficiently stable to be isolated, but this was not so with M226H, M226S or M226N mutations. However the enzyme kinetic properties of the M226L, M226A and M226I mutants were compromised and crystallographic studies indicated some alteration of the zinc binding histidines which might explain the effects of these mutations on the kinetic properties of the enzyme 40.

Substitution of the active site Met by selenomethionine (SeMet) in MMP-8 (human neutrophil collagenase) was also found to be somewhat perturbing. In the catalytic domain of MMP-8, SeMet appeared to decrease the protein's resistance to urea denaturation 41. This loss of stability was accompanied by a considerable decrease in activity and an increase in K_m towards the substrate (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3- [2,4-dinitrophenyl]-L- 2,3-diaminopropionyl)-Ala-Arg-NH₂. In a previous study using the substrate (dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH₂), however, k_cat and K_m values were unaffected by SeMet replacement in MMP-8 4243.

Evidently additional investigations into the properties of the metzincins through modification of the methionine position may be of importance. However the usual ensemble of potential replacements of amino acid residues through site-directed mutagenesis is limited in general to replacement with the other nineteen amino acids. In order to explore the effects of alterations of the critical methionine in the metzincin class of proteases, the introduction of a sterically and electronically subtle methionine analogue DFM, was the focus of the present study. For these studies the alkaline protease from Pseudomonas aeruginosa was initially investigated as it is representative of the metzincin family of proteases, is important in tissue invasion by this pathogen and has only two methionines in its structure, the N-terminal methionine and the Met-turn methionine, M214. The presence of only two methionine residues in this metzincin (and only the active site methionine if a mature secreted AprA is produced) facilitates bioincorporation of DFM into the protein without the use of complex cell free in-vitro translation system approaches 444546. As well, this choice of target protein for a preliminary study avoids complications with additional bioincorporation of the analogue into other methionine positions which may be present in other members of the metzincin superfamily.

DFM is a subtle derivative of methionine that has been shown to be useful as a ¹⁹F-NMR probe in proteins 2847. Unlike leucine and isoleucine, this unbranched analogue may only produce a moderate increase in the steric properties of the methyl group due to the incorporation of two fluorine atoms. In addition, fluorination could profoundly affect the electron density of the sulfur atom 27. As can be seen in Figure 5, the thiomethyl group of M214 is in close proximity to H176, H180, and H186 and may contribute to the proper positioning of these metal ligands. Hence it was of interest to explore the effects of this nonnatural amino acid on the catalytic activity and thermal stability of the modified protein by introduction of L-difluoromethionine into the critical methionine position in the Met-turn.

Previous studies on other metzincins such as stromelysin, MMP-8, and astacin have shown that the truncated enzymes containing only the catalytic domain of these enzymes could be expressed and were sufficiently stable to be studied 484950. In the current study, initial attempts were therefore focused on the overproduction of the smaller N-terminal catalytic domain of AprA (T40 to N258). Although evidence for cellular production of this fragment was observed, almost complete aggregation of this fragment occurred under a variety of experimental conditions designed for its control (Results). Previous reports by Guzzo and co-workers have shown that even minimal recombinant expression (1 μg/mL) of soluble, intact AprA protein required the co-expression of the entire secretion apparatus (AprD, AprE, AprF) 51. When the structural gene for AprA was expressed alone, however, intracellular accumulation of AprA was barely detectable, most likely due to rapid degradation inside the cell 52. In other reports, significant quantities of the enzyme could be found intracellularly, but only in the form of inclusion bodies, which are known to be highly resistant to degradation 245354.

However successful production of mature full length (G10 to V479) AprA, was accomplished without the co-expression of the entire secretion apparatus. Although insoluble protein aggregates were produced, it was possible to resolubilise and refold the overexpressed protein, yielding approximately 40 mg of high purity enzyme per 1 L of culture (Figure 2). The ESMS analysis revealed that although the majority of the protein isolated from this protocol had M_r = 49 500 Da (full length mature AprA, G10-V479), a small quantity of inseparable enzyme with an M_r = 49 632 Da had maintained its N-terminal methionine (Met-G10-V479) from the cloning procedure (Figure 3). Kinetic analysis of this recombinant mature AprA compared favourably with values previously reported 29 for the wild type AprA secreted by P. aeruginosa (k_cat/K_m of 2140 ± 540 M^-1s^-1 and 1800 ± 300 M^-1s^-1 for wild type and recombinant enzymes respectively).

An almost 100% incorporation level of the fluorinated probe into AprA was readily accomplished by induction of protein expression in minimal media supplemented with 2 mM DFM. The labelled protein was also expressed as intracellular inclusion bodies which were resolubilised and refolded using the same protocol as the unlabelled recombinant protein, yielding approximately 16 mg of nearly homogeneous protein per 1 L of culture (Figure 2). The ESMS analysis indicated that, unlike the unlabelled recombinant AprA, a majority of the DFM incorporated protein maintained the initiating residue (Mr = 49 702 Da), while this DFM was cleaved in only about one quarter of the sample (Mr = 49 535 Da) (Figure 4). The interesting observation that removal of the N-terminal DFM residue occurs to a lesser extent compared to methionine in this position indicates that the processing of fluorinated methionines by methionine aminopeptidase is a less efficient process. Related to this observation is a recent report by Budisa and coworkers indicating the lack of removal of trifluoromethionine from the N-terminus of a mutant green fluorescent protein55. It may be that the increased steric size of the fluorinated methyl groups in these analogues alters the correct positioning of the peptide bond of the substrate in the active site of the methionine aminopeptidase which could reduce hydrolytic efficiency by this enzyme. In any event, the presence of the N-terminal methionine (or DFM) is not expected to affect catalytic activity or thermal stability of the protein as the N-terminus is physically remote from the active site. The kinetic characteristics of the protein mixture with and without incorporated DFM were determined under the revised conditions described in the methods and results sections. The K_m was not significantly altered by replacement with DFM, the V_max and therefore k_cat were only slightly impaired, resulting in a decrease in k_cat/K_m from 620 ± 30 M^-1s^-1 to 530 ± 50 M^-1s^-1.

In order to compare the effects of DFM on thermal stability of the protein, preliminary differential scanning calorimetry studies were undertaken on AprA and DFM-AprA. Under the conditions utilized in these experiments, the thermal denaturations of AprA and DFM-AprA were found to be irreversible. As shown in Figure 6, the thermal stability in 20 mM HEPES (pH 7.8) was not significantly altered upon fluorination of the Met-turn methionine. The T_max for unlabelled and DFM-labelled AprA were determined to be 58.7 ± 0.1°C and 58.6 ± 0.2°C respectively. This would indicate that the introduction of two fluorine atoms into the methyl group, which is in close contact to the critical histidines in the active site, can be accommodated by the protein such that the catalytic properties and thermal stability of AprA are not drastically perturbed.

Due to the lack of background ¹⁹F resonances in most biological systems, the 100% natural abundance of the ¹⁹F isotope and its sensitivity to NMR detection of 83 % to that of ¹H, the application of ¹⁹F spectroscopy to studies in protein structure and function has been an important biophysical technique that has been shown to give critical information on the environment surrounding the fluorine nucleus 19565758. Previous studies on DFM incorporation into the transglycosylase of bacteriophage λ and the leucine-isoleucine-valine (LIV) binding protein from E. coli, have detected separate sets of resonances for each DFM residue present in each protein and their presence has been useful in detection of ligand binding 2847. As the two fluorine atoms in DFM are diastereotopic due to the presence of the chiral centre of the α-carbon of methionine, complex ¹⁹F resonances can result. In fact, it has been shown that a complex octet of ¹⁹F resonances is observed when a DFM residue is located in a conformationally restricted protein environment 28. It was therefore of interest to investigate the ¹⁹F NMR spectroscopy of DFM-AprA. The ¹H-coupled ¹⁹F NMR spectrum of DFM-AprA (Figure 7A) is a composite of the ¹⁹F resonances not only from the active site DFM at position 214, but also a contribution from the presence of protein molecules still maintaining the N-terminal DFM residue. Interestingly the two resonances overlap in the 564 MHz spectrum. It has previously been shown that application of paramagnetic line broadening agents, such as Gd(EDTA)^- will broaden the ¹⁹F NMR resonances from fluorine nuclei in a protein in a distance-dependent manner and can serve to indicate which ¹⁹F resonances originate from more solution exposed residues 5658. To possibly distinguish the signal contributions from each DFM, paramagnetic line broadening experiments utilizing Gd(EDTA)^- were undertaken. These experiments (Figure 7B) indicated that both DFM residues (DFM1 and DFM214) were equally susceptible to the line-broadening agent, indicating that close approach to the active site DFM by Gd(EDTA)^- was also possible in solution. Based on the crystal structure of AprA 1559, the active site is open to solvent and could be expected to reasonably accommodate the rather large Gd(EDTA)^- molecule resulting in little discrimination between the two DFM residues. Although proton decoupling simplified the ¹⁹F spectrum (Figure 7C), the resonances from the two DFM residues were still unresolved; the complexity of which may also result from contributions of the restricted environment of the active site DFM. Attempts to remove the N-terminal DFM with recombinant E. coli methionine aminopeptidase 60 under a variety of conditions resulted in complex mixtures due to proteolytic degradation by AprA. Additional attempts to produce a variant of AprA lacking the N-terminal DFM residue by introduction of the wild-type N-terminal self-cleaving propeptide sequence or a thrombin cleavage site resulted in extremely poor isolated yields of these forms of the protease. Further studies will be focused on the application of this NMR probe to investigating conformational changes in the active site upon ligand and inhibitor binding.

The ¹⁹F biophysical probe, DFM, was successfully incorporated into the critical Met-turn of AprA and resulted in minimal catalytic or structural alteration of the enzyme. These findings are encouraging for the further application of DFM as a subtle probe for the investigation of the metzincin family of proteases.

PW carried out the cloning and protein isolation and characterization. Both authors drafted the manuscript. JFH conceived of the study, and participated in its design and coordination. Both authors read and approved the final manuscript.