A secure future? Human urban and agricultural land use benefits a flightless island-endemic rail despite climate change.

Identifying environmental characteristics that limit species' distributions is important for contemporary conservation and inferring responses to future environmental change. The Tasmanian native hen is an island endemic flightless rail and a survivor of a prehistoric extirpation event. Little is known about the regional-scale environmental characteristics influencing the distribution of native hens, or how their future distribution might be impacted by environmental shifts (e.g. climate change). Using a combination of local fieldwork and species distribution modelling, we assess environmental factors shaping the contemporary distribution of the native hen, and project future distribution changes under predicted climate change. We find 37% of Tasmania is currently suitable for the native hens, owing to low summer precipitation, low elevation, human-modified vegetation and urban areas. Moreover, in unsuitable regions, urban areas can create 'oases' of habitat, able to support populations with high breeding activity by providing resources and buffering against environmental constraints. Under climate change predictions, native hens were predicted to lose only 5% of their occupied range by 2055. We conclude that the species is resilient to climate change and benefits overall from anthropogenic landscape modifications. As such, this constitutes a rare example of a flightless rail to have adapted to human activity.

Structure of a phosphonate-inhibited beta-lactamase. An analog of the tetrahedral transition state/intermediate of beta-lactam hydrolysis.

The crystal structure of beta-lactamase from Staphylococcus aureus inactivated by p-nitrophenyl[[N-(benzyloxycarbonyl)amino]methyl]phosphonate, a methylphosphonate monoester monoanion inhibitor, has been determined and refined at 2.3 A resolution. The structure reveals a tetrahedral phosphorus covalently bonded to the O gamma atom of the active site serine, Ser70. One of the oxygen atoms bonded to phosphorus is located in the oxyanion hole formed by the two main-chain nitrogen atoms of Ser70 and Gln237, and the second bonded oxygen is solvated. The (benzyloxycarbonyl)aminomethyl group is oriented towards the active site gully such that the peptide group forms compensating electrostatic interactions with polar groups on the enzyme. The benzyl group forms a hydrophobic interaction with Ile239 and an aromatic-aromatic edge-to-face interaction with Tyr105, which has undergone a conformational transition relative to the native structure. The mode of binding supports the proposal that on reaction with the enzyme, the phosphonate generates a structure analogous to the tetrahedral transition state/intermediate associated with the acylation step of a normal substrate. The disposition of the phosphonyl group in this complex is the same as that of the corresponding phosphoryl group in the complex resulting from the inhibition of trypsin by diisopropylphosphofluoridate. The structure is consistent with a mechanism of inactivation that follows an associative pathway, proceeding via a transition state/intermediate in which phosphorus is penta-co-ordinated, forming a trigonal bipyramidal geometry with the phosphonyl donor (p-nitrophenol) and acceptor (Ser70 O gamma atom) in apical positions. A model of this transition state can be accommodated in the active site of beta-lactamase without any steric hindrance. A model of the tetrahedral transition state associated with the acylation step by benzyl penicillin has been derived. Because of the conformational rigidity of the fused rings of penicillin molecules, the orientation of the substrate is fixed once the tetrahedral carbonyl carbon and its ligands are superimposed on the phosphonate group. The outcome is that the carboxylate substituent on the thiazolidine ring forms a salt bridge with Lys234, and the preferred puckering of the ring is that observed in the crystal structure of ampicillin, the so-called "open" conformer.

Structure-activity relationships in the inhibition of serine beta-lactamases by phosphonic acid derivatives.

A new series of phosphonyl derivatives has been prepared and tested for inhibition of serine (classes A and C) beta-lactamases. The results were compared with those previously acquired with aryl phosphonate monoesters and with alkaline hydrolysis rates. A methyl p-nitrophenyl phosphate monoanion was markedly poorer as an inhibitor of the class C beta-lactamase of Enterobacter cloacae P99 than a comparable p-nitrophenyl phosphonate. Phosphonyl fluorides, thiophenyl esters, N-phenylphosphonamidates and a p-nitrophenyl thionophosphonate were, in general, comparable with p-nitrophenyl phosphonates in inhibitory power. The incorporation of a specific amino side chain led to an increase in the rates of inhibition of around 10(4)-fold. Apparently unresponsive to the addition of the side chain to the enzyme was N-phenyl methylphosphonamidate, where binding of the side chain may interfere with access of the leaving group to a proton which is necessary to active-site phosphonylation and inhibition. Typical class A beta-lactamases were significantly more refractory than the class C enzyme to all of these reagents.

Phosphonate monoester inhibitors of class A beta-lactamases.

Phosphonate monoesters with the general structure: [formula: see text] are inhibitors of representative class A and class C beta-lactamases. This result extends the range of this type of inhibitor to the class A enzymes. Compounds where X is an electron-withdrawing substituent are better inhibitors than the unsubstituted analogue (X = H), and enzyme inhibition is concerted with stoichiometric release of the substituted phenol. Slow turnover of the phosphonates also occurs. These observations support the proposition that the mechanism of action of these inhibitors involves phosphorylation of the beta-lactamase active site. The inhibitory ability of these phosphonates suggests that the beta-lactamase active site is very effective at stabilizing negatively charged transition states. One of the compounds described also inactivated the Streptomyces R61 D-alanyl-D-alanine carboxypeptidase/transpeptidase.

Characterization of covalently bound enzyme inhibitors as transition-state analogs by protein stability measurements: phosphonate monoester inhibitors of a beta-lactamase.

An experimental method is described for determining whether a covalent enzyme-inhibitor complex has the properties expected of a transition-state analog. The method involves a comparison of the noncovalent interaction energies between the enzyme and the inhibitor on one hand (determined from protein denaturation thermodynamics) and the analogous transition state on the other (determined from kinetic measurements). These two quantities should presumably be large (in comparison with the interaction energies of substrates or reaction intermediates) and close to equal for a good transition state analog; the former is seen dramatically in a large increase in protein stability. The method is absolute in the sense that it does not require a crystal structure of the inhibited enzyme or any preconceptions as to the mechanism of action of the enzyme except those which led to adoption of the potential transition state analog and which might turn out to be right or wrong. In this paper the method is quantitatively applied to the inhibition of the Staphylococcus aureus PC1 beta-lactamase by phosphonate monoesters. It is concluded that the enzyme-inhibitor complex in this case is likely to be a good transition-state mimic. Therefore, mechanistic interpretation of the crystal structure of the complex can be made with more confidence. A semiquantitative assessment of the situation with serine proteinases is also made. It is concluded, in agreement with predictions based on the generally accepted mechanism and on crystal structures, that anionic, but not neutral, phosph(or/on)yl derivatives are good transition-state analogs.

Kinetics and mechanism of beta-lactamase inhibition by phosphonamidates: the quest for a proton.

Four phosphonamidates were synthesized as potential beta-lactamase inhibitors. Three were methanephosphonamidates [CH3PO2-NHR/Ar, where R/Ar = 4-methoxybenzyl (3a), phenyl (3b), and m-nitrophenyl (3c)], while the fourth, PhCH2OCONHCH2PO2-NHPh (2a), also contained a beta-lactamase active site-directed amido side chain. The pH-rate profiles for the hydrolyses of these compounds in the absence of enzyme demonstrated the necessity of nitrogen protonation in the transition state; the reactive neutral form was the zwitterion, IH. The four phosphonamidates irreversibly inhibited the class C beta-lactamase of Enterobacter cloacae P99 by phosphonylation of the active-site serine hydroxyl group, but they displayed strikingly different inhibition pH-rate profiles. The pH profile and inhibition rates of the N-alkyl derivative 3a could be understood in terms of a direct reaction between IH and EH, the form of the enzyme reactive with substrates and phosphonate monoester inhibitors. The pH profile for 2a also indicated that EH was the reactive enzyme form, but its direct reaction with IH is unlikely because of the low concentration of the latter, stemming from its low nitrogen pKa. In this case, proton uptake from solution subsequent to phosphonamidate anion binding probably accounts for the observed rates. The anilides 3b and 3c were weak inhibitors with respect to 2a and 3a. Their major inhibitory activity, observed at above neutral pH in contrast to that of 2a and 3a, probably involves modes of binding not typical of substrate analogs but which allow access to protons. Inhibition by 3c was interpreted to involve rate-determining protonation at high pH. At and above neutral pH, phosphonamidates will generally be less effective inhibitors than phosphonate p-nitrophenyl monoesters. Below pH 7, enzyme-specific phosphonamidates, especially N-alkyl derivatives, will become more effective than the esters. The results are consistent with the view that, because of the specific geometry of the phosphonyl-transfer transition state, the effectiveness of phosphonic acid derivatives as beta-lactamase inhibitors is limited by the absence of a suitably positioned general acid catalyst at the active site.

Mechanism of inhibition of the class C beta-lactamase of Enterobacter cloacae P99 by phosphonate monoesters.

The class C serine beta-lactamase of Enterobacter cloacae P99 was inhibited by a series of aryl methylphosphonate monoester monoanions. The effectiveness of these inhibitors was promoted by an acylamido substituent on the methyl group and a good leaving group at phosphorus. The former preference suggests that noncovalent interaction of these inhibitors with the enzyme resembles that of substrates, while the latter suggests that nucleophilic displacement at phosphorus occurs as part of the inhibition mechanism. The truth of the latter proposition was confirmed by observation of release of 1 equiv of phenol concomitant with inhibition and of the presence of an equivalent amount of 14C-label on the enzyme after inhibition by a 14C-labeled phosphonate. The hydrolytically inert nature of the enzyme-inhibitor adduct, and its 31P chemical shift, suggested that O-phosphonylation of the enzyme had occurred. Although, by analogy with substrates, one might expect that the hydroxyl of the active site serine residue would be covalently modified by these inhibitors, successive alkali and acid treatment of the enzyme-inhibitor adduct generated no pyruvate. Instead, 1 equiv of lysinoalanine was found. This product was rationalized to arise through intramolecular capture by an adjacent lysine amine group of the dehydroalanine residue produced by alkali treatment of an O-phosphonylated serine residue. One equivalent of lysinoalanine was also produced by alkali treatment of the enzyme that had been inhibited by 6 beta-bromopenicillanic acid, a mechanism-based inhibitor known to acylate the hydroxyl group of the active site serine residue. It is therefore likely that the aryl phosphonates phosphonylate this residue. These compounds should be useful as beta-lactamase active site titrants and as sources of fresh insight into the chemical properties of the active site. The significant mechanistic features of the inhibition, in particular its strong leaving group dependence and the distinctive ability of the beta-lactamase active site to stabilize a dianionic transition state containing a pentacoordinated phosphorus, are discussed with respect to the active site structure. The comparison with phosph(or/on)yl inhibitors of serine proteinases is made, and the mechanism-based features of inhibition of serine hydrolases by phosph(on)ates are noted.

Unambiguous stereochemical course of rabbit liver fructose bisphosphatase hydrolysis.

The stereochemical course of rabbit liver fructose bisphosphatase (EC 3.1.3.11) was determined by hydrolyzing the substrate analogue (Sp)-[1-18O]fructose 1-phosphorothioate 6-phosphate in H(2)17O, incorporating the chiral, inorganic phosphorothioate product into adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and analyzing the isotopic distribution of 18O in ATP beta S by 31P NMR. The result indicates that the 1-phosphoryl group is transferred with inversion of configuration. A series of single-turnover experiments ruled out an acyl phosphate intermediate in the hydrolysis. Consequently, fructose bisphosphatase catalyzes the hydrolysis of fructose 1,6-bisphosphate via a direct transfer of the phosphoryl moiety to water.

Structure-activity studies of the inhibition of serine beta-lactamases by phosphonate monoesters.

A new series of phosphonyl derivatives has been prepared and tested for inhibition of serine (class A and C) beta-lactamases. Variations of the leaving group in a series of methyl phosphonates showed that leaving groups better than the previously employed p-nitrophenoxide could give more effective inhibitors. Inclusion of a negative charge in the leaving group did not, per se, lead to better inhibitors. Aryl phosphonates appeared more effective than those with electronically comparable but smaller leaving groups. The combination of a good leaving group, 2,4-dinitrophenoxide, with an amido side-chain, phenylmethylsulfonamido--the latter rather than phenylacetamido in order to increase the stability of the compound with respect to intramolecular nucleophilic catalysis of hydrolysis by the amide group--did not yield overall a better inhibitor than previously employed p-nitrophenyl phosphonates. These results give the first indication of specific interactions between a beta-lactamase and the leaving group of a phosphonate inhibitor. Only one enantiomer of a chiral thiophosphonate, presumably the Rp isomer, was an effective inhibitor. Addition of either a D- or a L-methyl group to the methylene group of a p-nitrophenyl amidomethylphosphonate did not enhance the inhibitory ability of the phosphonate. Class A beta-lactamases remain refractory to phosphonates.

Rapid-quench and isotope-trapping studies on fructose-1,6-bisphosphatase.

Rapid-quench kinetic measurements yielded presteady-state rate data for rabbit liver fructose-1,6-bisphosphatase (FBPase) (a tetramer of four identical subunits) that are triphasic: the rapid release of Pi (complete within 5 ms), followed by a second reaction phase liberating additional Pi that completes the initial turnover of two or four subunits of the enzyme (requiring 100-150 ms), and a steady-state rate whose magnitude depends on the [alpha-Fru-1,6-P2]/[FBPase] ratio. With Mg2+ in the presence of excess alpha-fructose 1,6-bisphosphate (alpha-Fru-1,6-P2) all four subunits turn over in the pre steady state; with Mn2+ only two of the four are active. Thus the expression of half-site reactivity is a consequence of the nature of the metal ion and not a subunit asymmetry. In the presence of limiting alpha-anomer concentrations only two of the four subunits now remain active with Mg2+ as well as with Mn2+ in the pre steady state. However, so that the amount of Pi released can be accounted for, a beta leads to alpha anomerization or direct beta utilization is required at the active site of one subunit. Such behavior is consistent with the two-state conformational hysteresis displayed by the enzyme and altered affinities manifested within these states for alpha and beta substrate analogues. Under these limiting conditions the subsequent steady-state rate is limited by the beta leads to alpha solution anomerization. These data in combination with pulse--chase experiments permit evaluation of the internal equilibrium, which in the case of Mg2+ is unequivocally higher in favor of product complexes and represents a departure from balanced internal substrate-product complexes.

Crystallographic structure of a phosphonate derivative of the Enterobacter cloacae P99 cephalosporinase: mechanistic interpretation of a beta-lactamase transition-state analog.

The crystal structure of a complex formed on reaction of the Enterobacter cloacae P99 cephalosporinase (beta-lactamase) with a phosphonate monoester inhibitor, m-carboxyphenyl [[N-[(p-iodophenyl)acetyl]amino]methyl]phosphonate, has been obtained at 2.3-A resolution. The structure shows that the inhibitor has phosphonylated the active site serine (Ser64) with loss of the m-carboxyphenol leaving group. The inhibitor is positioned in the active site in a way that can be interpreted in terms of a transition-state analog. The arylacetamido side chain is placed as anticipated from analogous beta-lactamoyl complexes of penicillin-recognizing enzymes, with the amino group hydrogen-bonded to the backbone carbonyl of Ser318 (of the B3 beta-strand) and to the amides of Gln120 and Asn152. There is support in the asymmetry of the hydrogen bonding of this side chain to the protein and in the 2-fold disorder of the benzyl group for the considerable breadth in substrate specificity exhibited by class C beta-lactamases. One phosphonyl oxygen atom is in the oxyanion hole, hydrogen-bonded to main-chain NH groups of Ser318 and Ser64, while the other oxygen is solvated, not within hydrogen-bonding distance of any amino acid side chain. The closest active site functional group to the solvated oxygen atom is the Tyr150 hydroxyl group (3.4A); Lys67 and Lys315 are quite distant (4.3 and 5.7 A, respectively). Rather, Tyr150 and Lys67 are more closely associated with Ser64O gamma (2.9 and 3.3 A). This arrangement is interpreted in terms of the transition state for breakdown of the tetrahedral intermediate in the deacylation step of catalysis, where the Tyr150 phenol seems the most likely general acid. Thus, Tyr150, as the phenoxide anion, would be the general base catalyst in acylation, as proposed by Oefner et al. [Nature (1990) 343, 284-288]. The structure is compared with that of a similar phosphonate derivative of a class A beta-lactamase [Chen et al. (1993) J. Mol. Biol. 234, 165-178], and mechanistic comparisons are made. The sensitivity of serine beta-lactamases, as opposed to serine proteinases, toward inhibition by phosphonate monoanions is supported by electrostatic calculations showing a net positive potential only in the catalytic sites of the beta-lactamases.