Ionic effects on subunit interactions in a cold-active alkaline phosphatase from the marine bacterium Vibrio splendidus

Protein quaternary structures are fundament for life. Proteins have evolved towards large assemblies of subunits needed for the execution of complex chemistry, for building structural components or for compartmentalization. Most oligomeric enzymes exist as homodimers, having two subunits with identical active sites. It is largely unknown why enzymes have evolved in so many cases towards the homodimer structure (and larger oligomeric structures). Alkaline phosphatase (AP) is an excellent model enzyme for the large family of homodimeric hydrolases. Its activity is dependent on dimerization and correct metal occupancy of the three metal ions in the active site. The reason why the enzyme does not function in the monomeric form is still unknown. The focus of this Ph.D. work was on the AP ortholog from Vibrio splendidus (VAP), a cold-adapted marine bacterium. This enzyme is thermally unstable above room-temperature, but also one of the most active variants of its kind at low temperatures. These characteristics are very dependent on the solution conditions. Stability is only maintained at high ionic strength and pH. Here, the focus was on studying the importance of the dimeric structural state for the function, in particular the role of the metal ions for catalysis. The results suggested that the enzyme inactivates irreversibly to a dimeric intermediate state. This dimeric intermediate state was shown to be more dynamic than the native state, defined as measurable changes in the range of conformations of the ensemble of enzyme molecules, yet fully metalated. Furthermore, the enzyme lost its putative half-of-sites reactivity which is dependent on subunit interactions and communication between active sites. The effect of ionic strength on the activity and stability was studied and found to be positive for both features. It is proposed that a deprotonation step causes a conformational change in VAP, based on pH specific effects on physical parameters. Furthermore, the large interface loop, which characterizes VAP, was shown to be mostly important for stability but also needed for local structural rigidity close to the catalytically active residues. This thesis promotes the idea that the role of the homodimeric state is to provide a conformational energy for both stabilization and catalysis of the enzyme, through the interface, not available in the monomeric state.Fjórða stigs bygging próteina er grunnur alls lífs á jörðinni eins og við þekkjum það. Prótein hafa þróast í átt að stórum komplexum til að hvata flókin efnahvörf, til margvíslegra byggingahlutverka, eða hólfunar. Flest ensím eru virk sem einþátta tvíliður með tvær eins hvarfstöðvar. Hvers vegna svo mörg ensím finnast sem einþátta tvíliður er ekki vel rannsakað. Alkalískur fosfatasi (AP) er módel ensím fyrir hýdrólasa sem eru einþátta tvíliður. Virkni AP er háð tvíliðumyndun og málmjónum í hvarfstöð. Hvers vegna hvarfstöðvarnar hafa ekki hvarfgetu í einliðuforminu er ekki vitað. Rannsóknarefni þessarar doktorsritgerðar var AP úr Vibrio splendidus (VAP) kaldsjávarbakteríu. VAP er eitt hitaóstöðugasta ensím sem þekkist en einnig eitt það virkasta við lág hitastig miðað við sambærileg ensím. Áhersla var lögð á að skýra hlutverk tvíliðumyndunar hjá VAP og áhrif jóna á hvötun og stöðugleika, en einnig að útskýra hvers vegna AP hafa ekki virkar einliður. Þessir þættir eru mjög háðir lausnaaðstæðum. Niðurstöður bentu til þess að við afvirkjun ensímsins verði óafturkræf myndbreyting sem leiðir til óvirkrar tvíliðu. Óvirka tvíliðan var lausbundnari, innihélt allar þrjár málmjónirnar í hvarfstöð en hafði mögulega misst eiginleikann til þess að hvata með svokölluðum “half-of-sites” hvarfgangi. Áhrif jónastyrks voru mikil á virkni og stöðugleika og voru áhrifin háð pH, þar sem afprótónering óþekkts sýruhóp veldur myndbreytingu. Að auki var kannað hlutverk löngu yfirborðlykkjunnar sem einkennir VAP frá flestum AP. Meginhlutverk lykkjunna er að stuðla að stöðugleika hvarfstöðvar en einnig í að halda ákveðinni stífni í lykkjum nálægt virkum hliðarkeðjum. Niðurstaða vinnu þessarar ritgerðar bendir til þess að meginhlutverk tvíliðunnar sé það tillegg byggingarfríorku sem verður til á snertiflötum einliðanna sem stuðlar bæði að stöðugleika og hvötun, sem ekki eru til staðar í einliðunum.RANNÍ

Chloride promotes refolding of active Vibrio alkaline phosphatase through an inactive dimeric intermediate with an altered interface

Publisher's version (útgefin grein)Most enzymes are homodimers or higher order multimers. Cold‐active alkaline phosphatase from Vibrio splendidus (VAP) transitions into a dimer with very low activity under mild denaturation conditions. The desire to understand why this dimer fails to efficiently catalyse phosphomonoester hydrolysis led us to investigate interfacial communication between subunits. Here, we studied in detail the unfolding mechanism at two pH values and in the presence or absence of sodium chloride. At pH 8.0, the denaturation model had to include an inactive dimer intermediate and follow the pathway: N2 → I2 → 2U. At pH 10.5, the model was of a two‐state nature. Enzyme activity was not recovered under several examined refolding conditions. However, in the presence of 0.5 m NaCl, the enzyme was nearly fully reactivated after urea treatment. Thermal inactivation experiments were biphasic where the inactivation could be detected using CD spectroscopy at 190–200 nm. By incorporating a bimane fluorescence probe at the dimer interface, we could monitor inactivation/denaturation at two distinct sites at the dimer interface. A change in bimane fluorescence at both sites was observed during inactivation, but prior to the global unfolding event. Furthermore, the rate of change in bimane fluorescence correlated with inactivation rates at 40 °C. These results indicate and support the hypothesis that the subunits of VAP are only functional in the dimeric state due to the cooperative nature of the reaction mechanism when proper crosstalk between subunits is facilitated.Icelandic Research Fund. Grant Number: 141619‐051Peer reviewe

pH-Dependent Binding of Chloride to a Marine Alkaline Phosphatase Affects the Catalysis, Active Site Stability, and Dimer Equilibrium

Post-print (lokagerð höfundar)The effect of ionic strength on enzyme activity and stability varies considerably between enzymes. Ionic strength is known to affect the catalytic activity of some alkaline phosphatases (APs), such as Escherichia coli AP, but how ions affect APs is debated. Here, we studied the effect of various ions on a cold-adapted AP from Vibrio splendidus (VAP). Previously, we have found that the active form of VAP is extremely unstable at low ionic strengths. Here we show that NaCl increased the activity and stability of VAP and that the effect was pH-dependent in the range of pH 7–10. The activity profile as a function of pH formed two maxima, indicating a possible conformational change. Bringing the pH from the neutral to the alkaline range was accompanied by a large increase in both the Ki for inorganic phosphate (product inhibition) and the KM for p-nitrophenyl phosphate. The activity transitions observed as the pH was varied correlated with structural changes as monitored by tryptophan fluorescence. Thermal and urea-induced inactivation was shown to be accompanied by neither dissociation of the active site metal ions nor dimer dissociation. This would suggest that the inactivation involved subtle changes in active site conformation. Furthermore, the VAP dimer equilibrium was studied for the first time and shown to highly favor dimerization, which was dependent on pH and NaCl concentration. Taken together, the data support a model in which anions bind to some specific acceptor in the active site of VAP, resulting in great stabilization and catalytic rate enhancement, presumably through a different mechanism.Icelandic Research Fund 141619Peer reviewe

Cold-active alkaline phosphatase is irreversibly transformed into an inactive dimer by low urea concentrations

Post-print (lokagerð höfundar)Alkaline phosphatase is a homodimeric metallo-hydrolase where both Zn2+ and Mg2+ are important for catalysis and stability. Cold-adapted alkaline phosphatase variants have high activity at low temperatures and lower thermal stability compared with variants from mesophilic hosts. The instability, and thus inactivation, could be due to loose association of the dimers and/or loosely bound Mg2+ in the active site, but this has not been studied in detail for the cold-adapted variants. Here, we focus on using the intrinsic fluorescence of Trp in alkaline phosphatase from the marine bacterium Vibrio splendidus (VAP) to probe for dimerization. Trp → Phe substitutions showed that two out of the five native Trp residues contributed mostly to the fluorescence emission. One residue, 15 Å away from the active site (W460) and highly solvent excluded, was phosphorescent and had a distant role in substrate binding. An additional Trp residue was introduced to the dimer interface to act as a possible probe for dimerization. Urea denaturation curves indicated that an inactive dimer intermediate, structurally equivalent to the native state, was formed before dimer dissociation took place. This is the first example of the transition of a native dimer to an inactive dimer intermediate for alkaline phosphatase without using mutagenesis, ligands, or competitive inhibition.Financial support from the Icelandic Research Fund (project 141619-051) and the Science Institute of the University of Iceland is gratefully acknowledged. The authors also extend their gratitude to Tinna Pálmadóttir for performing the experiment of Fig. 2B.Peer reviewe

Structural Characterization of Functionally Important Chloride Binding Sites in the Marine Vibrio Alkaline Phosphatase

Enzyme stability and function can be affected by various environmental factors, such as temperature, pH, and ionic strength. Enzymes that are located outside the relatively unchanging environment of the cytosol, such as those residing in the periplasmic space of bacteria or extracellularly secreted, are challenged by more fluctuations in the aqueous medium. Bacterial alkaline phosphatases (APs) are generally affected by ionic strength of the medium, but this varies substantially between species. An AP from the marine bacterium Vibrio splendidus (VAP) shows complex pH-dependent activation and stabilization in the 0–1.0 M range of halogen salts and has been hypothesized to specifically bind chloride anions. Here, using X-ray crystallography and anomalous scattering, we have located two chloride binding sites in the structure of VAP, one in the active site and another one at a peripheral site. Further characterization of the binding sites using site-directed mutagenesis and small-angle X-ray scattering showed that upon binding of chloride to the peripheral site, structural dynamics decreased locally, resulting in thermal stabilization of the VAP active conformation. Binding of the chloride ion in the active site did not displace the bound inorganic phosphate product, but it may promote product release by facilitating rotational stabilization of the substrate-binding Arg129. Overall, these results reveal the complex nature and dynamics of chloride binding to enzymes through long-range modulation of electronic potential in the vicinity of the active site, resulting in increased catalytic efficiency and stability

X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine

Background - Para-nitrophenyl phosphate, the common substrate for alkaline phosphatase (AP), is available as a cyclohexylamine salt. Here, we report that cyclohexylamine is a non-competitive inhibitor of APs. Methods - Cyclohexylamine inhibited four different APs. Co-crystallization with the cold-active Vibrio AP (VAP) was performed and the structure solved. Results - Inhibition of VAP fitted a non-competitive kinetic model (Km unchanged, Vmax reduced) with IC50 45.3 mM at the pH optimum 9.8, not sensitive to 0.5 M NaCl, and IC50 27.9 mM at pH 8.0, where the addition of 0.5 M NaCl altered the inhibition to the level observed at pH 9.8. APs from E. coli and calf intestines were less sensitive to cyclohexylamine, whereas an Antarctic bacterial AP was similar to VAP in this respect. X-ray crystallography at 2.3 Å showed two binding sites, one in the active site channel and another at the surface close to dimer interface. Antarctic bacterial AP and VAP have Trp274 in common in their active-sites, that takes part in binding cyclohexylamine. VAP variants W274A, W274K, and W274H gave IC50 values of 179 mM, 188 mM and 187 mM, respectively, at pH 9.8. Conclusions - The binding of cyclohexylamine in locations at the dimeric interface and/or in the active site of APs may delay product release or reduce the rate of catalytic step(s) involving conformational changes and intersubunit communications. General significance - Cyclohexylamine is a common chemical in industries and used as a counterion in substrates for alkaline phosphatase, a clinically important and common enzyme in the biosphere