1 INTRODUCTION

Alkaline phosphatases(APs)(E.C 3.1.3.1)are orthophosphoric-monoester phosphohydrolases, and they catalyze phosphoryl ester hydrolysis in the presence of metal ions such as Zn ²⁺ and Mg ²⁺ . APs are widely distributed in the marine environment(Martinez et al., 1996). They play an important role in phosphorus uptake, which may be activated during rehydration. Algae and bacteria are dependent upon nutrients produced by ectohydrolytic APs(Hernandez et al., 1996; Hoppe and Ullrich, 1999). Photosynthetic autotrophs and cyanobacteria produce phosphatases in response to phosphorus deficiency in their external environment(Whitton et al., 2005). Most phosphatases are isolated from organisms living in neutral pH environments, and they usually have higher catalytic efficiency at pH>8(Hoppe, 2003). APs are widely distributed in nature, and they constitute a super family of conserved metalloenzymes that have broad substrate specificities. Microorganisms isolated from low-temperature habitats are natural resources for cold-adapted enzymes with potential industrial applications(Park et al., 2009). Psychrophilic APs have been widely applied to biotechnology because of their characteristic of catalytic efficiency at low temperatures(0–15℃)and inactivation at high temperatures. In comparison, mesophilic APs, such as mammalian calf intestine(CIAP), obtain their optima at 30–50℃, and thermophilic APs from Pyrococcus abyssi are still effective at 80–105℃(Janeway et al., 1993). Several thermolabile APs have been isolated and characterized from psychrophilic microorganisms(e.g., the Antarctic strain TAB 5, marine bacterium Vibrio G15-21, bacterium Shewanella sp. SIB1)(Hauksson et al., 2000; Rina et al., 2000; Suzuki et al., 2005). Psychrophilic APs are usually isolated from marine organisms living in cold environments, such as the Atlantic cod(Gadus morhua)(Ásgeirsson et al., 2003), marine Vibrio sp.(Ásgeirsson and Andrésson, 2001), bacterial strain HK47(Kobori et al., 1984), Shewanella sp.(Suzuki et al., 2005), and shrimp(Pandalus borealis)(de de Backer et al., 2002). Marine APs can be easily inactivated when they are put in warm environments(≥30℃), and heat denaturation of these enzymes is attributable to irretrievable structural changes caused by higher temperatures. Mesophilic and thermophilic APs from bacteria and mammals have higher thermo-stability, which is likely because of the arrangement of amino acid residues and the protein folding algorithm into secondary and tertiary structures. In eukaryotes, the proteins are frequently bound to the cytoplasmic membrane by a glycosyl-phosphatidylinositol(GPI)- modified amino acid in the proximity of the carboxyl terminus. In contrast, many bacterial APs are located in the periplasmic space. Many marine APs, e.g., SAP(shrimp AP)and VAP(Vibrio sp. AP), have been isolated and their structures have been investigated to some extent. In comparison with marine APs, ECAP(Escherichia coli AP)and PLAP(human placenta AP)are currently used extensively in biological research and clinical trials.

Most marine APs are dimers connected by a large α-β-α-sandwich. A few β-sheets exist in α-β-α- sandwich stretching through two subunit monomers, and they shield APs with α-helices(Le Du et al., 2001). Marine APs usually have three metal ions that bind to sites M1–M3, and the replacement of ions signifies APs to change their activity and stability to some degree. The M1 and M2 sites have received more attention because of their crucial roles compared with that of M3. M1 and M2 coordinate two zinc(Zn1 and Zn2)and one magnesium ion(rarely other metal ions)through water molecules(Zalatan et al., 2008). The substrate-binding amino acid residue Arg located in the active site takes part in substrate binding. The residue Ser located in the active site takes part in catalysis(Zalatan et al., 2008; Helland et al., 2009; Golotin et al., 2015). Marine TAP(AP from Antarctic bacterium strain TAB 5 )has two other metal-binding sites(M4 and M5), M4(Zn ²⁺)contributes to TAP’s higher thermo-stability, and M5(Mg ²⁺)facilitates dephosphorylation during the catalytic process(Wang et al., 2007; Lu et al., 2010).

Marine APs have extensive substrates including deoxyribonucleoside triphosphates, DNA, RNA, alkaloids, and flamotide, among others(Zalatan et al., 2006, 2008; Wang et al., 2007). There are two steps in the catalysis mechanism for all APs:(Ⅰ)a nucleophilic attack of a Ser102 alkoxide on the phospho-monoester substrate, forming the covalent phospho-enzyme;(Ⅱ)the hydrolysis of this phosphoseryl intermediate to the noncovalent enzyme-phosphate complex(Trentham and Gutfreund, 1968). Catalytic processes are performed simultaneously in the procedure. First, substrates combine with APs to release an alcohol molecule, generating a covalent phosphoserine intermediate. Second, either a nucleophilic water molecule or alcohol attacks the intermediate. Last, the intermediate changes its conformation to either release inorganic phosphate or form a new phosphoester. APs facilitate bone mineralization and the acquisition of phosphate in alkaline marine environments, and this process is accomplished by the removal/transfer of phosphoryl groups in cellular metabolism and regulation(Nilsen et al., 2001). Marine APs can be used as clinical diagnostic tools to determine various diseases including hypophosphatasia(Moss, 1992). We also use marine APs to assess lots of illnesses and ailments, such as tumors, liver disease, and vitamin D deficiency(Amin et al., 2004; Orimo, 2010). Because they are extremely efficient at low temperatures, marine APs could be used extensively in medical science in the future(Mornet, 2000).

2 FUNDAMENTAL TRAITS 2.1 Biological sources

Marine organisms have a variety of physiological traits, which include their need for several metal ions. APs from the halobios generally use zinc(Zn ²⁺)and magnesium(Mg ²⁺)as catalytic cofactors. In a few cases, cobalt(Co ²⁺)and iron(Fe ²⁺)are used(Wojciechowski and Kantrowitz, 2002; Wang et al., 2005). APs are named based on their biological sources, e.g., AP from human placenta is designated as PLAP, SAP from shrimp, ECAP from Escherichia coli, and VAP from Vibrio sp. This nomenclature method makes AP identification very convenient. All APs are non-specific monoester hydrolases, and their cleavage substrate preferences exhibit a range of catalytic rates. It is difficult to accurately identify marine AP catalytic mechanisms, and their instability at ambient temperatures contributes to the scarcity of information. There is far more information on thermophilic and mesophilic APs, e.g., four isozymes in the PLAP family have been separated, and they display apparent tissue specificities. PLAP changes configuration according to ambient conditions, and it mutates to another variant when it is placed in skeletal tissues, which helps bone mineralization. Marine APs have inferior thermo-stability, and their responses to inhibitors are different compared with mesophilic and thermophilic APs(Henthorn et al., 1992). Marine APs have evolved different characteristics at low temperatures because of their specific evolutionary history(Woolkalis and Baumann, 1981; Le Du and Millán, 2002; Llinas et al., 2005).

2.2 Temperature

APs have different temperature propensities, which has resulted in their widespread use in clinical diagnosis and molecular experiments. Psychrophilic, mesophilic, and thermophilic APs perform very well at low, medium, and high temperatures, respectively. Marine APs hydrolyze phosphomonoesters efficiently in cold environments, and their catalytic efficacies closely correlate with activation energies(AE). An increase in AE results in the reduction of catalytic efficacies. Marine TAP is a typical psychrophilic enzyme, it has excellent flexibility and thermostability, and its k _cat and AE are high and low at 25℃, respectively. In comparison, thermophilic ECAP acquires optimal activity at ~80℃, and mesophilic CIAP catalyzes phosphomonoester hydrolysis efficiently at ~40℃. Mesophilic and thermophilic APs have excellent thermo-stability and catalytic efficacy at higher temperatures, being in agreement with their lower AEs in hot environments(Suzuki et al., 2005). Marine organisms live in cold environments, and their APs are highly active in cold environments(even at ~0℃)(Lu et al., 2010), e.g., marine TAP retains 38% of its optimal activity at ~0℃. Wild-type Vibrio AP is very sensitive to heat inactivation(Gudjónsdóttir and Ásgeirsson, 2008). In some cases, catalytic efficiencies of marine APs increased significantly from 0 to 35℃, e.g., catalytic efficiency of TAP(k _cat)attains 1 212/s and 3 500/s at 10 and 25℃, respectively. Mesophilic and thermophilic APs have favorable efficacies under elevated temperatures, and their catalytic activities are considerably weak in cold environments, e.g., ECAP has little activity at 10℃, and its k _cat is approximately 80/s at 25℃, indicating that low temperatures inhibit thermophilic AP activity(Mavromatis et al., 2002).

It is very convenient for researchers to diagnose illness and perform molecular experiments using cold-adapted APs at room temperature, avoiding inactivation of other components in reaction solutions. It is preferable to separate cold-active APs from marine organisms. Therefore, many researchers have focused their attention on the exploitation of marine APs. To date, a few marine APs have been isolated and used, and preliminary reports on their mechanisms have been published. Marine APs are heat-sensitive enzymes, and they completely denature by heat inactivation. Marine APs have higher catalytic efficiency at low temperatures, suggesting that they could be used under ambient conditions(Feller and Gerday, 2003). A popular strategy is the use of psychrophilic APs in DNA and dNTP dephosphorylation(avoiding self-ligations)(Ruan et al., 1990). Considering that short-term incubations can denature psychrophilic APs at high temperatures and other components in the reaction mixture remain effective, marine APs, e.g., TAP, are recognized as ideal enzymes in molecular biology and clinical diagnosis(Rina et al., 2000). SAP exhibits moderate specific activity(>2 000 U/mg protein)and is irreversibly inactivated by a short heat-treatment at 65℃, indicating its commercial potential for DNA and dNTP dephosphorylation. Even though marine APs have been developed, mesophilic and thermophilic APs such as the commercially available CIAP and ECAP have more extensive applications, and they are traditionally used most in the clinical diagnosis of diseases such as hypophosphatasia. Considering that it is difficult to completely inactivate mesophilic and thermophilic APs and hightemperature deactivation destroys other active substances, we should exploit the more useful marine APs in biological research.

2.3 pH

APs have excellent catalytic efficacy in alkaline environments, and we can determine the scope of pH by inhibition of Tris on transphosphorylation. AP enzymatic activity is pH dependent(Murphy and Kantrowitz, 1994). Most APs remain active at pH 7.5–11.5, and their optimum pH change to some extent with substrate concentrations and components. Organisms in seas and oceans have similar marine surroundings, and in most cases they live in neutral environments. Marine organisms generate various APs, which have different activities under given pH environments. APs from marine microorganisms exhibit higher activity and efficiency than those of the marine animal APs at pH≥10.0(Hauksson et al., 2000; Ishibashi et al., 2005; Golotin et al., 2015), indicating that the pH specificities of marine APs are species specific. pH changes in turn cause variations in AP activity. With regard to vertebrate APs, they reduce their catalytic efficiencies at pH 8.0, which is similar to that of marine bacteria APs(Murphy and Kantrowitz, 1994). Although there are many reports of analogies in their active sites, marine APs have remarkable discrepancies in catalytic activities, indicating that minor divergences of conformation are responsible for their distinct catalytic efficacies(Murphy and Kantrowitz, 1994).

2.4 Molecular weight

Integral marine AP molecular weights(MW)are usually determined by gel chromatography, and their subunit MWs are detected by SDS-PAGE. Subunit sizes relate to structural differences, illustrating the distinct substrate specificities and catalytic efficacies of marine APs. Marine microbe APs have extensive size spectrums, e.g., TAP is small and VAP is comparably large. In comparison, mammalian APs are intermediate in size, close to that of ECAP. In nature, most APs are dimers, though there are a few monomer and trimer APs. For marine APs, natural monomer and dimer formulations have been reported, and they have different substrate specificities and catalytic efficiencies. Most APs are extracted from natural organisms, and some monomer APs have been acquired by gene cloning, e.g., Lysobacter enzymogenes are successfully used to express monomer APs(26 kDa)(von Tigerstrom, 1984). Dimer APs are double the MW of corresponding subunit monomers, e.g., AP from B . subtilis contains two subunit monomers(Hulett et al., 1990). Marine SAP occurs as both monomers and dimers in nature, and they both catalyze phosphoryl ester hydrolysis(Olsen et al., 1991). Dimer SAP is a homodimer with dimensions of approximately 95 Å×65 Å×50 Å, and can be divided into two asymmetric monomers by cleavage. However, SAP monomers from dimer cleavage produce little catalytic activity, and further investigation has revealed the asymmetric residue compositions of dimer SAP: 89.2% of the residues were located in favored regions, 10.3% in additional allowed regions, 0.2% in generously allowed regions, and 0.2% in disallowed regions(de Backer et al., 2002). Other monomer APs discovered have mainly been isolated from the Vibrio genus, e.g., marine VAP from Vibrio cholerae serogroup O1 strains. Compared with dimer APs, very little study has been carried out on monomer APs and so they have not been used much(Majumdar et al., 2005; Helland et al., 2009). AP dimerization of E . coli and its closest analogs as well as their activity depends on the metal ion fulfillment of the metal-binding site M3, while marine bacteria AP can be fully active as a monomer and forms dimers by other mechanisms(Helland et al., 2009; Golotin et al., 2015).

3 STRUCTURE AND CATALYSIS 3.1 Primary and secondary structures

Marine APs have 25%–30% structural identities with the APs from terrestrial bacteria, mammals, and other organisms, and they present unique characteristics. Among the marine APs, VAP has the longest amino acid sequence, while TAP has a very short chain. APs are evaluated based on the primary structure of ECAP. Studies have shown that most APs have three metal ion(one Mg ²⁺ and two Zn ²⁺)active sites, and their conserved residues, such as nucleophilic serine and arginine, facilitate the binding of substrate to phosphorous-oxygens. β-sheets in marine APs have similar structures and are situated in central sites, and they run through a dimeric structure, concomitant with the packing of helices in two sites. There are conspicuous differences between marine and other APs, and some marine APs have longer insertions and/or deletions of residues. Crown domains in marine APs have an important influence on the catalytic efficiency, and the loops in crown domains play key roles in catalytic processes. Loops in the crown domain create catalytically important conformational differences, and several amino acids and peptides have an effect on AP activity as noncompetitive inhibitors(Helland et al., 2009). In marine APs, the residues around M1 and M2 are highly conserved, indicating the similarity of conformations in active sites. In comparison with other APs, marine VAP has small displacements in M1 and M2:(Ⅰ)most APs are threonines at the VAP H118 site(except for PLAP Ser);(Ⅱ)residues in TAP, ECAP, SAP, and PLAP that correspond to the VAP W274 site are Trp, Lys, His, and His, respectively. The AP activities of psychrophilic strain TAB 5TAB 5 and the E . coli D153H mutant are both high(1 650 U´mg21 and 2 150 U´mg21)(Murphy and Kantrowitz, 1994). The geometry of the site formed in both the TAB 5 model and the D153H E . coli mutant can explain this(Rina et al., 2000).

The classical AP(from E . coli etc.)extraneous metal ions are needed to saturate an active site for dimerization of their subunits to be in the active form. However, extraneous metal ions are usually bound with the external binding sites in some marine AP molecules(CmAP, TAB, and VAP), therefore increasing their thermo-stability. At least some of the known marine APs exhibit 100% of their activity in the absence of metal ions from incubation medium(Kobori et al., 1984; Plisova et al., 2005; Golotin et al., 2015). Most APs have long N-terminal helices that link the subunits and strengthen their interactions. Marine VAP from the cold-adapted Vibrio strain G15- 21 has short N-terminal helices, which lose the subunit linkage function. Although VAP has a short N-terminal helix, it has other configurations that replenish the deficiency of connections between subunit monomers. VAP has four supernumerary longer inserts, which are not homologous with other APs: insert I(13-residue insert), insert Ⅱ(26-residue insert), insert Ⅲ(34-residue insert), and insert Ⅳ(46-residue insert). These four long inserts not only connect two subunit monomers, but they also enhance stability and catalytic efficiency by their stereoscopic configurations: insert I forms a three-turn helix; insert Ⅱ forms 11 intermolecular hydrogen bonds and extends along the surface of the other monomer; insert Ⅲ folds into two helices and stretches on the other side over the active site crevasse; insert Ⅳ has two helices and two short strands that constitute an antiparallel sheet(Helland et al., 2009). Among the four insertions, VAP insert Ⅱ plays a critical role in the cooperation of two subunit monomers, which conveys functions similar to those of VAP to other dimer APs. Marine APs have unique traits attributable to evolutionary pressure under specific environments.

3.2 Metal ions and ligands

Marine APs can be analyzed using X-ray diffraction techniques, and researchers have acquired their crystal structures at different resolution values, e.g., SAP was investigated at 1.9 Å(de Backer et al., 2002), TAP at 1.95 Å(Wang et al., 2007), and VAP at 1.4 Å(Helland et al., 2009). Other APs have been investigated at a similar resolution, e.g., ECAP at 2.0 Å(Kim and Wyckoff, 1991)and PLAP at 1.8 Å(Le Du et al., 2001). Investigations on catalytic efficiencies have shown that marine AP crystal structures have an intimate relationship with their specific catalytic rates and thermo-stabilities. Metal ions have highly conserved functions in marine APs, and two Zn ²⁺ and one Mg ²⁺ are situated in SAP, TAP, VAP, ECAP, and PLAP active sites(Zalatan et al., 2008). With the exception of ECAP, the abovementioned APs have tyrosines in the M1 site, which play an important role in their flexibility. Compared with the mesophilic and thermophilic enzymes, psychrophilic APs are highly flexible because they contain a lot of Gly residues, which results in a weak linkage between subunits. The distances of the hydroxyl group to Zn1 denote the locations of tyrosines, and they are quite different for these four APs: VAP(Y325), 4.69 Å; TAP(Y325), 5.94 Å; SAP(Y366), 5.64 Å; PLAP(Y367), 5.43 Å. Marine VAP is unique in that it has a second tyrosine residue in which the hydroxyl group is 5.99 Å from Zn1, thus increasing its flexibility(Helland et al., 2009).

In APs, Mg ²⁺ usually lies in the M3 site, one Zn ²⁺ binds with M1, and the other is located in M2, highlighting the different roles of the three metal ions in AP configuration. Many studies have reported the functions of metal ions: Zn ²⁺(M1)hydrolyzes a covalent intermediate to yield a free serine residue and a phosphate ion; Zn ²⁺(M2)activates the serine hydroxyl group and forms a covalent enzyme intermediate; Mg ²⁺(M3)facilitates deprotonation of the serine and releases the product(Bortolato et al., 1999; Wang et al., 2007). Zinc ions(M1 and M2)cooperate with each other during AP catalysis, enhancing thermo-stability and stabilizing catalytic efficiency. Residues in M1 and M2 are highly conserved in most APs, which might determine the propensity of zinc ions in M1 and M2. Several APs select other metal ions as catalytic cofactors, e.g., APs from T . maritima and B . subtilis use cobalt(Co ²⁺)rather than Zn ²⁺ and Mg ²⁺ ions, boosting their activity(Wang et al., 2005). With regard to marine APs, Mg ²⁺ binds with nucleophilic serine in M3, forming a metal-bound hydroxide, which can be replaced by Zn ²⁺, resulting in the elimination of superposition, e.g., substitution(Mg ²⁺ →Zn ²⁺)in M3(SAP, PLAP, TAP, and VAP)gives rise to the deflection of H149. For metal ions and their binding sites, Mg ²⁺ usually occupies the M3 active site in marine APs. However, there are some exceptions, e.g., in several APs M3 sites bind with other metal ions. W328 and H153(ECAP numbering)combine with residues selected for Co ²⁺ rather than Zn ²⁺(Wojciechowski and Kantrowitz, 2002). There are factors other than residues that affect the selection of metal ions, e.g., although marine VAP has the same metal ligands as TMAP, its metal ion in M3 is Mg ²⁺(Wojciechowski et al., 2002). In marine APs, Mg ²⁺ in M3 is octahedracoordinated, Zn ²⁺ in M1 is hexa-coordinated, and Zn ²⁺ in M2 is penta-coordinated. Therefore, metal ions promote binding with ligands by interacting with ambient residues. H₂O and phosphite are important ligands in ECAP and PLAP, respectively. Marine APs have specific ligands(sulphate in VAP and SAP, phosphite in TAP), which play key roles in catalytic processes(Helland et al., 2009). Nucleophile serine in M3 promotes the binding of Mg ²⁺ by the formation of ligands, inducing the nucleophilic attack of serine and providing excellent flexibility(Stec et al., 2000). Serine hydroxyl attacks the phosphorous center of substrates, and a nucleophilic hydroxide ion is subsequently formed. The product is released via protonation of the nucleophilic serine/phosphate group, and this is performed by coordination between magnesium and water molecules. For most marine APs, Mg ²⁺ in M3 coordinates with histidines. In comparison, D153 forms a ligand with Mg ²⁺ in ECAP. APs present selective preference of residues. However, marine APs usually prefer D153H, K328W, and K328H, conducing to high affinity of Co ²⁺ rather than Zn ²⁺(Wojciechowski and Kantrowitz, 2002). Studies have shown that metal ligands are usually short(≤0.3 Å)in marine APs(Helland et al., 2009). Besides the usual metal ligands(M1, M2, and M3), marine APs have other metal/compound binding sites(e.g., M4 and M5), which aid thermo-stability and catalytic efficiency(Llinas et al., 2005; Gudjónsdóttir and Ásgeirsson, 2008). Two additional sites have been observed in VAP, and they bind sulphate and ethylene glycol in solvents, facilitating the formation of an intermediate during substrate hydrolysis(Helland et al., 2009).

In marine APs, three metal ions(Zn ²⁺, Zn ²⁺, and Mg ²⁺)constitute a multi-ligand conformation, and the side chains present excellent superimposition conformation. Two dimer subunits have similar M3 ligands, and the superimpositions are analogous. Marine AP M3 sites have diverse residue conformations:(Ⅰ)the rings in both W274(VAP)and W260(TAP)are located in the same plane(satisfactory superimposition of nitrogen atoms)and correspond to the PLAP H317 imidazole ring, and H316(SAP)has a 45° rotation of imidazole ring through the Cβ-Cγ bond;(Ⅱ)there is a 55° rotation between the H116(VAP)imidazole ring and the D153 carboxyl group(ECAP), the H149(SAP)imidazole plane is rotated approximately 50° through the Cβ-Cγ bond compared with other APs [H135(TAP), H153(PLAP), and H116(ECAP)]. Helland et al.(2009)found that the distances between Mg ²⁺ and ligands in M3 sites are fixed(~4 Å). Substitution of residues in M3 sites changes the coordination formulation between Mg ²⁺ and ligands, resulting in alterations in M3 site conformations. Change in H135D in TAP M3 causes lower activity and higher stability compared with the wild type(Tsigos et al., 2001). To some extent, replacement of residues in M3 sites changes AP catalytic efficiency and thermo-stability. With regard to M1(Zn ²⁺)sites, ambient residues, such as histidine, play an important role in the hydrolysis of phosphoryl esters. In marine VAP(H465), histidine is located in a strand of the interface and strengthens stability; this is also observed in ECAP(H412). Zn ²⁺ in M1 forms various ligands with ambient residues and solvent molecules that are close to the crown domain and interface. AP activity and stability can be altered when ligands change, and genetic engineering might improve the properties of marine APs by displacement of ligands in M1. Zn ²⁺ in M1 coordinates with water molecules lowering the pKa of marine APs, and this coordination enhances the catalytic efficacy. An arginine side chain in active sites has some mobility, which facilitates the release of phosphate from noncovalent intermediates(Stec et al., 2000).

3.3 Interface

APs have various conformations that determine interface characteristics. AP interfaces share a close relationship with their crown domains. In marine APs, interface areas usually occupy a definite proportion(19%–22%)of the water-accessible surface area per monomer, with the exception of marine TAP(14.1%). TAP has a small crown domain, and it does not possess the N-terminal helix or N-terminal loop that PLAP, SAP, and ECAP do, resulting in the smallest interface area(~1 835 Å ²)among marine APs(Wang et al., 2007). VAP has a large interface area(~4 271 Å ²), which is attributable to the existence of insertions Ⅱ and Ⅳ. When these two insertions are deleted, the interface area is ~2 100 Å ² . Marine AP interface residues do not have obvious polarity distributions, non-polar percentages in VAP(40%)are less than that in PLAP(45%), and SAP and TAP have similar values to ECAP(30%–34%).

Two subunit monomers of dimer APs operate in coordination via their interfaces, and this synergy assists in product release and increases in thermostability, which coincides with changes in interface conformation(Coleman, 1992). Changes in subunit configuration facilitates the formation of intermediates and product release during catalytic processes, e.g., one VAP subunit transforms and some residues protrude into the active sites of the other subunit. Tyrosines are important marine AP residues, and they affect the binding of substrates and product release, e.g., some tyrosines in one monomer of dimer APs are extended into the active site of the second monomer, changing the conformation of the active site. Active sites approach AP interfaces and the interfaces strengthen the basic conformation of the active sites, guaranteeing substrate specificity. In marine dimer APs, there are intimate correlations between subunit monomers, and mutual forces are involved in the catalytic efficacies. With regard to marine APs, we should avoid the introduction of denaturants during substrate hydrolysis because many, such as urea and guanidinium, dissociate dimer APs. Monomers can be isolated by denaturalization, which is usually accompanied by a loss of activity(Ásgeirsson and Guðjónsdóttir, 2006). Marine APs interact weakly between two subunit monomers, resulting in outstanding plasticity and catalytic efficiency at low temperatures. VAP and TAP have similar structures at their interfaces, and weak interactions between monomers result from noncovalent connections. In ECAP and PLAP, extending loops or helices are observed at N-terminus; they interweave to form two subunit monomers, increasing the thermo-stability of these APs. The insertion and deletion of amino acids at the N-terminus rearranges secondary structures, which brings about changes in tertiary and quaternary structures(Tyler-Cross et al., 1989). In VAP, insert Ⅱ replaces the extending loops and helixes at the N-terminus, enhancing flexibility and improving activity. The changes in surface loops influence the overall stability of marine APs, which in turn changes the catalytic parameters. Therefore, modifying surface loops is an effective strategy to increase thermostability(Bossi et al., 1993).

3.4 Important residues

Some residues play key roles in marine AP M3 sites, and they interact with water molecules and substrates. Lysines bind phosphates vigorously by interacting with water, inhibiting product release. Replacement of lysines could improve product release, e.g., when histamines substitute lysines in M3 AP configuration AP, resulting in a reduction of the phosphate-binding constant. Metal ions in active sites coordinate with some residues, improving the construction of the catalytic locus and increasing the AP catalytic efficacy. The ECAP D153H/K328W mutant has a higher activity compared with the wild type, demonstrating that the mutant performs better in the catalysis of phosphoryl esters. Although different residues in marine APs(VAP, tryptophan; TAP, tryptophan; SAP, histidine)correspond to the ECAP K328 position, these residues have similar functions to K328(Helland et al., 2009). In VAP, serine 65 acts on substrates to form intermediates by nucleophilic attack, its orientation is comparable to ECAP serine 102, and both of these serines are located in the helixes of similar superimpositions. By comparing VAP serine 65 with TAP serine 84, a previous study has shown that oxygen atoms in these two APs have 2–3 steric positions, and they are located in the M2 ion coordination sphere(Helland et al., 2009). The multiple positions of oxygen atoms mean that both APs are highly flexible. Previous studies have shown that VAP R129 and ECAP R166 not only facilitate substrate binding, but they also improve product release. SAP R162 is flexible in orientation, and has two conformational positions(docked and nondocked). When SAP R162 is in the docked conformation, two elements(phosphate and Zn ²⁺)occupy M3, assisting in SAP binding either the substrate or inhibitors. In comparison, SAP R162 in the non-docked conformation helps the orientation of another two elements(sulphate and Mg ²⁺)in M3. SAP R162 presents two types of conformations that are related to product release rates, and the non-docked conformation corresponds to higher product release rates(de Backer et al., 2004). SAP R162 in the docked position is identical in configuration to VAP R129; this pattern is also observed in ECAP R166 and PLAP R166. These four arginines easily superimpose during catalytic processes, promoting AP hydrolytic efficacies. Anions, rather than metal ions, in M3 affect arginine conformation, and phosphites are frequently observed among them.

Conserved residues are usually located within or on the approach to secondary AP structures, and these structures play important roles during phosphoryl ester catalysis in marine APs. There are three kinds of completely conserved residues in APs:(Ⅰ)approximately 13 glycine residues;(Ⅱ)nucleophile serine;(Ⅲ)phosphoryl-arginine. Additionally, highly conserved residues are also observed in the M1–M3 sites(ECAP as the reference):(Ⅰ)Asp327, His331, and His412 in M1(Zn ²⁺);(Ⅱ)Asp369, His370, and Asp51 in M2(Zn ²⁺);(Ⅲ)Glu322 and Thr155 in M3(Mg ²⁺). Mg ²⁺ in the M3 of most APs generally forms four ligands with four coordinating residues(Glu322, Thr155, Asp153, and His 153)(Wang et al., 2005). However, VAP possesses another ligand(Trp328 in VAP corresponds to Lys328 in E . coli, and His116 in PLAP/bovine AP M3), which cooperates with residue—Trp328. In mammalian APs, there are no salt bridges between Asp153 and Lys328, resulting in excellent catalytic activity.

3.5 Surface potential

Several substrates, including phosphoryl esters, DNA, RNA, alkaloids, and flamotide, are catalyzed by APs, and negative potentials are detected on the surfaces of these compounds. Marine APs also have negative surface potentials. In marine VAP, inserts Ⅲ and Ⅳ provide the enzyme-specific traits, and positive potentials are observed in active sites rather than at the surface, dragging substrates into active sites. Negative potential is high on the surface of SAP, and generous positive charges exist in SAP active sites, resulting in high catalytic efficiency at low temperatures(de Backer et al., 2002). Negative potential on the surface of SAP interacts with the solvent by hydrogen bonding, which allows substrates to access SAP active sites(de Backer et al., 2002). There are two key steps in restricting rates of hydrolysis: hydrolysis of a covalent enzymephosphate intermediate and release of non-covalently bound phosphate, and the special potential at the surface and active sites affects the catalytic rates of the hydrolysis process. Substrate-negative potentials can be neutralized by positive ions in solution(e.g., Na + and K +), reducing rejection from negative surface potential. Negative potential at the surface cannot hamper substrate binding, and optimization of surface potentials is a cold adaptation strategy(de Backer et al., 2002).

3.6 Crown domain

Marine APs generally have crown domains, whose structure affects catalytic efficacies. For the dimer marine APs, there are two subunit monomers, which are connected by bonds in the crown domains. The sizes of crown domains are proportional to the interface area, which is important in the catalytic process. VAP has the largest interface area, and its crown domain is the biggest among APs. The VAP crown domain is similar to those of SAP and PLAP, and they all have a short antiparallel sheet and two conserved strands(VAP, 322–323 and 458–459 strands; PLAP, 362–364 and 391–395 strands; SAP, 360–363 and 390–392 strands). However, apparent disparities are observed in crown domains among PLAP, SAP, and VAP:(Ⅰ)PLAP has three small beta strands(362–364, 391–395, and 423–425), which form a six-stranded double-sheet layer with the other subunit monomer;(Ⅱ)SAP has one subunit monomer containing four small beta strands(360–363, 377– 380, 384–388, and 390–392), which form two adjacent hairpin motifs to cooperate with the other monomer during the catalytic process;(Ⅲ)in comparison with the hairpin strands(377–380 and 384–388)in SAP, insert strands(Ⅱ, Ⅲ, and Ⅳ)in VAP extend over the active site to access the opposite side of the crown domain(Helland et al., 2009). Residues in crown domains can be substituted, and residue replacements alter surface loop conformations, which in turn influence AP catalytic efficacy. Valines are key residues in marine AP crown domains, and their changes can reduce catalytic activity, which is attributable to crown-domain distortion and reduced AP activity(k _cat / K _m)(Helland et al., 2009). Valines in crown domains are replaced by other residues, resulting in the occurrence of defective mineralization(hypophosphatasia)in vertebrates. Alteration of other residues causes corresponding changes in enzymatic activity to varying degrees. Allosteric effects in crown regions can also change the conformation of each monomer, which in turn changes the activities of APs(Llinas et al., 2005).

3.7 N-terminus

The AP N-terminus is involved in dimer formation, e.g., Asn10 and Arg11 in the ECAP N-terminus connect two monomers by coordination. Monomer APs are mainly isolated from psychrophilic microorganisms, and they are highly active in cold environments. Dimer APs can be cleaved into two monomers. However, these monomers are often inactive in nature. The N-terminus enhances dimer AP stability by linking two monomers, minute changes can affect the coordination of two monomers, and truncation either decreases or ceases activity(Tyler-Cross et al., 1989). Among marine APs, that of VAP was originally thought to be monomeric; however, recent studies have proven it to be dimeric(Hauksson et al., 2000; Helland et al., 2009). Its short N-terminus cannot connect two monomers effectively. The VAP N-terminus contains 50 residues, and is located in the interface area. The VAP inserts form a larger extension loop, interrupting dimer formation by a loose conformation. VAP is very active at low temperatures, and the inserts play a surrogate(the other subunit)role. Long N-terminuses enhance stability in APs such as SAP, ECAP, and PLAP. Large inserts in VAP carry out the same function as the N-terminal region in other dimer APs, increasing VAP’s cold adaptation.

3.8 Structure and temperature

Marine APs often exhibit optimal activity at low temperatures, illustrating that they have an apparent temperature-dependence. Marine APs have similar traits according to their structures:(Ⅰ)a small number of hydrophobic residues;(Ⅱ)a large number of polar residues;(Ⅲ)a small amount of arginine and pralines(Helland et al., 2009). Glycine is an important amino acid residue in marine APs, and a cluster of glycines increases flexibility and cold adaptation, e.g., TAP and VAP have superb backbone mobility(Mavromatis et al., 2002). Temperature propensities involve some residues in active sites, where mutagenesis shifts catalytic temperatures(Koutsioulis et al., 2008). Reduction of thermo-stability and/or increase of flexibility increases a marine AP’s catalytic constant(k _cat), and a high k _cat can be acquired by a single mutation; e.g., compared with the wild type, the TAP mutant(S42G and S338T)has inferior stability, and its k _cat value is also lower, which is caused by a single modification. We could employ several approaches to improve marine AP activity. Many AP mutants exhibit changes in their catalytic constants caused by mutagenesis. The TAP mutant(D153G/D330N)has a high k _cat(seven-fold that of the wild type), which is attributed to the following factors:(Ⅰ)folded 330 N;(Ⅱ)a covalent phosphoseryl intermediate;(Ⅲ)optimal distance between the phosphoseryl intermediate and Zn ²⁺ in M2(Muller et al., 2001). The G330 in monomer VAP plays a critical role in catalytic performance(high k _cat), and its function is determined by three factors:(Ⅰ)optimal distances to the active site;(Ⅱ)enhanced coordination with metal ions;(Ⅲ)a strong interaction between the active site and substrates. When compared with their mesophilic/ thermophilic counterparts, marine APs have subtle structural differences, and they are all highly flexible, resulting in their low-temperature propensities. Based on their evolutionary history, most marine organisms undergo long periods of evolutionary pressure in cold environments, and they develop cold-adapted enzymes in vivo to sustain normal metabolism. Marine APs possess high catalytic mobility and inferior adhesion in the interior structure, illustrating their outstanding catalytic efficacy at low temperatures. At high temperatures, marine APs have poor thermo-stability and weak _catalytic function, which is involved in flexibility that can be acquired either by hydrogen-deuterium(H/D)exchange(Liang et al., 2004)or electron spin resonance(Columbus and Hubbell, 2002).

3.9 Disulfide bonds

Disulfide bonds increase AP thermo-stability; however, marine APs have few disulfides, resulting in inferior thermo-stability. Marine AP thermo-stability is determined by several weak interactions including hydrophobic interactions, noncovalent interactions(hydrogen bonds, van der Waals interactions, and ionpair networks), and covalent interactions(disulfide bonds). In marine AP cells, oxidation-reduction results in few disulfide bonds. However, APs contain several disulfide bonds in vitro, and thus strengthens the rigidity of active sites. In some cases, disulfide bonds are degraded by soluble enzymes and cellsurface receptors following the loss of enzymatic activity(Hogg, 2003).

In APs, cysteine sulfur atoms interact with aromatic rings, increasing their stability. Disulfide bonds increase entropy in main-chains, which in turn affects thermo-stability. A previous study has shown that cleavage of disulfide bonds uses 2–5 kcal/mol energy(Creighton, 1992). Many enzymes, including acetylcholinesterase(Dolginova et al., 1992), barnase(Clarke et al., 2000), beta-lactamase(Shimizu-Ibuka et al., 2006), and T4 lysozyme(Matsumura et al., 1989), have increased their thermo-stability by the introduction of disulfide bonds. Therefore, this strategy could be used to promote thermo-stability in marine APs by constructing disulfide bonds. However, compared with the wild type, some APs present mediocre flexibility when disulfide bonds are built, suggesting that we should consider two factors(flexibility and thermo-stability)to optimize marine APs(Almog et al., 2002). In general, four cysteine residues exist in mesophilic and thermophilic APs, and they could form two disulfide bridges, e.g., ECAP and vertebrate APs have two disulfide bridges. In marine APs, the removal of disulfide bridges induces local instability, accompanied by excellent catalytic efficiency, e.g., VAP has only one cysteine residue that approaches the nucleophilic serine in the active site and cannot form disulfide bridges with other residues, which results in improved catalytic activity at low temperatures(Siddiqui et al., 2005). Mesophilic ECAP has two disulfide bridges, and it has superb thermo-stability and catalytic efficiency at moderate temperatures, demonstrating that disulfide bridges are involved in thermo-stability and catalytic efficiency. Marine APs have superior k _cat and K _m in comparison with mesophilic and thermophilic APs at low temperatures. ECAP k _cat and K _m are 33/s and 0.06 mmol/L at 15℃, respectively. VAP has superior catalytic activity at 15℃, and its k _cat and K _m are 107/s(3-fold that of ECAP)and 0.11 mmol/L(2-fold that of ECAP), respectively(Hauksson et al., 2000).

A previous study demonstrated that there are seven residues between VAP two loops; they are located in 5Å radiuses and can be replaced by cysteines (Ásgeirsson et al., 2007). Engineered cysteines form four disulfide bridges, promoting thermal stability and decreasing k _cat and K _m(Ásgeirsson et al., 2007). New disulfide bridges induce changes in the covalent and/or noncovalent interactions between substrates and APs, resulting in unexpected stabilization and catalytic efficiency(Clarke et al., 2000; Mimura et al., 2005). Disulfide bonds enhance AP rigidity in the active sites, which in turn strengthens enzymatic thermo-stability. Cross-links have a close relationship with catalytic efficiency and stability, and they facilitate the hydrophobic packing and formation of new hydrogen bonds and other rearrangements(Mansfeld et al., 1997). Given that disulfide bridges bring about indeterminable effects, we generally develop our strategies based on four prerequisites:(Ⅰ)that the protein’s stereochemistry has been accurately described;(Ⅱ)disulfide bonds are situated in/near the surface of proteins with high mobility;(Ⅲ)the number of residues constituting disulfide bridges is equal to/less than that of original residues;(Ⅳ)the enclosed loop contains more residues(≥25)(Dani et al., 2003). TAP boosts catalytic efficiency by substituting residues appropriately, contributing to an increase in conformational freedom(Tsigos et al., 2001). Therefore, it is critical when introducing disulfide bridges to determine AP conformations. In eukaryotes and Gram-positive bacteria, APs are often membrane-bound. However, in Gram-negative bacteria, APs usually exist in the periplasmic space. The location of APs in organisms is conformationrelated, suggesting that APs from various organisms have different catalytic efficacies. 3.10 K _m and k _cat With regard to APs, K _m involves two factors: K s(substrate dissociation constant)and K _i(equilibrium constant of inorganic phosphate-competitive inhibitor). Compared with the wild type, marine VAP mutants with new disulfide bridges exhibit lower activity, which is attributable to a reduction in K _m . Disulfide bridges compact the loose structure and decrease flexibility in the binding site, and improved AP catalytic activity often results from just a few disulfide bridges in marine organisms. K _i and K _m indicate AP binding intensity, and VAP mutants have lower K _i values, suggesting that they can bind substrates better than wild types(Ásgeirsson et al., 2007). K cat, enzyme turnover rate, represents the catalytic efficiency of the active site, which closely correlates with dynamic network in the catalytic cycle(Eisenmesser et al., 2005). Marine APs have exceptional catalytic rates, and they catalyze phosphoryl esters efficiently at low temperatures. The dynamic movement of marine APs is restricted by disulfide bridges, and the enzymatic efficacies are mainly determined by cysteines in the amino acid residues(Siddiqui and Cavicchioli, 2006). Disulfide bridges have inverse effects on k _cat and AP stability, their formation decreases the former and increases the latter, and vice versa. Marine APs have evolved few disulfide bridges and cysteines, thus reducing the number of disulfide bonds. Marine TAP from the Antarctic strain TAB 5 has only one cysteine, and APs from the Shewanella strains SIB1 and SCAP have no cysteines in their amino acid sequences(Rina et al., 2000).

Compared with marine APs, thermo-stable homologs such as E . coli(Bradshaw et al., 1981)and mammalian(Le Du et al., 2001)APs have superior k _cat and stability at high temperatures because they have two disulfide bridges. The VAP mutant contains disulfide bridges, and its k _cat and K _m are reduced(Ásgeirsson et al., 2007). To obtain excellent k _cat and thermo-stability, disulfide bridges formulated with cysteines should be far away from active sites in marine APs. Product release is a rate-limiting step during conversion of phosphoryl esters to phosphorylated Tris, and it is an effective strategy for marine APs to facilitate product release to increase k _cat(Hoylartes et al., 2006). The removal of the N-terminal helix does not transform the geometric conformation of active sites. However, it inhibits the formation of phospho-serine and phosphate, accompanied by a significant decrease in k _cat and thermo-stability. The k _cat value is enhanced by increasing flexibility, which leads to thermo-stability in cold-active APs. 3.11 Hydrophobic interactions, hydrogen bonds, and salt bridges Hydrophobic residues in the protein core facilitate conformation packing, surface area reduction, and decrease enzyme-solvent interactions. Hydrophobic interactions in marine APs not only increase stability, but they also counteract the effects of intra-molecular networks of polar/charged residues. In general, marine APs are extracellular enzymes, and they are often purified by fragmenting cells. It is necessary to remove redundant fragments by N-terminal sequencing, avoiding incorrect comprehension on hydrophobic interactions. With regard to marine AP aliphatic side chains, they usually consist of alanine, valine, isoleucine, and leucine, which are involved in hydrophobic interactions. Compared with marine APs, those from thermophiles have more aliphatic side chains, contributing to core region packing and increasing thermo-stability. However, there are a few exceptions, e.g., (Ⅰ)marine TAP from Antarctic bacterium TAB 5 has a similar hydropathicity index to thermo-stable APs;(Ⅱ)ECAP has a lower percentage of aliphatic side chains compared with thermophilic APs. Marine VAP from Vibrio(G ^–)has very similar aliphatic side chains to APs from genera Bacillus, Lactobacilli, and Enterococcus(G ⁺)when compared with G ^– bacteria such as Escherichia and Serratia . Although VAP sits close to TAP in the dendrogram, its scores are similar to those of some extreme thermophilic APs such as those from Thermotoga maritima and Pyrococcus abyssi(Rina et al., 2000).

APs have different percentages of charged polar and hydrophobic residues. When compared with mesophilic/thermophilic APs, there are elevated proportions of charged polar and reduced hydrophobic residues in marine APs(Schrøder Leiros et al., 2000). Ile+Leu/Ile+Leu+Val can be used as indicators of hydrophobicity in APs, and marine APs have poor hydrophobicity, which is determined by the following factors:(Ⅰ)weak salt bridges, arginine-mediated hydrogen bonds, and prolines in loops;(Ⅱ)few extending surface loops;(Ⅲ)high proportions of glycines and methionines;(Ⅳ)very few interactions between aromatic-aromatic ring and/or intersubunit ion pairs(Ásgeirsson and Andrésson, 2001). In general, APs have a high proportion of acidic residues(Asp+Glu)(14±2%)and few basic residues(Arg+Lys)(11±3%). There are minute distinctions between the theoretical(calculated)and the actual p I s in marine APs, demonstrating that acidic residues(Asp+Glu)play a key role. Compared with thermophilic APs, marine and mesophilic counterparts have lower Arg/ Arg+Lys ratios. Marine APs have more prolines, fewer glycines, and a lower ratio(Arg/Arg+Lys), which results in their outstanding conformational flexibility.

High proportions of hydrogen bonds are observed in marine APs. These are mainly formed by amide hydrogen and carbonyl oxygen(accounting for 68% of total hydrogen bonds)(Tibbitts et al., 1996). With respect to enzymatic solutions, their pH dominates the charged state of ionization groups, which in turn affects the formation of salt bridges between APs and ionization groups. Extreme pH can decrease the formation of salt bridges by strengthening the unfolding tendency of marine APs. A great deal of hydrogen bonds and salt bridges are formulated in marine AP solution, strengthening their thermal stability and structural rigidity. Stabilization energy is generally 5–20 kcal/mol, and a single hydrogen bond can reduce free energy by 0.5–2 kcal/mol, and an ion pair by 0.4–1.0 kcal/mol, which is verified in marine APs. Additionally, long-range interactions play key roles in the overall protein stability of APs. Because hydrogen bonds are dipole–dipole attractions between hydrogen and electro-negative atoms such as oxygen and nitrogen, superior hydrogen bond density at the interface reinforces AP thermal stability, and hydrogen bonds in helices considerably enhance the local force. Temperature changes slightly affect electrostatic interactions in marine APs, which in turn changes the catalytic efficacies. High temperature weakens hydrogen bonds and strengthens hydrophobic interactions. Marine APs can be unfolded and/or denatured easily at temperatures between 50 and 80℃, which is involved in the weakening of salt bridges and hydrogen bonds. TAP has fewer hydrogen bonds, and no salt bridges have been observed in this AP, resulting in its lower thermal stability and structural rigidity.

4 RESTRICTION STEPS AND GENETIC REGULATION 4.1 Restriction steps

Marine APs catalyze phosphoryl ester hydrolysis at low temperatures; substrate accessibility and product release are key factors in limiting the catalytic rates. Excellent electrostatic attraction assists in the approach of substrates to the AP surface, and large active site entrances facilitate substrates access, which is evident from the results of previous studies in which potent attractive force facilitated the ingression of substrates to AP active sites(Smalås et al., 2000; Feller and Gerday, 2003). Marine APs have excellent structural flexibility, and their entrances can be altered. In VAP, attractive power drags large substrates into active sites, thus enlarging the entrance(Helland et al., 2009).

There are three actions in the catalytic process:(Ⅰ)phosphoryl ester hydrolysis;(Ⅱ)formation of phosphoryl-serine intermediates;(Ⅲ)product release. Product release is a key factor in evaluating the catalytic efficacies of APs, and its implementation allows the spaces to bind with substrates to accept other substrates, which in turn forms new intermediates in active sites. Of the known APs, ECAP K328A/ K328H is an exception; its transphosphorylation plays a key role in the reaction process, hindering phosphoryl-enzyme complex(covalently)hydrolysis(Xu and Kantrowitz, 1991). In general, product release rates and catalytic efficiencies are positively correlated in marine APs.

4.2 Genetic regulation

Many genes including phosphatase A(phoA, pho regulon), phosphatase B(phoB, transcriptional activator and inducer of pho regulon), and phosphatase R(phoR, an activator and a repressor)have been investigated to date(Wanner and Chang, 1987; Sola- Landa et al., 2003; Gristwood et al., 2009). The phoA gene encodes AP, and it is contained in an AP regulon, which dominates AP production(Wanner and Chang, 1987; Vershinina and Znamenskaya, 2002). Deficiency medium(not containing phosphate)induces the manufacture of marine APs, and the production is several hundred fold that of nondeficiency medium(containing phosphate). PhoB and phoR regulate phoA promoter transcription, which in turn affects AP expression. Other regulatory genes, such as phoE, psiB, psiC, psiD, and pstSCAB, are involved in expression and take part in phosphate assimilation. They are also regulated by phoB and phoR. With regard to gene locations, there are distinctions between marine APs and their counterparts, e.g., phoB and phoR are closely linked to phoA on the E . coli chromosome. Unlike the E . coli phoA, VAP phoA(phoA VC)is located on a separate chromosome(Majumdar et al., 2005). Marine APs have different regulatory mechanisms as a result of their marine environments, which induce AP mutations.

5 CONCLUSION

APs catalyze the hydrolytic cleavage of phosphate monoesters and play important roles in microbial ecology and molecular biology applications. Marine APs play an important role in phosphorus uptake in the marine environment. In this review, we have discussed the characteristics of AP, including structure, catalytic dynamic parameters, active sites, factors affecting catalytic rate(temperature, pH, etc.), and genetic regulation. Marine APs are structurally and functionally diverse in many respects, including subunits, metal ion requirements, disulfide bonds, and active sites when compared with mesophilic and thermophilic APs. This review provides insights into the psychrophilic characteristics of marine APs.