Cold Adaptation Of Metalloproteases In Thermolysin Family And Degradation Of Organic Nitrogen By Deep-Sea Psychrotrophic Bacterium Pseudoalteromonas Sp.SM9913

Partâ… .Cold Adaptation of Novel Cold-Adapted Metalloproteases MCP-02 and E495 in Thermolysin Family.Cold-adapted enzymes,produced by organisms thriving in permanently cold habitats(deep sea,polar regions and alpine regions),are characterized by high catalytic efficiencies at low temperatures and low stabilities at moderate and high temperatures.Different enzymes may adopt different strategies for cold adaptation. The prevailing hypothesis is that cold-adapted enzymes obtain high catalytic efficiencies by increasing conformational flexibility at the expense of stability. However,there are also reports about cold-adapted enzymes that possess both high activities and high stabilities.Laboratory evolution studies also show that high catalytic efficiency and high stability could be obtained simultaneously in a single molecule.Therefore,further studies are needed to gain new insights into the structural basis underlying cold adaptation.Thermolysin family(M4) is a family of zinc metalloproteases.The peptidases in this family are mainly produced by bacteria from various habitats.For example, thermolysin,the typical example of this family,is from the thermophilic bacterium Bacillus thermoproteolyticus,pseudolysin is from the mesophilic bacterium Pseudomonas aeruginosa PAO1 and a putative vibriolysin in this family is from the Antarctic bacterium strain 643(VAB).VAB is the only known enzyme in this family from a permanently cold habitat.Though its sequence has been deposited in the public database(GenBank^TM Accession # AF272770,NCBI Protein Database Accession # AAF78076),no biochemical data of this vibriolysin has been reported.Therefore, further studies are essential to understand the biomedical properties and cold adaptation strategies of the cold-adapted enzymes in thermolysin family.The psychrotrophic bacterial strain,Pseudoalteromonas sp.SM9913,is aγ-proteobacterium,which was isolated from deep-sea sediment.As aγ-proteobacterium,P sp.SM9913 secrets large amounts of extracellular proteases, and the metalloprotease MCP-02 is one of them.N-terminal sequencing showed that MCP-02 and VAB have a very similar N-terminal sequence(identity up to 95%), suggesting that MCP-02 is probably a member of thermolysin family.The metalloprotease E495,secreted by the Arctic sea ice bacterium P.sp.SM495,also has a very similar N-terminal sequence to VAB(identity up to 93%),suggesting that E495 is also a member of thermolysin family.Here,metalloproteases MCP-02 and E495 were used as models to study the biochemical and enzymatic characteristics and the cold adaptation of the cold-adapted metalloproteases in thermolysin family.The results are as follows.1.Gene cloning and characterization of novel metalloproteases in thermolysin family from a deep-sea bacterium and an Arctic sea-ice bacterium.(1) Gene cloning and characterization of extracellular metalloprotease MCP-02 from deep-sea bacterium P sp.SM9913.The full-length mcp-02 gene was cloned from P sp.SM9913 using nested PCR and TAIL PCR techniques.The nucleotide sequence contains 2184 bp,encoding a MCP-02 precursor of 727 amino acid residues.The nucleotide sequence was submitted to the GenBank^TM with accession number EF029091,and the amino acid sequence can be accessed through NCBI Protein Database under NCBI accession number ABL06977.Analysis of the amino acid sequence showed that MCP-02 is a zinc metalloprotease that belongs to Vibriolysin subfamily(MEROPS ID:M04.003) of Thermolysin family(M4).The full-length mcp-02 gene was cloned into the expression vector pET-22b,and then was transformed into Escherichia coli BL21(DE3).Crude recombinant MCP-02 was obtained by direct addition of solid ammonium sulfate powder to the culture supernatant. Recombinant MCP-02 in the crude proteins was purified using a DEAE Sepharose Fast Flow column with 50 mM Tris-HCl buffer(pH 8.0),and a linear NaCl gradient from 0 to 0.5 M. The purity was determined by 12.5%SDS-PAGE.The mcp-02 gene was confirmed by the N-terminal sequencing of the mature recombinant MCP-02,which showed that the recombinant MCP-02 has the same N-terminal sequence as the wild MCP-02 purified from P. sp.SM9913. The molecular mass of mature MCP-02 is 33929 Da,determined using mass spectroscopy.Mature MCP-02 contains 315 residues(A205 to D519),deduced based on its N-terminal sequence and the molecular mass.With casein as the substrate,MCP-02 has a pH optimum 8.0 and a temperature optimum 57℃.The half-life t_1/2 is 90 min at 55℃,and 23 min at 60℃.Using the synthetic dipeptides as substrates,MCP-02 preferred to catalyze the hydrolysis of the substrate that has a large and hydrophobic residue at the P1' subsite,and the preference order was Phe＞Leu＞Val.This study represents the first study on the biochemical properties of a protease in thermolysin family from a deep-sea bacterium.(2) Gene cloning and characterization of extracellular metalloprotease E495 from Arctic sea-ice bacterium P sp.SM495.The full-length E495 gene was cloned from P.sp.SM495 using nested PCR and TAIL PCR techniques.The nucleotide sequence contains 2193 bp,and encodes an E495 precursor of 730 amino acid residues.The nucleotide sequence was submitted to the GenBank^TM with accession number FJ211191,and the amino acid sequence can be accessed through NCBI Protein Database under NCBI accession number ACI28452. Analysis of the amino acid sequence showed that E495 is a zinc metalloprotease that belongs to Vibriolysin subfamily(MEROPS ID:M04.003) of Thermolysin family.The full-length E495 gene was cloned into the expression vector pET-22b,and then was transformed into E.coli BL21(DE3).Crude recombinant E495 were obtained by disruption of the cells using sonication and precipitation with solid ammonium sulfate. Recombinant E495 in the crude proteins was purified using a DEAE Sepharose Fast Flow column with 50 mM Tris-HCl buffer(pH 8.5),and a linear NaCl gradient from 0 to 0.5 M. The purity was determined by 12.5%SDS-PAGE.The E495 gene was confirmed by the N-terminal sequencing of the mature recombinant E495,which showed that the recombinant E495 has the same N-terminal sequence as the wild E495 purified from P.sp.SM495.The molecular mass of mature E495 is 33376 Da,determined using mass spectroscopy. Mature E495 contains 310 residues(A207 to T516),deduced based on its N-terminal sequence and molecular mass.E495 has similar biochemical and enzymatic properties to MCP-02.With casein as the substrate,E495 has a pH optimum 7.5 and a temperature optimum 57℃.The half-life t_1/2 is 82 min at 55℃,and 20 min at 60℃.Using the synthetic dipeptides as substrates,E495 preferred to catalyze the hydrolysis of the substrate that has a large and hydrophobic residue at the P1' subsite,and the preference order was Phe＞Leu＞Val. The study on E495 is the first report on the biochemical properties of a protease in thermolysin family from an Arctic sea-ice bacterium.2.Cold adaptation of the metalloproteases in thermolysin family.To investigate cold adaptation of the metalloproteases in thermolysin family, deep-sea MCP-02 and Arctic sea-ice E495,together with their mesophilic homolog, pseudolysin from terrestrial bacterium Pseudomonas aeruginosa PAO1,were systematically studied and compared.The gene of pseudolysin was cloned from Pseudomonas aeruginosa PAO1 genome,and then was expressed in E.coli BL21(DE3) strain.The recombinant pseudolysin was purified and the purity was confirmed by SDS-PAGE.The biochemical and enzymatic properties of pseudolysin was studied.And then the catalytic efficiency,thermostability,and flexibility of terrestrial pseudolysin,deep-sea MCP-02,and Arctic sea-ice E495 were carefully studied and compared.The activities of pseudolysin,MCP-02 and E495 toward synthetic FA-Gly-Leu-NH₂ was compared at pH 8.0.In the temperature range 10-40℃,the order of catalytic efficiency k_cat/K_m was pseudolysin＜MCP-02＜E495.The catalytic efficiency k_cat/K_m at 25℃was 0.64±0.01 s^-1 mM^-1 for pseudolysin,1.32±0.01 s^-1 mM^-1 for MCP-02,and 2.55±0.05 s^-1 mM^-1 for E495,with a ratio of approximately 1:2:4. The catalytic efficiency at low and medium temperatures increases as the temperature of the habitat decreases,suggesting different degrees of cold adaptation of the three proteases.The temperature optimum(T_opt) was 62℃for pseudolysin,57℃for MCP-02, and 57℃for E495.The half-life(t_1/2) was 530 min for pseudolysin,90 min for MCP-02,and 82 min for E495 at 55℃;and 160 min for pseudolysin,23 min for MCP-02,and 20 min for E495 at 60℃.The inactivation rate constant(k_inact) was 2.2×10^-5 s^-1 for pseudolysin,1.3×10^-4 s^-1 for MCP-02,and 1.4×10^-4 s^-1 for E495 at 55℃;and 7.3×10^-5 s^-1 for pseudolysin,5.1×10^-4 s^-1 for MCP-02,and 5.8×10^-4 s^-1 for E495 at 60℃.The heat-induced denaturation was monitored using fluorescence,and the apparent melting temperature(T_m) was 75℃for pseudolysin,64℃for MCP-02, and 63℃for E495.The heat-induced denaturation was also monitored using circular dichroism at 222 nm,and the resultant apparent T_m was 72℃for pseusolysin,65℃for MCP-02,and 65℃for E495.The analysis of T_opt,t_1/2(and k_inact),and apparent T_m comes to the same conclusion that the thermostability order is pseudolysin＞MCP-02≈E495.MCP-02 and E495 have a similar stability,both 5-10℃lower than pseudolysin.However,though slightly lower than pseudolysin,the relatively high thermostabilities of MCP-02 and E495(e.g.T_opt of 57℃) make them look like mesophilic enzymes.The fluorescence quenching experiment was used to compare the conformational flexibility of the three proteases.The Stern-Volmer quenching constants(K_SV) at 15℃and 40℃for pseudolysin,MCP-02 and E495 were determined,respectively.Because the numbers and local environments of tryptophans in these three enzymes are different,the absolute values of K_SV can not be compared directly.Instead,the difference in K_SV at different temperatures(40℃and 15℃in this study,i.e.ΔK_SV(40 - 15℃)) was used as a parameter to represent the conformational flexibility of the enzyme.TheΔK_SV(40℃- 15℃) value was 2.6±0.1 M^-1 for pseudolysin,5.3±0.1 M^-1 for MCP-02,and 5.7±0.1 M^-1 for E495,suggesting that pseudolysin has the most rigid structure,deep-sea MCP-02 has a more flexible structure and Arctic E495 has the most flexible structure.The difference in the flexibility of E495 and MCP-02 from cold habitats suggested that the Arctic E495 underwent further optimization of flexibility compared to deep-sea MCP-02.The above results showed that the catalytic efficiency(at low and medium temperatures) and the flexibility have the same order,both of which increase as the environment temperature of the habitat decreases.This result agrees with that of the reported "typical" cold-adapted enzymes.However,the stability of the enzyme does not decrease as the environment temperature of the habitat decreases,strongly suggesting that the increased flexibility(and catalytic efficiency) of the cold adapted vibriolysin(especially E495) is not obtained by sacrificing its stability.This conclusion differs from the prevailing hypothesis that "cold-adapted enzymes obtain high catalytic efficiencies by increasing conformational flexibility at the expense of stability".To further investigate this problem,long molecular dynamics(MD) simulations were conducted and detailed structural analyses were conducted together with Xie Bin-Bin(detailed results shown in the thesis of Xie Bin-Bin).The above results of experiments and the MD simulations suggest that the cold-adapted enzymes in thermolysin family obtain high catalytic efficiencies(at low temperature) by increasing the global conformational flexibility,and the increased conformational flexibility was not obtained at the expense of the stability.Finally,we proposed a model for cold adaptation of the cold-adapted enzymes in thermolysin family.This represents the first study on the cold adaptation strategy of the enzymes in thermolysin family.Partâ…¡,Enzymatic Hydrolysis of Organic Nitrogen by Deep-Sea Psychrotrophic Bacterium Pseudoalteromonas sp.SM9913Studies showed that someγ-proteobacteria secret large amounts of extracellular proteases to degrade environmental proteins or peptides to provide energy and carbon resources for the bacteria.The psychrotrophic bacterial strain,Pseudoalteromonas sp. SM9913 is aγ-proteobacterium.P.sp.SM9913 secrets several kinds of proteases, including serine protease MCP-01,metalloprotease MCP-02,and another kind of serine protease MCP-03.Currently,it is unclear how these proteases cooperate to degrade the environmental proteins.The study of this problem would facilitate our understanding of the adaptation mechanism of P.sp.SM9913 to the deep-sea environment and provide new insights into the biological degradation of deep-sea sedimentary organic nitrogen by bacteria.In this study,the oxidized insulin B chain(insulin B_ox) and bovine serum albumin (BSA) were used as substrates to identify the cleavage sites of the three extracellular proteases(MCP-01,MCP-02,and MCP-03) of P.sp.SM9913.The hydrolysis conditions were firstly optimized with the help of HPLC when using insulin B_ox as the substrate.Then,the hydrolysis products were analyzed using MALDI-TOF mass spectroscopy.The resultant molecular masses were then analyzed to find the corresponding cleavage sites.Five cleavage sites were identified for MCP-01 on insulin B_ox, and they were Gln(?)His,Leu(?)Tyr(?)Leu,Cya(?)Gly,and Phe(?)Phe,all of which are consistent with the reported corresponding sites of MCP-01 homolog subtilisin Carlsberg on insulin B_ox· Seven cleavage sites were identified for MCP-02 on insulin B_ox, and they were Gln(?)His,Leu(?)Cya,Ala(?)Leu(?)Tyr,Cya(?)Gly, Arg(?)Gly,and Phe(?)Phe,five of which are different from the reported sites of MCP-02 homolog thermolysin on insulin B_ox· Five cleavage sites were identified for MCP-03 on insulin B_ox,, and they were Leu(?)Cya,Glu(?)Ala,Leu(?)Tyr,Gly(?)Phe(?)Phe,one of which is different from the reported sites of MCP-03 homolog thermitase on insulin B_ox· It is noted that each enzyme has its distinctive cleavage site(s) on insulin B_ox, implying that each enzyme has its role in the degradation of the environmental proteins.Based on these results,it can be deduced that insulin B_ox can be degraded into oligopeptides of 2-7 amino acid residues and many single amino acids.When using BSA as the substrate,the hydrolysis products were firstly separated using SDS-PAGE,and then some clearly-separated peptide bands were identified by N-terminal sequencing.The results showed that different proteases tend to cleave different sites on BSA,and the three enzymes together could hydrolyze the BSA at the following sites,Thr83(?)Tyr84,Leu218(?)Ser219,Glu226(?)Phe227,Val234(?)Thr235, Leu422(?)Val423,Ala489(?)Leu490,all of which are exposed at the surface of the BSA molecule.The above results show that the extracellular proteases of P.sp.SM9913 have both same cleavage sites and distinctive cleavage sites on proteins,suggesting that they can collaborate with each other in degrading the environmental proteins.This study would facilitate our understanding on the adaptation mechanism of P.sp. SM9913 to the deep sea environment.