Thermolysin家族金属蛋白酶的适冷机制及深海适冷菌Pseudoalteromonas sp.SM9913对有机氮的降解
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摘要
一、Thermolysin家族金属蛋白酶的适冷机制
适冷酶由生存于永久低温环境(如深海、极地和高山地区等)的生物所生产,兼有低温下高的催化效率和高温下的热不稳定性双重特点。虽然对适冷酶的适冷机制有着不同的解释,但目前最流行的解释是,适冷酶在低温下高的催化效率来自于高的酶蛋白柔性,而高的蛋白柔性则源于适冷酶的热不稳定性。但是,这一解释仍有局限性,因为,目前有报道发现适冷酶可以同时具有高的活性和高的热稳定性。在实验室内通过定向进化对酶分子的改造实验也证实,有些情况下,少数氨基酸残基的突变也可以产生同时具备高催化活性和高热稳定性的酶。因此,适冷酶的适冷机制以及适冷的结构基础有待进一步研究。
嗜热菌蛋白酶家族(thermolysin family,也称M4 family)中的金属蛋白酶多来源于细菌,其中的典型代表有来自嗜热菌Bacillus thermoproteolyticus的嗜热thermolysin和来自Pseudomonas aeruginosa PAO1的中温pseudolysin,以及来自南极细菌643的(适冷)vibriolysin(VAB)。VAB是目前所知的M4家族中唯一来源于永久低温环境的蛋白酶,但是目前关于VAB的信息仅有NCBI公共数据库中的核酸序列(AF272770)和预测的氨基酸序列(AAF78076),对于其来源菌643的生理特征以及VAB的生化性质并没有任何报道。因此,发现Thermolysin家族的(新的)适冷酶并对其性质进行系统研究,对于提高对蛋白质结构—功能之间的关系的认识,以及对蛋白酶温度(低温)适应进化规律的认识具有重要意义。
适冷菌Pseudoalteromonas sp.SM9913是分离自深海沉积物的一株γ-proteobacterium。P.sp.SM9913能够分泌大量蛋白酶,MCP-02是P.sp.SM9913分泌的一种金属蛋白酶。MCP-02的N端序列与VAB具有很高的同源性(identity为95%),北极海冰细菌P.sp.SM495所分泌的胞外酶E495的N端序列也与VAB具有很高的同源性(identity为93%),表明这两种酶都很可能属于Thermolysin家族。本论文选用金属蛋白酶MCP-02和E495为研究对象,对它们进行了基因克隆和表达、性质研究和序列与结构分析,并以中温的pseudolysin为对照,研究了MCP-02和E495的适冷机制。取得了如下研究结果:
(一)深海来源和北极海冰来源的Thermolysin家族的新型蛋白酶的基因克隆与表达及其生化和酶学性质研究。
(1)深海适冷菌P.sp.SM9913所产胞外金属蛋白酶MCP-02的研究。
本论文通过使用巢式PCR和TAIL PCR等技术手段,克隆了深海适冷菌P.sp.SM9913所产胞外金属蛋白酶MCP-02的全基因序列。mcp-02基因全长2184 bp,编码含有727个氨基酸残基的蛋白酶前体。基因序列已提交GenBank,序列号为EF029091(核酸序列),ABL06977(氨基酸序列)。序列分析表明,MCP-02为Zn金属蛋白酶,属于Thermolysin家族,Vibriolysin亚家族(Vibriolysin subfamily,M04.003)。
将MCP-02在E.coli中进行异源表达,使用硫酸铵沉淀、阴离子交换层析等技术,从胞外分离纯化得到了电泳纯的重组MCP-02酶。N端测序表明重组酶和野生酶具有相同的N端序列,表明获得的mcp-02基因序列是正确的。MALDI-TOF质谱分析表明,成熟的重组酶MCP-02分子量为33929 Da。综合酶分子量和N端序列,推断MCP-02成熟酶长315氨基酸残基(A205-D519)。以酪蛋白为底物,MCP-02最适催化pH为8.0,最适催化温度为57℃。55℃半衰期t_(1/2)为90 min,60℃半衰期t_(1/2)为23 min。对二肽合成底物的降解(pH 8.0)表明,MCP-02偏好降解P1'位是大的疏水残基的二肽底物,顺序为Phe＞Leu＞Val。对MCP-02的研究是对深海来源的Thermolysin家族适冷酶生化和酶学性质的首次研究。
(2)北极海冰细菌P.sp.SM495所产胞外金属蛋白酶EA95的研究。
本论文通过使用巢式PCR和TAIL PCR等技术手段,克隆了北极海冰细菌P.sp.SM495所产胞外金属蛋白酶E495的全基因序列。蛋白酶E495的基因全长2193 bp,编码含有730个氨基酸残基的蛋白酶前体。基因序列已提交GenBank,序列号为FJ211191(核酸序列),ACI28452(氨基酸序列)。序列分析表明,E495为Zn金属蛋白酶,属于Thermolysin家族的Vibriolysin亚家族。
将E495在E.coli中进行异源表达,使用超声波破碎、硫酸铵沉淀、阴离子交换层析、Native-PAGE等技术,从周质空间分离纯化得到了电泳纯的重组E495酶。N端测序表明重组酶和野生酶具有相同的N端序列,表明获得的E495基因序列是正确的。MALDI-TOF质谱分析表明,成熟E495分子量为33376 Da。综合酶分子量和N端序列,推测E495成熟酶含有310个氨基酸残基(A207-T516)。E495的酶学性质与MCP-02相似。以酪蛋白为底物,E495最适催化pH为7.5,最适催化温度为57℃。55℃半衰期t_(1/2)为82 min,60℃半衰期t_(1/2)为20 min。对二肽合成底物的降解(pH 8.0)表明,E495偏好降解P1'位是大的疏水残基的二肽底物,顺序为Phe＞Leu＞Val。对E495性质的研究是对极地海冰来源的Thermolysin家族适冷酶生化和酶学性质的首次研究。
(二)对Thermolysin家族适冷酶的适冷机制的研究,揭示适冷酶“活性—柔性—稳定性”三者之间的关系。
以Thermolysin家族的适冷酶——深海MCP-02和北极海冰E495为研究材料,以MCP-02和E495的中温同源酶——来自陆地细菌Pseudomonas aeruginosaPAO1的中温pseudolysin,作为中温对照,研究Thermolysin家族适冷酶的适冷进化机制,以揭示适冷酶“活性—柔性—稳定性”三者之间的关系。
首先从Pseudomonas aeruginosa PAO1的基因组中克隆了pseudolysin的全基因序列,并将其在E.coli中进行异源表达,分离纯化并获得了电泳纯度的重组pseudolysin,详细研究了pseudolysin的性质。在此基础上,本论文系统比较了pseudolysin、MCP-02和E495的催化效率、热稳定性和柔性。
结果表明,以合成二肽FA-Gly-Leu-NH_2为底物,在Tris-HCl缓冲液(pH 8.0)中,在低温和中温(10-40℃)条件下,三个蛋白酶的催化效率顺序(k_(cat)/K_m)为pseudolysin＜MCP-02＜E495,其中在25℃下,pseudolysin的k_(cat)/K_m为0.64±0.01 s~(-1)mM~(-1),MCP-02的k_(cat)/K_m为1.32±0.01 s~(-1) mM~(-1),E495的k_(cat)/K_m为2.55±0.05 s~(-1)mM~(-1),三个酶的催化效率接近1:2:4。可见在低温和中温条件下,三个酶的催化效率随着生存环境温度的降低而升高,表明三个酶因生存环境温度不同而表现不同程度的冷适应性。
三个酶催化酪蛋白水解的最适酶活温度分别为62℃(pseudolysin),57℃(MCP-02),和57℃(E495)。55℃的半衰期t_(1/2)分别为530 min(pseudolysin),90 min(MCP-02),和82 min(E495);60℃的半衰期t_(1/2)分别为160 min(pseudolysin),23 min(MCP-02)和20 min(E495)。三个酶在55℃下的失活速率常数k_(inact)分别为2.2×10~(-5) s~(-1)(pseudolysin),1.3×10~(-4) s~(-1)(MCP-02)和1.4×10~(-4) s~(-1)(E495);在60℃的失活速率常数k_(inact)分别为7.3×10~(-5) s~(-1)(pseudolysin),5.1×10~(-4) s~(-1)(MCP-02)和5.8×10~(-4) s~(-1)(E495)。使用荧光法监测酶的热变性过程,获得了三个酶的表观变构温度(T_m)分别为75℃(pseudolysin),64℃(MCP-02)和63℃(E495);使用圆二色谱监测α螺旋的解螺旋过程,获得了三个酶的表观变构温度T_m分别为72℃(pseudolysin),65℃(MCP-02)和65℃(E495)。分析最适酶活温度、半衰期(和失活速率常数)和表观变构温度的数据都得到一致的结论,三个酶的热稳定性顺序为pseudolysin＞MCP-02≈E495。MCP-02和E495的热稳定性相当,都比pseudolysin降低5～10℃,但是,总体上讲MCP-02和E495的热稳定性仍然很高,如最适酶活温度都约为57℃,仅从热稳定性上判断两酶都类似于中温酶。
以丙烯酰胺为淬灭剂,研究了三个酶在低温(15℃)和中温(40℃)下的动态荧光淬灭,计算了各酶在不同温度下的Stern-Volmer淬灭常数K_(SV),和不同温度下的K_(SV)的差值,△K_(SV)(40-15℃),并用△K_(SV)(40-15℃)来表征酶的柔性。三个酶的△K_(SV)(40-15℃)分别为2.6±0.1 M~(-1)(pseudolysin),5.3±0.1 M~(-1)(MCP-02)和5.7±0.1 M~(-1)(E495),表明三个酶存在以下柔性顺序,pseudolysin＜MCP-02＜E495。
上述结果中三个酶的催化效率和柔性具有相同的顺序,都随生存环境温度的降低而升高,这与已报道的“典型”适冷酶相似。但是,热稳定却并不随生存环境温度的降低而降低,适冷酶E495的催化效率和柔性都高于MCP-02,但是其热稳定性却与MCP-02几乎相同,这表明,至少E495的高的柔性的获得并不是通过降低本身的热稳定性来实现的。这个结论与目前的流行观点“适冷酶在低温下高的催化效率来自于高的柔性,而高的柔性则来源于低的热稳定性”不同。为了进一步研究MCP-02和E495的适冷机制,与解彬彬博士一起对三个酶进行了分子动力学模拟以及模拟过程中的氢键、盐键数目的统计分析(详细结果见解彬彬博士论文)。
根据上述实验结果以及分子动力学模拟结果,我们提出了Thermolysin家族适冷酶的适冷进化机制模型。Thermolysin家族的适冷酶通过提高整体柔性来提高其低温下的催化能力,稳定性的降低并非柔性提高的前提。这是对Thermolysin家族适冷酶适冷机制的首次报道。
二、深海适冷菌P.sp.SM9913对有机氮的降解
研究表明,深海中一些γ-proteobacteria通过分泌大量的蛋白酶将环境中的蛋白降解为短肽,并最终运输到胞内为菌体提供能量和碳源。深海适冷菌Pseudoalteromonas sp.SM9913属于γ-proteobacterium。P.sp.SM9913能够分泌大量蛋白酶,目前的研究表明,这些蛋白酶中至少包括两种不同的丝氨酸蛋白酶,MCP-01和MCP-03,和一种金属蛋白酶MCP-02。这些胞外蛋白酶如何协同作用对胞外蛋白进行充分降解目前还不清楚。而这个问题的阐明不仅有助于阐明深海适冷菌P.sp.SM9913对深海极端环境的适应机制,而且对阐明深海沉积物中有机氮的降解和氮循环机制具有重要意义。
本论文分别以多肽——氧化型的胰岛素B链(insulin B_(ox),含30个氨基酸残基)和大分子蛋白——牛血清白蛋白(BSA,含583个氨基酸残基)为底物,研究了P.sp.SM9913的三种胞外蛋白酶MCP-01、MCP-02和MCP-03对它们的酶切位点,并分析了它们潜在的协同作用,为阐明深海适冷菌P.sp.SM9913对有机氮的酶解机制奠定基础。
以insulin B_(ox)为底物,首先使用HPLC分离肽段的方法优化了各酶酶解条件,然后将降解后的混合肽段使用MALDI-TOF质谱分析获得产物中的各肽段的分子量信息,通过分析分子量找到丰度高的肽段所对应的序列,从而获得酶切位点。结果表明,以insulin B_(ox)为底物,鉴定到了MCP-01的5个切点,为Gln+His、Leu+Tyr+Leu、Cya+Gly和Phe+Phe,这些切点均与已报道的subtilisin Carlsberg的相应切点一致;鉴定到了MCP-02的7个切点,为Gin+His、Leu+Cya、Ala+Leu+Tyr、Cya+Gly、Arg+Gly和Phe+Phe,其中5个切点与已报道的thermolysin的切点不同;鉴定到了MCP-03的5个切点,为Leu+Cya、Glu+Ala、Leu+Tyr、Gly+Phe+Phe,其中1个切点与subtilisn Carlsberg和Thermitase不同。三种酶在insulin B_(ox)上既有相同的切点,也有不同的切点。将三种酶在insulin B_(ox)上的切点叠加在一起进行分析表明,P.sp.SM9913的三种胞外蛋白酶MCP-01、MCP-02和MCP-03可以将30个残基的insulin B_(ox)切成2～7个残基长度不等的肽段或多种游离氨基酸。
以BSA为底物,首先用SDS-PAGE技术分离三种酶酶解所产生的大分子肽段,优化了酶解条件,在此基础上,选取高峰度的大分子量肽段,通过使用Edman降解法测定肽段N端序列,分析了三种酶在BSA上的部分酶切位点。结果表明,三种酶在有限降解大分子蛋白BSA时,具有不同的切点。三种酶的切点叠加在一起共有6个,为Thr83+Tyr84,Leu218+Ser219,Glu226+Phe227,Val234+Thr235,Leu422+Val423,Ala489+Leu490,这些切点均位于BSA蛋白质外表面的α-螺旋或者loop上。
上述结果表明,深海适冷菌P.sp.SM9913所产的三种胞外蛋白酶MCP-01、MCP-02和MCP-03降解蛋白质时既具有相同的酶切位点,又具有不同的酶切位点,表明它们在降解胞外蛋白或多肽底物中可能有协同性,这为进一步研究P.sp.SM9913在深海沉积物中对有机氮的降解机制和生态适应机制奠定了基础。
PartⅠ.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-NH_2 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.
PartⅡ,Enzymatic Hydrolysis of Organic Nitrogen by Deep-Sea Psychrotrophic Bacterium Pseudoalteromonas sp.SM9913
Studies 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.
适冷酶由生存于永久低温环境(如深海、极地和高山地区等)的生物所生产,兼有低温下高的催化效率和高温下的热不稳定性双重特点。虽然对适冷酶的适冷机制有着不同的解释,但目前最流行的解释是,适冷酶在低温下高的催化效率来自于高的酶蛋白柔性,而高的蛋白柔性则源于适冷酶的热不稳定性。但是,这一解释仍有局限性,因为,目前有报道发现适冷酶可以同时具有高的活性和高的热稳定性。在实验室内通过定向进化对酶分子的改造实验也证实,有些情况下,少数氨基酸残基的突变也可以产生同时具备高催化活性和高热稳定性的酶。因此,适冷酶的适冷机制以及适冷的结构基础有待进一步研究。
嗜热菌蛋白酶家族(thermolysin family,也称M4 family)中的金属蛋白酶多来源于细菌,其中的典型代表有来自嗜热菌Bacillus thermoproteolyticus的嗜热thermolysin和来自Pseudomonas aeruginosa PAO1的中温pseudolysin,以及来自南极细菌643的(适冷)vibriolysin(VAB)。VAB是目前所知的M4家族中唯一来源于永久低温环境的蛋白酶,但是目前关于VAB的信息仅有NCBI公共数据库中的核酸序列(AF272770)和预测的氨基酸序列(AAF78076),对于其来源菌643的生理特征以及VAB的生化性质并没有任何报道。因此,发现Thermolysin家族的(新的)适冷酶并对其性质进行系统研究,对于提高对蛋白质结构—功能之间的关系的认识,以及对蛋白酶温度(低温)适应进化规律的认识具有重要意义。
适冷菌Pseudoalteromonas sp.SM9913是分离自深海沉积物的一株γ-proteobacterium。P.sp.SM9913能够分泌大量蛋白酶,MCP-02是P.sp.SM9913分泌的一种金属蛋白酶。MCP-02的N端序列与VAB具有很高的同源性(identity为95%),北极海冰细菌P.sp.SM495所分泌的胞外酶E495的N端序列也与VAB具有很高的同源性(identity为93%),表明这两种酶都很可能属于Thermolysin家族。本论文选用金属蛋白酶MCP-02和E495为研究对象,对它们进行了基因克隆和表达、性质研究和序列与结构分析,并以中温的pseudolysin为对照,研究了MCP-02和E495的适冷机制。取得了如下研究结果:
(一)深海来源和北极海冰来源的Thermolysin家族的新型蛋白酶的基因克隆与表达及其生化和酶学性质研究。
(1)深海适冷菌P.sp.SM9913所产胞外金属蛋白酶MCP-02的研究。
本论文通过使用巢式PCR和TAIL PCR等技术手段,克隆了深海适冷菌P.sp.SM9913所产胞外金属蛋白酶MCP-02的全基因序列。mcp-02基因全长2184 bp,编码含有727个氨基酸残基的蛋白酶前体。基因序列已提交GenBank,序列号为EF029091(核酸序列),ABL06977(氨基酸序列)。序列分析表明,MCP-02为Zn金属蛋白酶,属于Thermolysin家族,Vibriolysin亚家族(Vibriolysin subfamily,M04.003)。
将MCP-02在E.coli中进行异源表达,使用硫酸铵沉淀、阴离子交换层析等技术,从胞外分离纯化得到了电泳纯的重组MCP-02酶。N端测序表明重组酶和野生酶具有相同的N端序列,表明获得的mcp-02基因序列是正确的。MALDI-TOF质谱分析表明,成熟的重组酶MCP-02分子量为33929 Da。综合酶分子量和N端序列,推断MCP-02成熟酶长315氨基酸残基(A205-D519)。以酪蛋白为底物,MCP-02最适催化pH为8.0,最适催化温度为57℃。55℃半衰期t_(1/2)为90 min,60℃半衰期t_(1/2)为23 min。对二肽合成底物的降解(pH 8.0)表明,MCP-02偏好降解P1'位是大的疏水残基的二肽底物,顺序为Phe＞Leu＞Val。对MCP-02的研究是对深海来源的Thermolysin家族适冷酶生化和酶学性质的首次研究。
(2)北极海冰细菌P.sp.SM495所产胞外金属蛋白酶EA95的研究。
本论文通过使用巢式PCR和TAIL PCR等技术手段,克隆了北极海冰细菌P.sp.SM495所产胞外金属蛋白酶E495的全基因序列。蛋白酶E495的基因全长2193 bp,编码含有730个氨基酸残基的蛋白酶前体。基因序列已提交GenBank,序列号为FJ211191(核酸序列),ACI28452(氨基酸序列)。序列分析表明,E495为Zn金属蛋白酶,属于Thermolysin家族的Vibriolysin亚家族。
将E495在E.coli中进行异源表达,使用超声波破碎、硫酸铵沉淀、阴离子交换层析、Native-PAGE等技术,从周质空间分离纯化得到了电泳纯的重组E495酶。N端测序表明重组酶和野生酶具有相同的N端序列,表明获得的E495基因序列是正确的。MALDI-TOF质谱分析表明,成熟E495分子量为33376 Da。综合酶分子量和N端序列,推测E495成熟酶含有310个氨基酸残基(A207-T516)。E495的酶学性质与MCP-02相似。以酪蛋白为底物,E495最适催化pH为7.5,最适催化温度为57℃。55℃半衰期t_(1/2)为82 min,60℃半衰期t_(1/2)为20 min。对二肽合成底物的降解(pH 8.0)表明,E495偏好降解P1'位是大的疏水残基的二肽底物,顺序为Phe＞Leu＞Val。对E495性质的研究是对极地海冰来源的Thermolysin家族适冷酶生化和酶学性质的首次研究。
(二)对Thermolysin家族适冷酶的适冷机制的研究,揭示适冷酶“活性—柔性—稳定性”三者之间的关系。
以Thermolysin家族的适冷酶——深海MCP-02和北极海冰E495为研究材料,以MCP-02和E495的中温同源酶——来自陆地细菌Pseudomonas aeruginosaPAO1的中温pseudolysin,作为中温对照,研究Thermolysin家族适冷酶的适冷进化机制,以揭示适冷酶“活性—柔性—稳定性”三者之间的关系。
首先从Pseudomonas aeruginosa PAO1的基因组中克隆了pseudolysin的全基因序列,并将其在E.coli中进行异源表达,分离纯化并获得了电泳纯度的重组pseudolysin,详细研究了pseudolysin的性质。在此基础上,本论文系统比较了pseudolysin、MCP-02和E495的催化效率、热稳定性和柔性。
结果表明,以合成二肽FA-Gly-Leu-NH_2为底物,在Tris-HCl缓冲液(pH 8.0)中,在低温和中温(10-40℃)条件下,三个蛋白酶的催化效率顺序(k_(cat)/K_m)为pseudolysin＜MCP-02＜E495,其中在25℃下,pseudolysin的k_(cat)/K_m为0.64±0.01 s~(-1)mM~(-1),MCP-02的k_(cat)/K_m为1.32±0.01 s~(-1) mM~(-1),E495的k_(cat)/K_m为2.55±0.05 s~(-1)mM~(-1),三个酶的催化效率接近1:2:4。可见在低温和中温条件下,三个酶的催化效率随着生存环境温度的降低而升高,表明三个酶因生存环境温度不同而表现不同程度的冷适应性。
三个酶催化酪蛋白水解的最适酶活温度分别为62℃(pseudolysin),57℃(MCP-02),和57℃(E495)。55℃的半衰期t_(1/2)分别为530 min(pseudolysin),90 min(MCP-02),和82 min(E495);60℃的半衰期t_(1/2)分别为160 min(pseudolysin),23 min(MCP-02)和20 min(E495)。三个酶在55℃下的失活速率常数k_(inact)分别为2.2×10~(-5) s~(-1)(pseudolysin),1.3×10~(-4) s~(-1)(MCP-02)和1.4×10~(-4) s~(-1)(E495);在60℃的失活速率常数k_(inact)分别为7.3×10~(-5) s~(-1)(pseudolysin),5.1×10~(-4) s~(-1)(MCP-02)和5.8×10~(-4) s~(-1)(E495)。使用荧光法监测酶的热变性过程,获得了三个酶的表观变构温度(T_m)分别为75℃(pseudolysin),64℃(MCP-02)和63℃(E495);使用圆二色谱监测α螺旋的解螺旋过程,获得了三个酶的表观变构温度T_m分别为72℃(pseudolysin),65℃(MCP-02)和65℃(E495)。分析最适酶活温度、半衰期(和失活速率常数)和表观变构温度的数据都得到一致的结论,三个酶的热稳定性顺序为pseudolysin＞MCP-02≈E495。MCP-02和E495的热稳定性相当,都比pseudolysin降低5～10℃,但是,总体上讲MCP-02和E495的热稳定性仍然很高,如最适酶活温度都约为57℃,仅从热稳定性上判断两酶都类似于中温酶。
以丙烯酰胺为淬灭剂,研究了三个酶在低温(15℃)和中温(40℃)下的动态荧光淬灭,计算了各酶在不同温度下的Stern-Volmer淬灭常数K_(SV),和不同温度下的K_(SV)的差值,△K_(SV)(40-15℃),并用△K_(SV)(40-15℃)来表征酶的柔性。三个酶的△K_(SV)(40-15℃)分别为2.6±0.1 M~(-1)(pseudolysin),5.3±0.1 M~(-1)(MCP-02)和5.7±0.1 M~(-1)(E495),表明三个酶存在以下柔性顺序,pseudolysin＜MCP-02＜E495。
上述结果中三个酶的催化效率和柔性具有相同的顺序,都随生存环境温度的降低而升高,这与已报道的“典型”适冷酶相似。但是,热稳定却并不随生存环境温度的降低而降低,适冷酶E495的催化效率和柔性都高于MCP-02,但是其热稳定性却与MCP-02几乎相同,这表明,至少E495的高的柔性的获得并不是通过降低本身的热稳定性来实现的。这个结论与目前的流行观点“适冷酶在低温下高的催化效率来自于高的柔性,而高的柔性则来源于低的热稳定性”不同。为了进一步研究MCP-02和E495的适冷机制,与解彬彬博士一起对三个酶进行了分子动力学模拟以及模拟过程中的氢键、盐键数目的统计分析(详细结果见解彬彬博士论文)。
根据上述实验结果以及分子动力学模拟结果,我们提出了Thermolysin家族适冷酶的适冷进化机制模型。Thermolysin家族的适冷酶通过提高整体柔性来提高其低温下的催化能力,稳定性的降低并非柔性提高的前提。这是对Thermolysin家族适冷酶适冷机制的首次报道。
二、深海适冷菌P.sp.SM9913对有机氮的降解
研究表明,深海中一些γ-proteobacteria通过分泌大量的蛋白酶将环境中的蛋白降解为短肽,并最终运输到胞内为菌体提供能量和碳源。深海适冷菌Pseudoalteromonas sp.SM9913属于γ-proteobacterium。P.sp.SM9913能够分泌大量蛋白酶,目前的研究表明,这些蛋白酶中至少包括两种不同的丝氨酸蛋白酶,MCP-01和MCP-03,和一种金属蛋白酶MCP-02。这些胞外蛋白酶如何协同作用对胞外蛋白进行充分降解目前还不清楚。而这个问题的阐明不仅有助于阐明深海适冷菌P.sp.SM9913对深海极端环境的适应机制,而且对阐明深海沉积物中有机氮的降解和氮循环机制具有重要意义。
本论文分别以多肽——氧化型的胰岛素B链(insulin B_(ox),含30个氨基酸残基)和大分子蛋白——牛血清白蛋白(BSA,含583个氨基酸残基)为底物,研究了P.sp.SM9913的三种胞外蛋白酶MCP-01、MCP-02和MCP-03对它们的酶切位点,并分析了它们潜在的协同作用,为阐明深海适冷菌P.sp.SM9913对有机氮的酶解机制奠定基础。
以insulin B_(ox)为底物,首先使用HPLC分离肽段的方法优化了各酶酶解条件,然后将降解后的混合肽段使用MALDI-TOF质谱分析获得产物中的各肽段的分子量信息,通过分析分子量找到丰度高的肽段所对应的序列,从而获得酶切位点。结果表明,以insulin B_(ox)为底物,鉴定到了MCP-01的5个切点,为Gln+His、Leu+Tyr+Leu、Cya+Gly和Phe+Phe,这些切点均与已报道的subtilisin Carlsberg的相应切点一致;鉴定到了MCP-02的7个切点,为Gin+His、Leu+Cya、Ala+Leu+Tyr、Cya+Gly、Arg+Gly和Phe+Phe,其中5个切点与已报道的thermolysin的切点不同;鉴定到了MCP-03的5个切点,为Leu+Cya、Glu+Ala、Leu+Tyr、Gly+Phe+Phe,其中1个切点与subtilisn Carlsberg和Thermitase不同。三种酶在insulin B_(ox)上既有相同的切点,也有不同的切点。将三种酶在insulin B_(ox)上的切点叠加在一起进行分析表明,P.sp.SM9913的三种胞外蛋白酶MCP-01、MCP-02和MCP-03可以将30个残基的insulin B_(ox)切成2～7个残基长度不等的肽段或多种游离氨基酸。
以BSA为底物,首先用SDS-PAGE技术分离三种酶酶解所产生的大分子肽段,优化了酶解条件,在此基础上,选取高峰度的大分子量肽段,通过使用Edman降解法测定肽段N端序列,分析了三种酶在BSA上的部分酶切位点。结果表明,三种酶在有限降解大分子蛋白BSA时,具有不同的切点。三种酶的切点叠加在一起共有6个,为Thr83+Tyr84,Leu218+Ser219,Glu226+Phe227,Val234+Thr235,Leu422+Val423,Ala489+Leu490,这些切点均位于BSA蛋白质外表面的α-螺旋或者loop上。
上述结果表明,深海适冷菌P.sp.SM9913所产的三种胞外蛋白酶MCP-01、MCP-02和MCP-03降解蛋白质时既具有相同的酶切位点,又具有不同的酶切位点,表明它们在降解胞外蛋白或多肽底物中可能有协同性,这为进一步研究P.sp.SM9913在深海沉积物中对有机氮的降解机制和生态适应机制奠定了基础。
PartⅠ.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-NH_2 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.
PartⅡ,Enzymatic Hydrolysis of Organic Nitrogen by Deep-Sea Psychrotrophic Bacterium Pseudoalteromonas sp.SM9913
Studies 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.
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