Preparation and properties of recombinant corynebacterial sarcosine oxidase: evidence for posttranslational modification during turnover with sarcosine.
Preparation and properties of recombinant corynebacterial sarcosine oxidase: evidence for posttranslational modification during turnover with sarcosine.
复制标题
重组棒状杆菌肌氨酸氧化酶的制备和特性：肌氨酸周转过程中翻译后修饰的证据。
DOI：
10.1021/bi00092a024
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Jorns,MS
中科院分区：
文献类型：
--
作者：
Chlumsky,LJ;Zhang,L;Ramsey,AJ;Jorns,MS
1993年7月20日接收的修订版Mandalpt ®摘要：编码来自棒状杆菌P-1的肌氨酸氧化酶的四个亚基的基因被分离并在单一步骤中过表达，通过使用指示板筛选基因组文库中在肌氨酸依赖性反应中产生过氧化氢的菌落。通过将预先用Sau 3Al部分消化的大小分级的基因组DNA插入pBluescript IISK（+）中构建基因组文库。至少1.0 kb，但小于4.0 kb，可以从原始玉米细菌插入物（7.3 kb）的3 '端缺失而不影响肌氨酸氧化酶表达，与估计的5.0-kb操纵子大小一致。重组肌氨酸氧化酶被分离为含有等摩尔量的共价和非共价黄素的异源四聚体，与从棒状杆菌P-1分离的酶所观察到的相同。尽管其类似的黄素含量，但重组酶具有与来自棒杆菌属P-1的酶显著不同的光谱特性（括号中显示的值）[ε 450= 9.7（12.7）mM-1 cm-1; λ/λ q= 1.0（0.83）; λ 280/λ 450= 16.9（12.2）]。这种差异是由于重组酶中约一半的共价黄素与半胱氨酸残基形成可逆的共价4 α-加合物（λ max = 383 nm; λ 383= 7.3 mM-1 cm-1）。通过用过氧化氢氧化半胱氨酸残基或通过在加入过量二硫苏糖醇后完全可逆的反应中用甲硫基磺酸甲酯烷基化，平衡向有利于加合物解离的方向移动。半胱氨酸残基在有氧代谢过程中也被肌氨酸氧化。半胱氨酸残基与在周转期间形成的过氧化氢（或前体）的反应部分地与过氧化氢释放到溶液中竞争，如通过过氧化氢酶对该反应的影响所判断的。虽然重组酶和棒状杆菌P-1的酶观察到相同的比活性，但重组酶在NADH过氧化物酶偶联测定中表现出明显的滞后。通过与过氧化氢或甲烷硫代磺酸甲酯反应预先破坏4 α-硫醇盐加合物来消除滞后。结果表明，4a-硫醇盐加合物是肌氨酸氧化酶的一种非活性形式，可以通过与肌氨酸反应来激活，这似乎是与营业额相关的翻译后修饰的第一个例子。当在棒状杆菌属P-1中产生肌氨酸氧化酶时，体内发生完全活化，其中酶合成由以肌氨酸作为碳和能量来源的生物体的生长诱导。当棒状杆菌P-1以肌氨酸作为碳源和能量源生长时，肌氨酸氧化酶作为诱导酶产生（Kvalnes-Krick & Jorns，1986）。该酶催化肌氨酸的氧化去甲基化以产生甘氨酸、甲醛和过氧化氢（方程式1）。
Revised Manuscript Received July 20, 1993® abstract: The genes encoding the four subunits of sarcosine oxidase from Corynebacterium sp. P-1 were isolated and overexpressed in a single step by using indicator plates to screen a genomic library for colonies that generated hydrogen peroxide in a sarcosine-dependent reaction. The genomic library was constructed by inserting size-fractionated genomic DNA, previously subjected to partial digestion by Sau3Al, into pBluescript IISK (+). At least 1.0 kb, but less than 4.0 kb, can be deleted from the 3'end of the original cornyebacterial insert (7.3 kb) without affecting sarcosine oxidase expression, consistent with the estimated 5.0-kb operon size. Recombinant sarcosine oxidase is isolated as a heterotetramer containing equimolar amounts of covalent and noncovalent flavin, identical to that observed for enzyme isolated from Corynebacterium sp. P-1. Despite its similar flavin content, recombinant enzyme exhibitssignificantly different spectral properties than enzyme from Corynebacterium sp. P-1 (values shown in parentheses)[£ 450= 9.7 (12.7) mM" 1 cm-1; A^/A^ q= 1.0 (0.83);^ 280/^ 450= 16.9 (12.2)]. This difference is dueto the fact that about half of the covalent flavin in recombinant enzyme forms a reversible covalent 4a-adduct with a cysteine residue (Xmax= 383 nm;€ 383= 7.3 mM-1 cm-1). The equilibrium is shifted in favor of adduct dissociation by oxidizing the cysteine residue with hydrogen peroxide or by alkylation with methyl methanethiosulfonate in a reaction that is fully reversible upon addition of excess dithiothreitol. The cysteine residue is also oxidized during aerobic turnover with sarcosine. Reaction of the cysteine residue with hydrogen peroxide (or a precursor) formed during turnover partially competes with the release of hydrogen peroxide into solution, as judged by the effect of catalase on this reaction. Although the same specific activity is observed for recombinant enzyme and enzyme from Corynebacterium sp. P-1, the recombinant enzyme exhibits a pronounced lag in an NADH peroxidase-coupled assay. The lag is eliminated by prior disruption of the 4a-thiolate adduct via reaction with hydrogen peroxide or methyl methane-thiosulfonate. The resultsshow that the 4a-thiolate adduct is an inactive form of sarcosine oxidase that can be activated by reaction with sarcosine in what appears to be the first example of a posttranslational modification associated with turnover. Complete activation occurs in vivo when sarcosine oxidase is produced in Corynebacterium sp. P-1, where enzyme synthesis is induced by growth of the organism with sarcosine as the source of carbon and energy. The opportunity for in vivo activation is diminished when the recombinant enzyme is produced in Escherichia coli under the control of a vector-encoded lac promoter.Sarcosine oxidase is produced as an inducible enzyme when Corynebacterium sp. P-1 is grown with sarcosine as the source of carbon and energy (Kvalnes-Krick & Jorns, 1986). The enzyme catalyzes the oxidative demethylation of sarcosine to yield glycine, formaldehyde, and hydrogen peroxide (eq 1).