Chloroplast NADP-malate dehydrogenase: structural basis of light-dependent regulation of activity by thiol oxidation and reduction
Chloroplast NADP-malate dehydrogenase: structural basis of light-dependent regulation of activity by thiol oxidation and reduction
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DOI：
10.1016/s0969-2126(99)80058-6
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发表时间：
1999-04-15
期刊：
影响因子：
5.7
通讯作者：
Ollis, DL
中科院分区：
文献类型：
--
作者：
Carr, PD;Verger, D;Ollis, DL
背景：NADP依赖的苹果酸脱氢酶（EC 1.1.1.82）是一种光激活的叶绿体酶，在光合作用的C-4途径中起作用。光调节被认为是通过硫氧还蛋白催化的胱氨酸残基的还原和再氧化在体内介导的。TI的酶的可逆激活和失活的速率强烈影响的辅酶底物，似乎最终确定在vivo.Results的激活的稳定状态的程度：X-射线结构的非活性，氧化酶被确定在2.8埃分辨率。核心结构与NAD依赖性苹果酸脱氢酶同源。存在两个表面暴露和硫氧还蛋白可接近的二硫键，一个在N端延伸中，另一个在C端延伸中。无活性的氧化酶的C-末端肽受其二硫键限制，折叠到NADP（+）上的活性位点，与催化性His 225形成氢键，并阻碍C-4酸底物的进入。两个环侧翼的活性位点，被称为Arg（2）和色氨酸环，其中包含C-4酸底物结合残基被阻止关闭的C-末端extendation.Conclusions：结构解释的C-末端延伸在抑制活性的作用。负C端与NADP（+）中带正电荷的烟酰胺的相互作用比与NADPH的相互作用更强，这解释了为什么该酶的辅酶结合亲和力与所有其他同源α-羟基酸脱氢酶的辅酶结合亲和力如此显著不同。NADP（+）也可以减缓C末端在还原时的解离，提供了NADP（+）而不是NADPH抑制活化的机制。
Background: NADP-dependent malate dehydrogenase (EC 1.1.1.82) is a light-activated chloroplast enzyme that functions in the C-4 pathway of photosynthesis. The light regulation is believed to be mediated in vivo by thioredoxin-catalyzed reduction and re-oxidation of cystine residues. TI The rates of reversible activation and inactivation of the enzyme are strongly influenced by the coenzyme substrates that seem to ultimately determine the steady-state extent of activation in vivo.Results: The X-ray structure of the inactive, oxidized enzyme was determined at 2.8 Angstrom resolution. The core structure is homologous to NAD-dependent malate dehydrogenases. Two surface-exposed and thioredoxin-accessible disulfide bonds are present, one in the N-terminal extension and the other in the C-terminal extension. The C-terminal peptide of the inactive, oxidized enzyme is constrained by its disulfide bond to fold into the active site over NADP(+), hydrogen bonding to the catalytic His225 as well as obstructing access of the C-4 acid substrate. Two loops flanking the active site, termed the Arg(2) and Trp loops, that contain the C-4 acid substrate binding residues are prevented from closing by the C-terminal extension.Conclusions: The structure explains the role of the C-terminal extension in inhibiting activity. The negative C terminus will interact more strongly with the positively charged nicotinamide of NADP(+) than NADPH, explaining why the coenzyme-binding affinities of the enzyme differ so markedly from those of all other homologous alpha-hydroxy acid dehydrogenases. NADP(+) may also slow dissociation of the C terminus upon reduction, providing a mechanism for the inhibition of activation by NADP(+) but not NADPH.