Modulating the function of human serine racemase and human serine dehydratase by protein engineering.-猫眼课题宝

通过蛋白质工程调节人丝氨酸消旋酶和人丝氨酸脱水酶的功能。

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发表时间：

2012

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Protein engineering, design & selection : PEDS

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作者：

Cy Wang;S. Ku;Cheng;A. H. Wang

D-丝氨酸是N-甲基-D-天冬氨酸的联合激动剂，N-甲基-D-天冬氨酸是一种谷氨酸受体，是大脑中一种主要的兴奋性神经递质受体。人丝氨酸消旋酶(HSR)和丝氨酸脱水酶(HSDH)是合成和降解D-丝氨酸的两种重要的5‘-磷酸吡哆醛依赖酶。HSR和hSDH具有显著的序列同源性(28%的同源性)，并且在结构折叠方面相似(均方根偏差，1.12？)。HSR和hSDH的序列比对和结构比较表明，HSR中的S84和hSDH中的A65对各自的酶活性起着重要的作用。我们推测，通过引入S84A HSR和A65S hSDH突变体来交换这两个氨基酸可能会导致它们的蛋白质功能发生改变。为了了解关键残基的调控机制，构建了hsr中的突变体S84A和hSDH中的突变体A65S来监测其活性的变化。对A65S hSDH突变体的结构进行了测定，确定了这一关键氨基酸的作用。我们的研究表明，S84A HSR突变体的行为类似于hSDH，而A65S hSDH突变体获得了以D-丝氨酸为底物的额外功能。

D-Serine is a co-agonist of N-methyl D-aspartate, a glutamate receptor, which is a major excitatory neurotransmitter receptor in the brain. Human serine racemase (hSR) and serine dehydratase (hSDH) are two important pyridoxal-5'-phosphate-dependent enzymes that synthesize and degrade D-serine, respectively. hSR and hSDH have significant sequence homology (28% identity) and are similar in their structural folds (root-mean-square deviation, 1.12 Å). Sequence alignment and structural comparison between hSR and hSDH reveal that S84 in hSR and A65 in hSDH play important roles in their respective enzyme activities. We surmise that exchange of these two amino acids by introducing S84A hSR and A65S hSDH mutants may result in switching their protein functions. To understand the modulating mechanism of the key residues, mutants S84A in hSR and A65S in hSDH were constructed to monitor the change of activities. The structure of A65S hSDH mutant was determined at 1.3 Å resolution (PDB 4H27), elucidating the role of this critical amino acid. Our study demonstrated S84A hSR mutant behaved like hSDH, whereas A65S hSDH mutant acquired an additional function of using D-serine as a substrate.