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首页> 外文期刊>Biochemistry >Identification of a Novel Pyridoxal 5'-Phosphate Binding Site in Adenosylcobalamin-Dependent Lysine 5,6-Aminomutase from Porphyromonas gingivalis.
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Identification of a Novel Pyridoxal 5'-Phosphate Binding Site in Adenosylcobalamin-Dependent Lysine 5,6-Aminomutase from Porphyromonas gingivalis.

机译：在来自齿龈卟啉单胞菌的腺苷钴胺依赖性赖氨酸5，6-氨基变位酶中新型吡咯醛5'-磷酸结合位点的鉴定。

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摘要

Lysine 5,6-aminomutase (5,6-LAM) catalyzes the interconversion of D-lysine with 2,5-diaminohexanoate and of L-beta-lysine with 3,5-diaminohexanoate. The coenzymes for 5,6-LAM are adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP). In the proposed chemical mechanism, AdoCbl initiates the formation of substrate radicals, and PLP facilitates the radical rearrangement by forming an external aldimine linkage with the epsilon-amino group of a substrate, either D-lysine or L-beta-lysine. In the resting enzyme, an internal aldimine between PLP and an essential lysine in the active site facilitates productive PLP binding and catalysis. We present here biochemical, biophysical, and site-directed mutagenesis experiments, which document the existence of an essential lysine residue in the active site of 5,6-LAM from Porphyromonas gingivalis. Reduction of 5,6-LAM with NaBH(4) rapidly inactivates the enzyme and shifts the electronic absorption band from 420 to 325 nm. This is characteristic of the reduction of an aldimine linkage between the carbonyl group of PLP and the epsilon-amino group of a lysine residue. The reduced peptide was identified by Q-TOF/MS and further confirmed by Q-TOF/MS/MS sequencing. We show that lysine 144 in the small subunit of 5,6-LAM is the essential lysine residue. Lysine 144(beta) is separated by only 11 amino acids from histidine 133(beta), which forms a part of the base-off motif is conserved in 5,6-LAM from Clostridium sticklandii and P. gingivalis, and it is distinct from all known PLP-binding motifs. Mutation of lysine 144(beta) to glutamine led to K144Q(beta)-5,6-LAM, which displayed no enzymatic activity and no absorption band corresponding to an internal PLP-aldamine. In summary, we introduce a novel PLP-binding motif, the first to be discovered in an AdoCbl-dependent enzyme.
机译：赖氨酸5,6-氨基变位酶（5,6-LAM）催化D-赖氨酸与2,5-二氨基己酸酯和L-β-赖氨酸与3,5-二氨基己酸酯的相互转化。 5,6-LAM的辅酶是腺苷钴胺素（AdoCbl）和吡ido醛5'-磷酸（PLP）。在提出的化学机理中，AdoCbl启动底物自由基的形成，而PLP通过与底物的ε-氨基或D-赖氨酸或L-β-赖氨酸形成外部醛亚胺键来促进自由基重排。在静止酶中，PLP和活性位点中的赖氨酸之间的内部醛亚胺促进了有效的PLP结合和催化作用。我们在这里介绍了生化，生物物理和定点诱变实验，该实验记录了牙龈卟啉单胞菌的5,6-LAM活性位点中存在必需的赖氨酸残基。用NaBH（4）还原5,6-LAM会迅速使酶失活，并将电子吸收带从420 nm移至325 nm。这是PLP的羰基和赖氨酸残基的ε-氨基之间的醛亚胺键减少的特征。通过Q-TOF / MS鉴定还原的肽，并通过Q-TOF / MS / MS测序进一步证实。我们显示5,6-LAM小亚基中的赖氨酸144是必需的赖氨酸残基。赖氨酸144β与组氨酸133β仅相隔11个氨基酸，形成了碱基关闭基序的一部分，在粘胶梭状芽胞杆菌和牙龈假单胞菌的5,6-LAM中是保守的，与所有已知的PLP结合基序。赖氨酸144β突变为谷氨酰胺导致K144Qβ-5,6-LAM，它没有酶活性，也没有对应于内部PLP-醛胺的吸收带。总之，我们介绍了一种新的PLP结合基序，这是第一个在AdoCbl依赖性酶中发现的基序。

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