Mutagenesis of Glycine 179 modulates both catalytic efficiency and reduced pyridine nucleotide specificity in cytochrome b5 reductase.
Mutagenesis of Glycine 179 modulates both catalytic efficiency and reduced pyridine nucleotide specificity in cytochrome b5 reductase.
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DOI：
10.1021/bi051165t
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发表时间：
2005-10
期刊：
影响因子：
2.9
通讯作者：
Glenn W. Roma;Louis J. Crowley;C. Davis;Michael J. Barber
中科院分区：
文献类型：
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作者：
Glenn W. Roma;Louis J. Crowley;C. Davis;Michael J. Barber
细胞色素 b5 还原酶 (cb5r) 是黄素蛋白转氢酶铁氧还蛋白：NADP+ 还原酶家族的成员，可催化细胞色素 b5 的 NADH 依赖性还原。在该家族中，保守的“GxGxxP”序列基序与还原的吡啶核苷酸的结合有关。然而，甘氨酸 179（cb5r 一级结构中的保守残基）位于该六残基“180GxGxxP185”基序之前，该基序已被鉴定为结合 NADH 的腺苷部分。为了研究 G179 在 NADH 复合物形成和 NAD(P)H 特异性中的作用，产生了一系列大鼠 cb5r 变体，对应于 G179A、G179P、G179T 和 G179V，在大肠杆菌中重组表达并纯化至同质。发现每个突变蛋白以 1:1 辅因子/蛋白质化学计量掺入 FAD，并表现出与野生型 cb5r 相同的吸收和 CD 光谱，表明正确的蛋白质折叠和相似的黄素环境，而 FAD/FADH2 对 (n = 2) 的氧化还原电位也与野生型蛋白质相当 (E(o)' = -272 mV)。所有四种突变体均表现出NADH：铁氰化物还原酶活性降低，kcat 按WT > G179A > G179P > G179T > G179V 的顺序降低，其中G179V 变体仅保留野生型活性的1.5%。对NADH的亲和力也按WT>G179A>G179P>G179T>G179V的顺序降低，G179V的Km(NADH)比野生型大180倍。 Ks(H4NAD) 和 Ks(NAD+) 值均证实 G179 突变体的 NADH 和 NAD+ 结合亲和力均受到损害。对各种突变体的 NADH/NADPH 特异性常数的测定表明，G179 也参与吡啶核苷酸选择性，其中 G179V 变体对 NADPH 的偏好比野生型 cb5r 大约高 8000 倍。这些结果表明，虽然 G179 对于黄素掺入或维持 cb5r 中适当的黄素环境并不重要，但 G179 对于有效的 NADH/NADPH 选择性和维持富含脯氨酸的“CGpppM”基序中保守半胱氨酸的正确方向和位置是必需的，这对于最佳 NADH 结合和有效的氢化物转移至关重要。
Cytochrome b5 reductase (cb5r), a member of the ferredoxin:NADP+ reductase family of flavoprotein transhydrogenases, catalyzes the NADH-dependent reduction of cytochrome b5. Within this family, a conserved "GxGxxP" sequence motif has been implicated in binding reduced pyridine nucleotides. However, Glycine 179, a conserved residue in cb5r primary structures, precedes this six-residue "180GxGxxP185" motif that has been identified as binding the adenosine moiety of NADH. To investigate the role of G179 in NADH complex formation and NAD(P)H specificity, a series of rat cb5r variants were generated, corresponding to G179A, G179P, G179T, and G179V, recombinantly expressed in Escherichia coli and purified to homogeneity. Each mutant protein was found to incorporate FAD in a 1:1 cofactor/protein stoichiometry and exhibited absorption and CD spectra that were identical to those of wild-type cb5r, indicating both correct protein folding and similar flavin environments, while oxidation-reduction potentials for the FAD/FADH2 couple (n = 2) were also comparable to the wild-type protein (E(o)' = -272 mV). All four mutants showed decreased NADH:ferricyanide reductase activities, with kcat decreasing in the order WT > G179A > G179P > G179T > G179V, with the G179V variant retaining only 1.5% of the wild-type activity. The affinity for NADH also decreased in the order WT > G179A > G179P > G179T > G179V, with the Km(NADH) for G179V 180-fold greater than that of the wild type. Both Ks(H4NAD) and Ks(NAD+) values confirmed that the G179 mutants had both compromised NADH- and NAD+-binding affinities. Determination of the NADH/NADPH specificity constant for the various mutants indicated that G179 also participated in pyridine nucleotide selectivity, with the G179V variant preferring NADPH approximately 8000 times more than wild-type cb5r. These results demonstrated that, while G179 was not critical for either flavin incorporation or maintenance of the appropriate flavin environment in cb5r, G179 was required for both effective NADH/NADPH selectivity and to maintain the correct orientation and position of the conserved cysteine in the proline-rich "CGpppM" motif that is critical for optimum NADH binding and efficient hydride transfer.