Neeli Rajasekhar, Sabri Muna, McLean Kirsty J, Dunford Adrian J, Scrutton Nigel S, Leys David, Munro Andrew W
Manchester Interdisciplinary Biocentre, The University of Manchester, Faculty of Life Sciences, 131 Princess Street, Manchester M1 7DN, UK.
Biochem J. 2008 May 1;411(3):563-70. doi: 10.1042/bj20071298.
Mtb (Mycobacterium tuberculosis) FprA (flavoprotein reductase A) is an NAD(P)H-dependent FAD-binding reductase that is structurally related to mammalian adrenodoxin reductase, and which supports the catalytic function of Mtb cytochrome P450s. Trp(359), proximal to the FAD, was investigated in light of its potential role in controlling coenzyme interactions, as observed for similarly located aromatic residues in diflavin reductases. Phylogenetic analysis indicated that a tryptophan residue corresponding to Trp(359) is conserved across FprA-type enzymes and in adrenodoxin reductases. W359A/H mutants of Mtb FprA were generated, expressed and the proteins characterized to define the role of Trp(359). W359A/H mutants exhibited perturbed UV-visible absorption/fluorescence properties. The FAD semiquinone formed in wild-type NADPH-reduced FprA was destabilized in the W359A/H mutants, which also had more positive FAD midpoint reduction potentials (-168/-181 mV respectively, versus the standard hydrogen electrode, compared with -230 mV for wild-type FprA). The W359A/H mutants had lower ferricyanide reductase k(cat) and NAD(P)H K(m) values, but this led to improvements in catalytic efficiency (k(cat)/K(m)) with NADH as reducing coenzyme (9.6/18.8 muM(-1).min(-1) respectively, compared with 5.7 muM(-1).min(-1) for wild-type FprA). Stopped-flow spectroscopy revealed NAD(P)H-dependent FAD reduction as rate-limiting in steady-state catalysis, and to be retarded in mutants (e.g. limiting rate constants for NADH-dependent FAD reduction were 25.4 s(-1) for wild-type FprA and 4.8 s(-1)/13.4 s(-1) for W359A/H mutants). Diminished mutant FAD content (particularly in W359H FprA) highlighted the importance of Trp(359) for flavin stability. The results demonstrate that the conserved Trp(359) is critical in regulating FprA FAD binding, thermodynamic properties, catalytic efficiency and coenzyme selectivity.
结核分枝杆菌（Mtb）黄素蛋白还原酶A（FprA）是一种依赖NAD(P)H的FAD结合还原酶，其结构与哺乳动物肾上腺皮质铁氧化还原蛋白还原酶相关，并支持Mtb细胞色素P450的催化功能。鉴于黄素腺嘌呤二核苷酸（FAD）附近的色氨酸（Trp）359在控制辅酶相互作用方面的潜在作用，对其进行了研究，这在双黄素还原酶中位于类似位置的芳香族残基中也有观察到。系统发育分析表明，对应于Trp359的色氨酸残基在FprA型酶和肾上腺皮质铁氧化还原蛋白还原酶中是保守的。构建、表达了Mtb FprA的W359A/H突变体，并对这些蛋白质进行了表征，以确定Trp359的作用。W359A/H突变体表现出受干扰的紫外可见吸收/荧光特性。在野生型NADPH还原的FprA中形成的FAD半醌在W359A/H突变体中不稳定，其FAD中点还原电位也更正（相对于标准氢电极，分别为-168/-181 mV，而野生型FprA为-230 mV）。W359A/H突变体的铁氰化物还原酶催化常数（k(cat)）和NAD(P)H米氏常数（K(m)）较低，但这导致以NADH作为还原辅酶时催化效率（k(cat)/K(m)）提高（分别为9.6/18.8 μM(-1)·min(-1)，而野生型FprA为5.7 μM(-1)·min(-1)）。停流光谱显示，在稳态催化中，依赖NAD(P)H的FAD还原是限速步骤，并且在突变体中受到阻碍（例如，野生型FprA依赖NADH的FAD还原的极限速率常数为25.4 s(-1)，W359A/H突变体为4.8 s(-1)/13.4 s(-1)）。突变体FAD含量减少（特别是在W359H FprA中）突出了Trp359对黄素稳定性的重要性。结果表明，保守的Trp359在调节FprA的FAD结合、热力学性质、催化效率和辅酶选择性方面至关重要。
