Structural and catalytic properties of the peroxygenase P450 enzyme CYP152K6 from Bacillus methanolicus.
Structural and catalytic properties of the peroxygenase P450 enzyme CYP152K6 from Bacillus methanolicus.
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DOI：
10.1016/j.jinorgbio.2018.08.002
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发表时间：
2018-11
影响因子：
3.9
通讯作者：
Munro AW
中科院分区：
文献类型：
--
作者：
Girvan HM;Poddar H;McLean KJ;Nelson DR;Hollywood KA;Levy CW;Leys D;Munro AW
细胞色素P450酶（P450或CYP）的CYP 152家族是细菌过氧合酶，其使用过氧化氢来驱动脂肪酸底物的羟基化和脱羧。我们从甲醇芽孢杆菌MGA 3中表达并纯化了一个新的CYP 152家族成员-CYP 152 K6。CYP 152 K6的特征在于使用光谱，分析和结构方法。与少动鞘氨醇单胞菌的过氧合酶对应物P450 SP α（CYP 152 B1）一样，CYP 152 K6不会对血红素谱产生显著的脂肪酸诱导干扰，但在结合十二烷酸时观察到轻微的Soret位移。然而，从E.大肠杆菌表达系统进行结晶，并确定其结构为1.3kDa，与十四烷酸结合。在用于晶体发生的条件下不存在脂质，因此CYP 152 K6必须通过掺入来自E. coli细胞。十二烷酸的周转研究揭示了几种产物，2-羟基十二烷酸为主要产物，3-羟基十二烷酸的量少得多。次级周转产物为十一碳-1-烯-1-醇、2-羟基十二碳-2-烯酸和2，3-二羟基十二烷酸。这是首次报道由过氧合酶P450产生的2，3-羟基化脂肪酸产物，其中二羟基化产物由CYP 152 K6催化的2-羟基十二烷酸的3-羟基化形成，而不是由3-羟基十二烷酸的2-羟基化形成。甲醇芽孢杆菌CYP 152 K6是一种新的细胞色素P450（P450或CYP 152）环加氧酶家族成员。其与十四烷酸结合的晶体结构表明，底物通过其羧酸盐与保守的精氨酸残基结合。过氧化氢取代血红素上的远端水，形成催化底物氧化的活性铁氧物种。对甲醇芽孢杆菌细胞色素P450（P450或CYP 152 K6）进行了表征。CYP 152 K6是一种过氧合酶，催化十二烷酸的有效氧化。解得了与十四烷酸结合的CYP 152 K6的1.3 μ m晶体结构。EPR揭示了三个低自旋CYP 152 K6物种，其种群在脂质结合上发生变化。CYP 152 K6产生2，3-二羟基十二烷酸作为一种新的过氧合酶产物。
The CYP152 family of cytochrome P450 enzymes (P450s or CYPs) are bacterial peroxygenases that use hydrogen peroxide to drive hydroxylation and decarboxylation of fatty acid substrates. We have expressed and purified a novel CYP152 family member – CYP152K6 from the methylotroph Bacillus methanolicus MGA3. CYP152K6 was characterized using spectroscopic, analytical and structural methods. CYP152K6, like its peroxygenase counterpart P450SPα (CYP152B1) from Sphingomonas paucimobilis, does not undergo significant fatty acid-induced perturbation to the heme spectrum, with the exception of a minor Soret shift observed on binding dodecanoic acid. However, CYP152K6 purified from an E. coli expression system was crystallized and its structure was determined to 1.3 Å with tetradecanoic acid bound. No lipids were present in conditions used for crystallogenesis, and thus CYP152K6 must form a complex by incorporating the fatty acid from E. coli cells. Turnover studies with dodecanoic acid revealed several products, with 2-hydroxydodecanoic acid as the major product and much smaller quantities of 3-hydroxydodecanoic acid. Secondary turnover products were undec-1-en-1-ol, 2-hydroxydodec-2-enoic acid and 2,3-dihydroxydodecanoic acid. This is the first report of a 2,3-hydroxylated fatty acid product made by a peroxygenase P450, with the dihydroxylated product formed by CYP152K6-catalyzed 3-hydroxylation of 2-hydroxydodecanoic acid, but not by 2-hydroxylation of 3-hydroxydodecanoic acid. Bacillus methanolicus CYP152K6 is a novel cytochrome P450 (P450 or CYP) peroxygenase family member. Its tetradecanoic acid-bound crystal structure reveals that the substrate binds through its carboxylate to a conserved arginine residue. Hydrogen peroxide displaces the distal water on the heme, forming a reactive iron-oxo species that catalyzes substrate oxidation. The cytochrome P450 (P450 or CYP) CYP152K6 from Bacillus methanolicus was characterized. CYP152K6 is a peroxygenase that catalyzes efficient oxidation of dodecanoic acid. A 1.3 Å crystal structure was solved for CYP152K6 bound to tetradecanoic acid. EPR reveals three low-spin CYP152K6 species whose populations change on lipid binding. CYP152K6 produces 2,3-dihydroxydodecanoic acid as a novel peroxygenase product.
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影响因子：
3.9
作者：
Munro AW;McLean KJ;Grant JL;Makris TM
通讯作者：
Makris TM
影响因子：
3.9
作者：
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Makris, Thomas M.
影响因子：
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作者：
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通讯作者：
Poulos, Thomas L.
影响因子：
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作者：
Matsunaga, I;Yokotani, N;Ichihara, K
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Ichihara, K