Song Min-Sun, Chen Weiguo, Zhang Min, Napoli Joseph L
Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California 94720, USA.
J Biol Chem. 2003 Oct 10;278(41):40079-87. doi: 10.1074/jbc.M304910200. Epub 2003 Jul 10.
We report a mouse short-chain dehydrogenase/reductase (SDR), retinol dehydrogenase-similar (RDH-S), with intense mRNA expression in liver and kidney. The RDH-S gene localizes to chromosome 10D3 with the SDR subfamily that catalyzes metabolism of retinoids and 3 alpha-hydroxysteroids. RDH-S has no activity with prototypical retinoid/steroid substrates, despite 92% amino acid similarity to mouse RDH1. This afforded the opportunity to analyze for functions of non-catalytic SDR residues. We produced RDH-S Delta 3 by mutating RDH-S to remove an "additional" Asn residue relative to RDH1 in its center, to convert three residues into RDH1 residues (L121P, S122N, and Q123E), and to substitute RDH1 sequence G208FKTCVTSSD for RDH-S sequence F208-FLTGMASSA. RDH-S Delta 3 catalyzed all-trans-retinol and 5 alpha-androstane-3 alpha,17 alpha-diol (3 alpha-adiol) metabolism 60-70% as efficiently (Vm/Km) as RDH1. Conversely, substituting RDH-S sequence F208FLTGMASSA into RDH1 produced a chimera (viz. C3) that was inactive with all-trans-retinol, but was 4-fold more efficient with 3 alpha-adiol. A single RDH1 mutation in the C3 region (K210L) reduced efficiency for all-trans-retinol by >1250-fold. In contrast, the C3 area mutation C212G enhanced efficiency with all-trans-retinol by approximately 2.4-fold. This represents a >6000-fold difference in catalytic efficiency for two enzymes that differ by a single non-catalytic amino acid residue. Another chimera (viz. C5) retained efficiency with all-trans-retinol, but was not saturated and was weakly active with 3 alpha-adiol, stemming from three residue differences (K224Q, K229Q, and A230T). The residues studied contribute to the substrate-binding pocket: molecular modeling indicated that they would affect orientation of substrates with the catalytic residues. These data report a new member of the SDR gene family, provide insight into the function of non-catalytic SDR residues, and illustrate that limited changes in the multifunctional SDR yield major alterations in substrate specificity and/or catalytic efficiency.
我们报道了一种小鼠短链脱氢酶/还原酶（SDR），即视黄醇脱氢酶类似物（RDH-S），其在肝脏和肾脏中具有强烈的mRNA表达。RDH-S基因定位于10D3染色体上，属于催化视黄醇和3α-羟基类固醇代谢的SDR亚家族。尽管RDH-S与小鼠RDH1的氨基酸相似度达92%，但它对典型的视黄醇/类固醇底物没有活性。这为分析非催化性SDR残基的功能提供了契机。我们通过突变RDH-S产生了RDH-S Delta 3，相对于RDH1，去除了其中心的一个“额外”Asn残基，将三个残基转变为RDH1的残基（L121P、S122N和Q123E），并用RDH1的序列G208FKTCVTSSD替换了RDH-S的序列F208-FLTGMASSA。RDH-S Delta 3催化全反式视黄醇和5α-雄甾烷-3α,17α-二醇（3α-二醇）代谢的效率（Vm/Km）为RDH1的60 - 70%。相反，将RDH-S的序列F208FLTGMASSA替换到RDH1中产生了一种嵌合体（即C3），它对全反式视黄醇无活性，但对3α-二醇的催化效率提高了4倍。C3区域的单个RDH1突变（K210L）使全反式视黄醇的催化效率降低了>12,500倍。相比之下，C3区域的突变C212G使全反式视黄醇的催化效率提高了约2.4倍。这代表了两种仅相差一个非催化性氨基酸残基的酶在催化效率上>60,000倍的差异。另一种嵌合体（即C5）对全反式视黄醇仍保持催化效率，但不饱和且对3α-二醇的活性较弱，这源于三个残基的差异（K224Q、K229Q和A230T）。所研究的这些残基构成了底物结合口袋：分子模拟表明它们会影响底物与催化残基的取向。这些数据报道了SDR基因家族的一个新成员，深入了解了非催化性SDR残基的功能，并表明多功能SDR中有限的变化会导致底物特异性和/或催化效率的重大改变。
