Monroe Jonathan D, Pope Lauren E, Breault Jillian S, Berndsen Christopher E, Storm Amanda R
Department of Biology, James Madison University, Harrisonburg, VA, United States.
Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, VA, United States.
Front Plant Sci. 2018 Aug 14;9:1176. doi: 10.3389/fpls.2018.01176. eCollection 2018.
The β-amylase family in has nine members, four of which are both plastid-localized and, based on active-site sequence conservation, potentially capable of hydrolyzing starch to maltose. We recently reported that one of these enzymes, BAM2, is catalytically active in the presence of physiological levels of KCl, exhibits sigmoidal kinetics with a Hill coefficient of over 3, is tetrameric, has a putative secondary binding site (SBS) for starch, and is highly co-expressed with other starch metabolizing enzymes. Here we generated a tetrameric homology model of Arabidopsis BAM2 that is a dimer of dimers in which the putative SBSs of two subunits form a deep groove between the subunits. To validate this model and identify key residues, we generated a series of mutations and characterized the purified proteins. (1) Three point mutations in the putative subunit interfaces disrupted tetramerization; two that interfered with the formation of the starch-binding groove were largely inactive, whereas a third mutation prevented pairs of dimers from forming and was active. (2) The model revealed that a 30-residue N-terminal acidic region, not found in other BAMs, appears to form part of the putative starch-binding groove. A mutant lacking this acidic region was active and did not require KCl for activity. (3) A conserved tryptophan residue in the SBS is necessary for activation and may form π-bonds with sugars in starch. (4) Sequence alignments revealed a conserved serine residue next to one of the catalytic glutamic acid residues, that is a conserved glycine in all other active BAMs. The serine side chain points away from the active site and toward the putative starch-binding groove. Mutating the serine in BAM2 to a glycine resulted in an enzyme with a similar to that of the wild type enzyme but with a 7.5-fold lower for soluble starch. Interestingly, the mutant no longer exhibited sigmoidal kinetics, suggesting that allosteric communication between the putative SBS and the active site was disrupted. These results confirm the unusual structure and function of this widespread enzyme, and suggest that our understanding of starch degradation in plants is incomplete.
中的β-淀粉酶家族有九个成员，其中四个定位于质体，并且基于活性位点序列保守性，有可能将淀粉水解为麦芽糖。我们最近报道，这些酶之一BAM2在生理水平的KCl存在下具有催化活性，呈现出希尔系数超过3的S形动力学，是四聚体，具有一个假定的淀粉二级结合位点（SBS），并且与其他淀粉代谢酶高度共表达。在这里，我们生成了拟南芥BAM2的四聚体同源模型，它是一个二聚体的二聚体，其中两个亚基的假定SBS在亚基之间形成一个深沟。为了验证该模型并鉴定关键残基，我们进行了一系列突变并对纯化的蛋白质进行了表征。（1）假定亚基界面的三个点突变破坏了四聚化；两个干扰淀粉结合凹槽形成的突变体基本上没有活性，而第三个突变阻止了二聚体对的形成但具有活性。（2）该模型表明，在其他BAM中未发现的一个30个残基的N端酸性区域似乎构成了假定淀粉结合凹槽的一部分。缺乏该酸性区域的突变体具有活性，并且活性不需要KCl。（3）SBS中一个保守的色氨酸残基对于激活是必需的，并且可能与淀粉中的糖形成π键。（4）序列比对揭示了一个催化谷氨酸残基之一旁边的保守丝氨酸残基，在所有其他活性BAM中它是一个保守的甘氨酸。丝氨酸侧链指向远离活性位点并朝向假定的淀粉结合凹槽。将BAM2中的丝氨酸突变为甘氨酸产生了一种酶，其与野生型酶的 相似，但对可溶性淀粉的 低7.5倍。有趣的是，突变体不再表现出S形动力学，这表明假定的SBS和活性位点之间的变构通讯被破坏。这些结果证实了这种广泛存在的酶的异常结构和功能，并表明我们对植物中淀粉降解的理解是不完整的。
