The carrageenophyte red alga Chondrus crispus produces three family 16 glycoside hydrolases (CcGH16-1, -2 and -3). Phylogenetically, the red algal GH16 members are closely related to bacterial GH16 homologues from subfamilies 13 and 14, which have characterized marine bacterial β-carrageenase and β-porphyranase activities, respectively, yet the functions of these CcGH16 hydrolases have not been determined. Here, we first confirmed the gene locus of the ccgh16-3 gene in the alga to facilitate further investigation. Next, our biochemical characterization of CcGH16-3 revealed an unexpected β-porphyranase activity, since porphyran is not a known component of the C. crispus extracellular matrix. Kinetic characterization was undertaken on natural porphyran substrate with an experimentally determined molecular weight. We found CcGH16-3 has a pH optimum between 7.5-8.0; however, it exhibits reasonably stable activity over a large pH range (pH 7.0-9.0). CcGH16-3 has a K_M of 4.0 ± 0.8 μM, a kcat of 79.9 ± 6.9 s^-1 and a kcat/K_M of 20.1 ± 1.7 μM^-1.s^-1. We structurally examined fine enzymatic specificity by performing a subsite dissection. CcGH16-3 has a strict requirement for D-galactose and L-galactose-6-sulfate in its -1 and +1 subsites, respectively, whereas the outer subsites are less restrictive. CcGH16-3 is one of a handful of algal enzymes characterized with a specificity for a polysaccharide unknown to be found in their own extracellular matrix. This β-porphyranase activity in a carrageenophyte red alga may provide defense against red algal pathogens or, alternatively, provide a competitive advantage in niche colonization.

角叉藻红藻Chondrus crispus产生三种家族 16 糖苷水解酶（CcGH16-1、-2 和 -3）。在系统发育上，红藻 GH16 成员与来自亚科 13 和 14 的细菌 GH16 同系物密切相关，它们分别具有海洋细菌 β-卡拉胶酶和 β-卟啉酶活性的特征，但这些 CcGH16 水解酶的功能尚未确定。在这里，我们首先确定了藻类中ccgh16-3基因的基因位点，以方便进一步研究。接下来，我们对 CcGH16-3 的生化表征揭示了意想不到的 β-卟啉酶活性，因为卟啉不是C. crispus的已知成分细胞外基质。对具有实验确定的分子量的天然卟啉底物进行动力学表征。我们发现 CcGH16-3 的最佳 pH 值在 7.5-8.0 之间；然而，它在较大的 pH 范围 (pH 7.0-9.0) 内表现出相当稳定的活性。CcGH16-3 的K _M为 4.0 ± 0.8 μM，k cat 为 79.9 ± 6.9 s ^-1和k cat/ K _M为 20.1 ± 1.7 μM ^-1 .s ^-1. 我们通过执行亚位点解剖在结构上检查了精细的酶特异性。CcGH16-3 分别在其 -1 和 +1 子位点中对 D-半乳糖和 L-半乳糖-6-硫酸盐有严格要求，而外部子位点的限制较少。CcGH16-3 是少数藻类酶中的一种，其特征是对在其自身细胞外基质中未知的多糖具有特异性。角叉藻红藻中的这种 β-卟啉酶活性可以提供对红藻病原体的防御，或者在生态位定植中提供竞争优势。