Division of Gene Research, Department of Life Sciences, Research Center for Human and Environmental Sciences, Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, Japan.
Present address: Fornia Biosolutions, Inc., 3876 Bay Center Place, Hayward, CA 94545, USA.
Microbiology (Reading). 2018 Mar;164(3):277-286. doi: 10.1099/mic.0.000609. Epub 2018 Jan 30.
Biopolymers on the cell surface are very important for protecting microorganisms from environmental stresses, as well as storing nutrients and minerals. Synthesis of biopolymers is well studied, while studies on the modification and degradation processes of biopolymers are limited. One of these biopolymers, poly-γ-glutamic acid (γ-PGA), is produced by Bacillus species. Bacillus subtilis PgdS, possessing three NlpC/P60 domains, hydrolyses γ-PGA. Here, we have demonstrated that several dl-endopeptidases with an NlpC/P60 domain (LytE, LytF, CwlS, CwlO, and CwlT) in B. subtilis digest not only an amide bond of d-γ-glutamyl-diaminopimelic acid in peptidoglycans but also linkages of γ-PGA produced by B. subtilis. The hydrolase activity of dl-endopeptidases towards γ-PGA was inhibited by IseA, which also inhibits their hydrolase activity towards peptidoglycans, while the hydrolysis of PgdS towards γ-PGA was not inhibited. PgdS hydrolysed only the d-/l-Glu‒d-Glu linkages of d-Glu-rich γ-PGA (d-Glu:l-Glu=7 : 3) and l-Glu-rich γ-PGA (d-Glu:l-Glu=1 : 9), indicating that PgdS can hydrolyse only restricted substrates. On the other hand, the dl-endopeptidases in B. subtilis cleaved d-/l-Glu‒d-/l-Glu linkages of d-Glu-rich γ-PGA (d-Glu:l-Glu=7 : 3), indicating that these enzymes show different substrate specificities. Thus, the dl-endopeptidases digest γ-PGA more flexibly than PgdS, even though they are annotated as "dl-endopeptidase, digesting the d-γ-glutamyl-diaminopimelic acid linkage (d‒l amino acid bond)".
细胞表面的生物聚合物对于保护微生物免受环境压力以及储存营养物质和矿物质非常重要。生物聚合物的合成研究得很好，而生物聚合物的修饰和降解过程的研究则有限。这些生物聚合物之一，聚-γ-谷氨酸（γ-PGA），是由芽孢杆菌属产生的。芽孢杆菌枯草亚种 PgdS 具有三个 NlpC/P60 结构域，可水解 γ-PGA。在这里，我们已经证明了枯草芽孢杆菌中的几种具有 NlpC/P60 结构域的 dl-内肽酶（LytE、LytF、CwlS、CwlO 和 CwlT）不仅可以水解肽聚糖中 d-γ-谷氨酸-d-二氨基庚二酸的酰胺键，还可以水解枯草芽孢杆菌产生的 γ-PGA 的键。dl-内肽酶对 γ-PGA 的水解酶活性被 IseA 抑制，IseA 也抑制其对肽聚糖的水解酶活性，而 PgdS 对 γ-PGA 的水解则不受抑制。PgdS 仅水解富含 d-谷氨酸的 γ-PGA（d-Glu:l-Glu=7∶3）和富含 l-谷氨酸的 γ-PGA（d-Glu:l-Glu=1∶9）中的 d-/l-Glu‒d-Glu 键，表明 PgdS 只能水解有限的底物。另一方面，枯草芽孢杆菌中的 dl-内肽酶可切割富含 d-谷氨酸的 γ-PGA（d-Glu:l-Glu=7∶3）中的 d-/l-Glu‒d-/l-Glu 键，表明这些酶具有不同的底物特异性。因此，即使被注释为“dl-内肽酶，可水解 d-γ-谷氨酸-d-二氨基庚二酸键（d‒l 氨基酸键）”，dl-内肽酶对 γ-PGA 的水解也比 PgdS 更灵活。
