Abstract
Bacterial extracellular proteases are important for bacterial nutrition and marine sedimentary organic nitrogen degradation. However, only a few proteases from marine sedimentary bacteria have been characterized. Some subtilases have a protease-associated (PA) domain inserted in the catalytic domain. Although structural analysis and deletion mutation suggests that the PA domain in subtilases is involved in substrate binding, direct evidence to support this function is still absent. Here, a protease, P57, secreted by Photobacterium sp. A5-7 isolated from marine sediment was characterized. P57 could hydrolyze casein, gelatin and collagen. It showed the highest activity at 40°C and pH 8.0. P57 is a new subtilase, with 63% sequence identity to the closest characterized protease. Mature P57 contains a catalytic domain and an inserted PA domain. The recombinant PA domain from P57 was shown to have collagen-binding ability, and Phe349 and Tyr432 were revealed to be key residues for collagen binding in the PA domain. This study first shows direct evidence that the PA domain of a subtilase can bind substrate, which provides a better understanding of the function of the PA domain of subtilases and bacterial extracellular proteases from marine sediment.
Highlights
Organic nitrogen degradation is an important part of marine nitrogen cycle (Aluwihare et al, 2005)
Sequence comparison showed that strain A5– 7 shared the highest 16S rRNA gene sequence identities with known Photobacterium species, suggesting an affiliation with the genus Photobacterium
In the neighbor-joining and maximumlikelihood trees (Figures 1A,B) based on the 16S rRNA gene sequences, strain A5–7 fell within the clade of the genus Photobacterium and formed a distinct intra-branch with type strain of Photobacterium aplysiae supported by high bootstrap values (>85%), indicating its close phylogenetic relationship to the latter
Summary
Organic nitrogen degradation is an important part of marine nitrogen cycle (Aluwihare et al, 2005). Particulate organic nitrogen (PON) that deposits to marine sediments is mainly decomposed by bacterial extracellular proteases, which is generally considered to be the initial and rate-limiting step of nitrogen cycle in marine sediments (Talbot and Bianchi, 1997; Brunnegård et al, 2004). It has been found that protease-producing bacteria and their extracellular proteases are rich and diverse in marine sediments (Olivera et al, 2007; Zhou et al, 2009, 2013). Peptidase family S8, known as the subtilisin or subtilase family, is the second-largest family of serine proteases (Rawlings et al, 2010).
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