Elsevier

Marine Chemistry

Volume 233, 20 June 2021, 103973
Marine Chemistry

The effect of chemical structure on hydrolysis pathways of small peptides in coastal seawater

https://doi.org/10.1016/j.marchem.2021.103973Get rights and content

Highlights

  • The effect of chemical structure on peptide hydrolysis pathways was evaluated.

  • Aminopeptidases preferred hydrophobic or positive-charged N-terminal amino acids.

  • Hydrolysis rates of different peptides were similar although their pathways differed.

  • Extracellular enzymes were induced more easily under oligotrophic conditions.

Abstract

The hydrolysis pathway of a given peptide or protein in seawater, or how it is hydrolyzed by extracellular enzymes, may depend on its chemical structure or specific amino acid composition. This knowledge is important for a better understanding of the first steps on decomposition of labile organic matter that is often dominated by proteins. The hydrolysis pathway can be estimated from the hydrolyzed fragments of small peptides through incubation experiments. Here we incubated a group of small peptides with different amino acid compositions in coastal seawater, including tetrapeptides alanine-valine-phenylalanine-alanine (AVFA) and its modifications of the N terminal A with valine (V), serine (S), arginine (R) and aspartic acid (D) (i.e., peptides VVFA, SVFA, RVFA, DVFA). Through these incubations, we evaluated the effect of chemical structure or specific amino acid composition on peptide hydrolysis pathways and the roles of different types of peptidases. Our results showed that aminopeptidases preferentially hydrolyzed tetrapeptides with N terminal A and R (29–100% of the total peptidases), followed by V (30–90%) and S (20–51%), but were ineffective with acidic amino acid D (0–14%). This pattern indicates that the N-terminal amino acid affects how a peptide is hydrolyzed in a given environment. For N-terminal amino acids with uncharged side chains, aminopeptidases preferred hydrophobic A, V to polar uncharged S, and for those with charged side chains, they preferred positively-charged R to negatively-charged D. Our results also suggest that if aminopeptidases could not access the N-terminal amino acid of a peptide, carboxypeptidases and/or endopeptidases will hydrolyze it, leading to similar peptide hydrolysis rates among different peptides although their hydrolysis pathways may differ. The effect of amino acid composition on peptide hydrolysis pathways is further demonstrated by comparing trialanine (AAA), AVF, AVFA, VFA, phenylalanine-alanine-serine-tryptophan-glycine-alanine (FASWGA) and SWGA. Overall, results from this study show that the chemical structure of a peptide affects its hydrolysis pathway in coastal seawater, and shed new lights on the early steps of organic nitrogen degradation and further nitrogen cycling in marine environments.

Introduction

Proteins and peptides account for a major fraction of marine biota biomass, and their hydrolysis is a fundamental process in marine carbon and nitrogen cycles (Pantoja et al., 1997; Findlay and Sinsabaugh, 2003; Nagata, 2008). To be available to bacteria, proteins and peptides need to be hydrolyzed to small peptides (<600 Da) by extracellular enzymes dissolved freely in the water, or by ectoenzymes attached to the cell surface or within the periplasmic space of bacteria (Chróst, 1991; Weiss et al., 1991). Small peptides have been detected as important intermediates during protein and peptide decomposition (Hollibaugh and Azam, 1983; Nunn et al., 2003; Roth and Harvey, 2006). As the first step in peptide decomposition, enzymatic hydrolysis is usually considered to be rate-limiting (Hoppe, 1991; Meyer-Reil and Koster, 1992; Davey et al., 2001), although in certain cases hydrolysis can outpace other factors such as enzyme production and the hydrolysis can be instant (Arnosti, 2004; Liu and Liu, 2018).

The pathway of peptide hydrolysis, i.e., how a peptide is cleaved by enzymes and where the peptide chain is cleaved, remains key to understanding factors controlling hydrolysis and interactions between enzymes and peptides. Hydrolysis pathways also provide insights into the types of peptidases synthesized by microbes, including amino-, carboxy- and endopeptidase present in seawater (Chróst, 1991). Aminopeptidases cleave peptides from the N-terminus, carboxypeptidases from the C-terminus, and endopeptidases from internal peptide bonds. The type of peptidases that are active can be inferred through the investigation of peptide hydrolysis pathways. For example, an N-terminus hydrolysis pathway points to the role of aminopeptidases. Knowing the relative roles of these different peptidases during peptide hydrolysis is also a fundamental step needed to understand the controlling mechanisms of enzyme synthesis by microbes, such as at the gene level (Chróst, 1991).

Previous studies on hydrolysis pathways or the roles of peptidases have mainly relied on proteins or peptide analogs with fluorophores that can be easily detected (Hoppe, 1983; Pantoja et al., 1997; Obayashi and Suzuki, 2005; Steen and Arnosti, 2013). The dominant role of aminopeptidases, rather than endopeptidases, in peptide hydrolysis was suggested by modeling hydrolysis data of radiolabeled proteins in aquatic environments (Billen, 1991). However, significant contributions of carboxypeptidases and endopeptidases have also been observed using a series of targeted peptide analogs (Hashimoto et al., 1985; Obayashi and Suzuki, 2005; Obayashi and Suzuki, 2008). In these studies, peptide analogs were targeted by specific kinds of peptidases through blocking N-terminus, C-terminus, or both of peptides, making it difficult to simultaneously compare the relative roles of different peptidases. In contrast, using plain peptides without fluorogenic tags allows examination of the relative roles of three types of peptidases with all peptide bonds available. Using small peptides with similar structures, Liu et al. (2013) suggested that the hydrolysis by aminopeptidases is the dominant pathway, but their conclusion was based on only two peptides, alanine-valine-phenylalanine-alanine (AVFA) and VFA. Considering the enormous possible combinations of amino acids in peptides, there is a need to expand the range of peptides to examine the role of chemical structure in determining peptide hydrolysis pathways.

In this study, our main objective was to evaluate how amino acid composition in a peptide affects its hydrolysis pathway in coastal seawater. We first derived hydrolysis pathways of different small peptides from their hydrolyzed fragments, which implied the relative roles of different peptidases, and then compared their hydrolysis pathways to assess the effect of chemical structure. A series of small peptides from tri- to hexapeptides were examined, including trialanine (AAA), alanine-valine-phenyalanine (AVF), VFA, AVFA, VVFA, serine-valine-phenylalanine-alanine (SVFA), arginine-valine-phenylalanine-alanine (RVFA), aspartic acid-valine-phenylalanine-alanine (DVFA), serine-tryptophan-glycine-alanine (SWGA), and FASWGA. These peptides were mainly modified from AVFA, a fragment of ribulose-1, 5 bisphosphate carboxylase oxygenase (RuBisCO), and SWGA, a peptide with fluorescence due to the W in the structure; we have gained hydrolysis and decomposition patterns of these two peptides in coastal seawater (Liu et al., 2010; Liu et al., 2013; Liu and Liu, 2015; Liu and Liu, 2016). We chose two contrasting coastal waters for the experiments, one the western Gulf of Mexico coastal water without much riverine influence which is relatively oligotrophic, and the other the Mississippi River plume in northern Gulf of Mexico as the eutrophic water. Results of this work showed that hydrolysis by the types of peptidases was highly selective, depending on the amino acid composition of a peptide, and it was also affected by environmental factors such as the quality of ambient organic matter and microbial community.

Section snippets

Seawater sampling and chemical analysis of initial water

Coastal seawater for incubation was collected from two sampling sites: less river-influenced station SC (27.84°N, 97.05°W) in Port Aransas, Texas, in the western Gulf of Mexico and station C6 (28.86°N, 90.45°W) in the northern Gulf of Mexico that is largely influenced by Mississippi River discharge. At station SC, surface seawater was manually collected using a 2-L acid-cleaned polyethylene bottle in March 2013, April 2013, and June 2014. Temperature and salinity of the seawater were measured

Testing small peptides differing in the N-terminal amino acid at stations SC and C6

Peptides AVFA, RVFA, VVFA, SVFA, and DVFA were hydrolyzed generally in a linear mode with rates ranging over 0.017–0.071 μmol L−1 h−1 at station SC (Fig. 1, rates difference non-significant, ANOVA p > 0.5). The production of VFA from AVFA and RVFA hydrolysis, as high as 0.27 μmol L−1, outcompeted those of other peptide fragments (Fig. 1a, b). Amino acid A increased by 0.16 μmol L−1 at 4 h and then decreased afterwards during the AVFA incubation, and R increased by 0.34 μmol L−1 at 12 h followed

Discussion

In this study we conducted peptide incubations within a relatively short-time period (19–27 h). Bacterial abundances increased at different levels (<85%) over 21–24 h as shown in Supplementary Figs. S2b and S3b. However, the concentrations of peptide decreased generally in a linear manner, indicating their hydrolysis rates were constant throughout the incubation time and not affected much by the bacterial abundance change. The linearity of peptide hydrolysis within 24 h is consistent with our

Funding

This work was funded by the Chemical Oceanography Program of the National Science Foundation (OCE-1129659 to Z. Liu).

Author contributions

SL and ZL conceived of the study and designed the experiments. SL conducted the experiments and analyzed samples and data. SL and ZL wrote the manuscript.

Declaration of Competing Interest

None declared.

Acknowledgement

We thank the help from the crew of R/V Pelican. We appreciate Dr. C. Shank for analyzing DOC samples and Dr. T. Villareal for access to the flow cytometer. We are grateful for the help from N. Reyna, Dr. H. Bacosa and K. Halim with the incubation experiments. We thank the comments from Drs. D. Erdner, W. Gardner, D. Kirchman, J. McClelland, and K. Lu on earlier versions of this manuscript. We are also grateful for the ship time shared by Dr. W. Gardner (NOAA) and Dr. L. Hamdan (BOEM).

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    Current address: Marine Science Institute/Department of Ecology, Evolution and Marine Biology, University of California Santa Barbara, California 93106, USA

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