JMLS 2021 June;6(1):1-8. Epub 2021 May 31
Copyright © 2021 by The Korean Society of Marine Life Science
Enzymes and Their Reaction Mechanisms in Dimethylsulfoniopropionate Cleavage and Biosynthesis of Dimethylsulfide by Marine Bacteria
Hackwon Do; Research Unit of Cryogenic Novel Material, Korea Polar Research Institute, Incheon 21990, Korea
Jisub Hwang; Research Unit of Cryogenic Novel Material, Korea Polar Research Institute, Incheon 21990, Korea; Department of Polar Sciences, University of Science and Technology, Incheon 21990, Korea
Sung Gu Lee; Research Unit of Cryogenic Novel Material, Korea Polar Research Institute, Incheon 21990, Korea; Department of Polar Sciences, University of Science and Technology, Incheon 21990, Korea
Jun Hyuck Lee1; Research Unit of Cryogenic Novel Material, Korea Polar Research Institute, Incheon 21990, Korea; Department of Polar Sciences, University of Science and Technology, Incheon 21990, Korea
In marine ecosystems, the biosynthesis and catabolism of dimethylsulfoniopropionate (DMSP) by marine bacteria is critical to microbial survival and the ocean food chain. Furthermore, these processes also influence sulfur recycling and climate change. Recent studies using emerging genome sequencing data and extensive bioinformatics analysis have enabled us to identify new DMSP-related genes. Currently, seven bacterial DMSP lyases (DddD, DddP, DddY, DddK, DddL, DddQ and DddW), two acrylate degrading enzymes (DddA and DddC), and four demethylases (DmdA, DmdB, DmdC, and DmdD) have been identified and characterized in diverse marine bacteria. In this review, we focus on the biochemical properties of DMSP cleavage enzymes with special attention to DddD, DddA, and DddC pathways. These three enzymes function in the production of acetyl coenzyme A (CoA) and CO2 from DMSP. DddD is a DMSP lyase that converts DMSP to 3-hydroxypropionate with the release of dimethylsulfide. 3-Hydroxypropionate is then converted to malonate semialdehyde by DddA, an alcohol dehydrogenase. Then, DddC transforms malonate semialdehyde to acetyl-CoA and CO2 gas. DddC is a putative methylmalonate semialdehyde dehydrogenase that requires nicotinamide adenine dinucleotide and CoA cofactors. Here we review recent insights into the structural characteristics of these enzymes and the molecular events of DMSP degradation.
Keywords: Dimethylsulfide Dimethylsulfoniopropionate Molecular modeling Methylmalonate semialdehyde dehydrogenase Sulfur recycle
Correspondence to: Jun Hyuck Lee; Research Unit of Cryogenic Novel Material, Korea Polar Research Institute, Incheon 21990, Korea; E-mail : junhyucklee@kopri.re.kr
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1. Introduction
The movement and cycling of sulfur through the geosphere and biosphere is facilitated by weathering, microbes, and plants. Sulfur is released from the geosphere by volcanic activity and then flows into the ocean with rain. In marine ecosystems, sulfur exists mainly in its dimethylsulfoniopropionate (DMSP) form in micro- algae, macroalgae, and fish (Fig. 1) (Kiene et al., 2000; Simo, 2001; Howard et al., 2008; Carslaw et al., 2010). DMSP functions as an osmolyte and antioxidant and its degraded products are major sources of carbon and sulfur for marine microbes. The biosyn- thesis of DMSP occurs primarily in marine phytoplankton and macroalgae. The sulfur-containing amino acid methionine is the DMSP precursor. The backbone for methionine is derived from the aspartate family, but sulfur may also be derived from cysteine,methanethiol, and hydrogen sulfide (Fig. 2) (Kiene et al., 2000). Additional studies are required to understand the specific mech- anism of DMSP biosynthesis at the molecular level.
Several marine bacteria can degrade DMSP into dimethylsulfide (DMS), a volatile sulfur compound that contributes significantly to the global sulfur cycle. Specifically, DMS molecules influence cloud formation by acting as cloud condensation nuclei (CCN), and they move into the atmosphere, thereby returning to land with rain (Reisch et al., 2011; Moran et al., 2012; Song et al., 2020). Moreover, DMS may be involved in the food web. DMS dissolved in the ocean as well as its gaseous form attracts sea birds and copepod crustaceans so that sea animals can locate food sources such as marine phytoplankton and macroalgae (Kiene et al., 2000; Simo, 2001; Nevitt and Haberman, 2003).
Marine bacteria species have their own unique DMSP degradation pathways. The degradation of DMSP by marine bacteria can be categorized into two competing pathways: the DMSP cleavage pathway and the DMSP demethylation pathway. To date, seven bacterial DMSP lyases (DddD, DddP, DddY, DddK, DddL, DddQ and DddW) have been identified as participants in the DMSP cleavage pathway (Reisch et al., 2011; Moran et al., 2012; Schnicker et al., 2017; Peng et al., 2019). Notably, Alma1 is a phytoplankton specific DMSP lyase but it is not found in marine bacteria. Thus, it is not the subject of this study. DMSP lyase activity has been observed in crude extracts of the macrophyte Polysiphonia lanosa and the heterotrophic dinoflagellate Gyrodinium cohnii and, more recently, in whole cells of axenic Phaeocystis sp. cultures (Anderson and Cantoni, 1956; Van Boekel and Stefels, 1993). DMSP lyase cleaves DMSP into DMS and acrylate, and the latter is further degraded by the DddA and DddC enzymes. In the DMSP demethylation pathway, DMSP demethylases (DmdA, DmdB, DmdC, and DmdD) convert DMSP into methyl mercaptopropionate with tetrahydro- folate as the methyl acceptor (Fig. 3) (Reisch et al., 2011; Moran et al., 2012; Schuller et al., 2012). Oceans are the main reservoir and source of atmospheric DMS. Many studies have focused on the DMSP lyases because they produce DMS and 3-hydroxypropionate (3HP) directly from DMSP.
2. Catabolism and Cleavage of DMSP
DMSP can be catalyzed with enzymes for the cleavage or demethylation pathways, which are found in phytoplankton or marine bacteria. Bacterial activity is the main contributor to DMS production in seawater (Kiene et al., 2000). DMSP can be con- verted into acrylic acid or acrylate and DMS by DMSP lyases. Seven DMSP lyases—DddD, DddP, DddY, DddK, DddL, DddQ and DddW—have been identified, four of which—DddY, DddP, DddQ, and DddW—have been purified and characterized for their activities in DMS production (Kirkwood et al., 2010; Curson et al., 2011; Hehemann et al., 2014; Li et al., 2014; Brummett et al., 2015; Schnicker et al., 2017; Peng et al., 2019). Even though the functions of these enzymes are similar, their genes show low sequential similarity (Reisch et al., 2011; Moran et al., 2012). Among them, the structures of DddQ (Li et al., 2014) and DddP (Hehemann et al., 2014) were recently resolved with X-ray crystallography, and their DMSP binding sites and reaction mechanisms were investigated.
dddQ, the most abundant gene in marine metagenomes, is a DMSP lyase gene found in diverse microorganisms (Todd et al., 2009; Todd et al., 2011). DddQ has been identified as a zinc metal- loenzyme and, compared with non-metal ion-binding enzymes, shows four- or five-fold increased activity when Mn or Co is added, respectively. Recently, a high-resolution structure of the DddQ from Ruegeria lacuscaerulensis was shown to include five α-helices and eight β-strands (Li et al., 2014). These eight antiparallel β-strands form a β-barrel fold structure, which is typical for members of the cupin superfamily. Incidentally, Li et al. (2014) also found the active site through co-crystallization with 2-(N-morpholino)- ethanesulfonic acid, which is derived from the crystallization buffer. 2-(N-Morpholino)ethanesulfonic acid has a sulfate and carbon backbone structure similar to that of DMSP. Furthermore, the authors confirmed this active site with a DMSP complex structure and a single mutagenesis experiment. DMSP binds to the cupin motif in the active pocket. The entrance of the active site tunnel is formed by two main loops called loop 1 (residues 74~78) and loop 2 (residues 184~180). DMSP may migrate through the tunnel into the active pocket and bind with zinc ion. Notably, the Tyr131 residue has two conformations and may have an important role in the cleavage of DMSP (Li et al., 2014).
The crystal structure of DddP from Roseobacter denitrificans (RdDddP) also has been determined at a resolution of 2.15 Å (Hehemann et al., 2014). RdDddP is a homodimeric metalloprotein, and each monomer can be divided into N- and C-terminal domains. The N-terminal domain consists of six β-strands sur- rounded by five α-helices. In the C-terminal domain, six anti-parallel β-strands are surrounded by four α-helices (Hehemann et al., 2014). The metal ions bind closely to the substrate-binding site, and the entrance of the substrate binding pocket is surrounded by loops: residues 116~120, 135~139, 364~369, and 383~401 (Hehemann et al., 2014). Even with a structure for RdDddP in hand, the specific mechanism at the molecular level remains unclear. The results of this study raise the question of whether the mechanism of metalloprotein DddP or DMSP demethylation is metal depen- dent.
3. DddD (DMSP lyase)
DddD is sporadically found in α- and β-proteobacteria and is more frequently observed in γ-proteobacteria (Curson et al., 2012; Alcolombri et al., 2014). This enzyme cleaves DMSP to DMS and 3HP. Recently, the biochemical characterization of DddD (UniProtKB code A6W2K8, 837 amino acids) from the marine bacterium Marinomonas sp. MWYL1 was reported by Alcolombri et al. (2014) (Fig. 4). The authors suggested that this DddD protein has dual enzymatic functions as a CoA transferase and DMSP lyase. The authors also demonstrated that DddD preferentially uses acetyl-CoA as a CoA donor and produces 3HP-CoA.
When the sequence of Alcaligenes faecalis DddD (UniProtKB code E7DDH7) was used to search databases of microbial genomes, close homologs of DddD (>75% identical at the amino acid level) were found in a large number of divergent marine bacteria. The highly conserved putative active site Asp602 was identified with multiple sequence alignment and homology modeling, and its mutation to alanine significantly reduced the DMSP lyase activity of DddD. This result indicated that Asp602 is a critical and plausible active site for DddD enzyme activity (Alcolombri et al., 2014). DMS, one of the reaction products of DddD, is released into the marine environment as a gas, whereas another product, 3HP-CoA, is further processed by two additional enzymes, DddA and DddC.
4. DddA (putative 3HP dehydrogenase)
DddA is a member of the alcohol dehydrogenase superfamily. This enzyme catalyzes the oxidation of 3HP-CoA in the DMSP catabolism pathway, forming malonate semialdehyde (Reisch et al., 2011; Moran et al., 2012). Multiple sequence alignments of DddA from Alcaligenes faecalis (AfaDddA; UniProtKB code E7DDH7) and other dehydrogenases including DddA from Halomonas sp. HTNK1 (UniProtKB code C8YX89) and a choline oxidase from Arthrobacter globiformis (UniProtKB code Q7X2H8, PDB code 2JBV) (Quaye et al., 2008) were determined using the ClustalX program (Fig. 5A). The results showed that AfaDddA has the conserved and cata- lytically important residues H381 and H491. In the Arthrobacter globiformis choline oxidase structure, the E312 residue is involved in substrate binding (Quaye et al., 2008). However, AfaDddA and Halomonas sp. HTNK1 DddA proteins have a glutamine residue in the corresponding position, thereby supporting the hypothesis that compared with choline oxidases, DddA proteins have different substrate binding sites such as 3HP.
DddA is a member of the alcohol dehydrogenase superfamily. This enzyme catalyzes the oxidation of 3HP-CoA in the DMSP catabolism pathway, forming malonate semialdehyde (Reisch et al., 2011; Moran et al., 2012). Multiple sequence alignments of DddA from Alcaligenes faecalis (AfaDddA; UniProtKB code E7DDH7) and other dehydrogenases including DddA from Halomonas sp. HTNK1 (UniProtKB code C8YX89) and a choline oxidase from Arthrobacter globiformis (UniProtKB code Q7X2H8, PDB code 2JBV) (Quaye et al., 2008) were determined using the ClustalX program (Fig. 5A). The results showed that AfaDddA has the conserved and cata- lytically important residues H381 and H491. In the Arthrobacter globiformis choline oxidase structure, the E312 residue is involved in substrate binding (Quaye et al., 2008). However, AfaDddA and Halomonas sp. HTNK1 DddA proteins have a glutamine residue in the corresponding position, thereby supporting the hypothesis that compared with choline oxidases, DddA proteins have different substrate binding sites such as 3HP.
5. DddC (methylmalonate semialdehyde dehydrogenase)
The dddC gene is located near the DMSP lyase genes and encodes methylmalonate semialdehyde dehydrogenase (MMSDH) (Curson et al., 2012). MMSDH belongs to the CoA-dependent aldehyde dehydrogenase subfamily and is involved in the de- carboxylation of malonate semialdehyde downstream of the DMSP cleavage pathway (Todd et al., 2010). DddC, an MMSDH, cata- lyzes the transformation of malonate semialdehyde to acetyl-CoA and CO2 gas. The newly classified gram-negative marine γ-proteobacterium Oceanimonas doudoroffii (ATCC 27123) was isolated from seawater and has multiple DMSP lyase genes (Fig. 4).
Recently, our group successfully determined the crystal structure of the dddC (UniProtKB code G5CZI2) product from O. doudoroffii (OdoMMSDH). The molecular weight of OdoMMSDH was esti- mated to be approximately 120 kDa according to analytic size-exclusion chromatography. These results revealed that OdoMMSDH in solution undergoes self-association to form a dimer because the calculated molecular weight of the monomer was ~54 kDa (Do et al., 2016).
The results of multiple sequence alignments of OdoMMSDH with other MMSDH enzymes revealed that the R103, R279, and C280 residues are highly conserved (Fig. 6A). The conservation of amino acid residues across several species suggests that these residues have critical roles in enzyme activity or stability. The results of sequence and structural comparison studies suggested that the C280 residue is a catalytic site and that the highly conserved arginine residues may be crucial for substrate binding (Talfournier et al., 2011; Bchini et al., 2012; Do et al., 2016). Recent structural studies combined with the result of size-exclusion chromato- graphy have shown that OdoMMSDH is a dimeric structure and have determined the important residues involved in the substrate binding (Do et al., 2016) (Fig. 6B).
Conclusively, this review summarizes and discusses the structure, function, and enzymatic mechanisms of DddD, DddA, and DddC as participants in the pathways of DMSP catabolism. Additional structural and functional studies with site-directed mutagenesis may elicit additional information about key residues in these important enzymes.