Phosphoadenosine 5'-phosphosulfate Research Articles

Polysaccharide sulfotransferases: the identification of putative sequences and respective functional characterisation.

The vast structural diversity of sulfated polysaccharides demands an equally diverse array of enzymes known as polysaccharide sulfotransferases (PSTs). PSTs are present across all kingdoms of life, including algae, fungi and archaea, and their sulfation pathways are relatively unexplored. Sulfated polysaccharides possess anti-inflammatory, anticoagulant and anti-cancer properties and have great therapeutic potential. Current identification of PSTs using Pfam has been predominantly focused on the identification of glycosaminoglycan (GAG) sulfotransferases because of their pivotal roles in cell communication, extracellular matrix formation and coagulation. As a result, our knowledge of non-GAG PSTs structure and function remains limited. The major sulfotransferase families, Sulfotransfer_1 and Sulfotransfer_2, display broad homology and should enable the capture of a wide assortment of sulfotransferases but are limited in non-GAG PST sequence annotation. In addition, sequence annotation is further restricted by the paucity of biochemical analyses of PSTs. There are now high-throughput and robust assays for sulfotransferases such as colorimetric PAPS (3'-phosphoadenosine 5'-phosphosulfate) coupled assays, Europium-based fluorescent probes for ratiometric PAP (3'-phosphoadenosine-5'-phosphate) detection, and NMR methods for activity and product analysis. These techniques provide real-time and direct measurements to enhance the functional annotation and subsequent analysis of sulfated polysaccharides across the tree of life to improve putative PST identification and characterisation of function. Improved annotation and biochemical analysis of PST sequences will enhance the utility of PSTs across biomedical and biotechnological sectors.

Anthraquinone biocolourant dermocybin is metabolized whereas dermorubin is not in in vitro liver fractions and recombinant metabolic enzymes.

Fungal anthraquinones dermocybin and dermorubin are attractive alternatives for synthetic dyes but their metabolism is largely unknown. We conducted a qualitative in vitro study to identify their metabolism using human liver microsomes and cytosol, as well as recombinant human cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) enzymes. Additionally, liver microsomal and cytosolic fractions from rat, mouse and pig were used. Following incubations of the biocolourants with the enzymes in the presence of nicotinamide adenine dinucleotide phosphate, UDP-glucuronic acid, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) or S-adenosyl methionine (SAM) to enable CYP oxidation, glucuronidation, sulfonation or methylation, we observed several oxidation and conjugation metabolites for dermocybin but none for dermorubin. Human CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A7 catalysed dermocybin oxidation. The formation of dermocybin glucuronides was catalysed by human UGT1A1, 1A3, 1A7, 1A8, 1A9, 1A10 and 2B15. Human SULT1B1, 1C2 and 2A1 sulfonated dermocybin. Dermocybin oxidation was faster than conjugation in human liver microsomes. Species differences were seen in dermocybin glucuronidation between human, rat, mouse and pig. In conclusion, many CYP and conjugation enzymes metabolized dermocybin, whereas dermorubin was not metabolized in human liver fractions in vitro. The results indicate that dermocybin would be metabolized in humans in vivo.

Sulfate: a neglected (but potentially highly relevant) anion.

Sulfate is an important anion as sulfonation is essential in modulation of several compounds, such as exogens, polysaccharide chains of proteoglycans, cholesterol or cholesterol derivatives and tyrosine residues of several proteins. Sulfonation requires the presence of both the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and a sulfotransferase. Genetic disorders affecting sulfonation, associated with skeletal abnormalities, impaired neurological development and endocrinopathies, demonstrate the importance of sulfate. Yet sulfate is not measured in clinical practice. This review addresses sulfate metabolism and consequences of sulfonation defects, how to measure sulfate and why we should measure sulfate more often.

Enhancing the expression of chondroitin 4-O-sulfotransferase for one-pot enzymatic synthesis of chondroitin sulfate A

Chondroitin sulfate (CS) stands as a pivotal compound in dietary supplements for osteoarthritis treatment, propelling significant interest in the biotechnological pursuit of environmentally friendly and safe CS production. Enzymatic synthesis of CS for instance CSA has been considered as one of the most promising methods. However, the bottleneck consistently encountered is the active expression of chondroitin 4-O-sulfotransferase (C4ST) during CSA biosynthesis. This study meticulously delved into optimizing C4ST expression through systematic enhancements in transcription, translation, and secretion mechanisms via modifications in the 5′ untranslated region, the N-terminal encoding sequence, and the Komagataella phaffii chassis. Ultimately, the active C4ST expression escalated to 2713.1 U/L, representing a striking 43.7-fold increase. By applying the culture broth supernatant of C4ST and integrating the 3′-phosphoadenosine-5′-phosphosulfate (PAPS) biosynthesis module, we constructed a one-pot enzymatic system for CSA biosynthesis, achieving a remarkable sulfonation degree of up to 97.0 %. The substantial enhancement in C4ST expression and the development of an engineered one-pot enzymatic synthesis system promises to expedite large-scale CSA biosynthesis with customizable sulfonation degrees.

Identification and genomic analysis of a novel temperate bacteriophage infecting Labrenzia aggregata isolated from the Mariana Trench

In marine environments, viruses play a pivotal role, yet deep-sea bacteriophages remains largely uncharacterized. The bacterium Labrenzia aggregata RF14, isolated from the Mariana Trench at a depth of 4,000 meters, harbors prophage regions based on a previous study. In this study, we induced a temperate bacteriophage from it using mitomycin C. The bacteriophage exhibited an icosahedral structure with a non-extendable tail and was named vB_LagS-V1. The genome size of it is 39,329 bps with a 59.46% G+C content, encoding 60 putative open reading frames. Genomic and phylogenetic analyses demonstrated that vB_LagS-V1 along with many bacteriophages infecting Hyphomicrobiales, constituted a newly unclassified family, which we designated as Hyphoviridae. Within this novel family, vB_LagS-V1 is distinct with isolated phages and clustered with two uncultured prophages within Labrenzia, forming an unclassified new genus, given a name of Labrenmarinevirus. The codon usage correlation and absence of tRNAs found in vB_LagS-V1, also prevail in some deep-sea bacteriophages, highlighting their adaptations to the deep-sea prokaryotic hosts. Moreover, vB_LagS-V1 encoded two auxiliary metabolic genes, cysteine dioxygenase and phosphoadenosine phosphosulfate reductase, which might help the phage and its host adapt to high hydrostatic pressure in the deep-sea environments. Our study will significantly contribute to the understanding of deep-sea bacteriophages and their interactions with hosts in extreme environments.

Bioinformatic Analysis of Sulfotransferases from an Unexplored Gut Microbe, Sutterella wadsworthensis 3_1_45B: Possible Roles towards Detoxification via Sulfonation by Members of the Human Gut Microbiome.

Sulfonation, primarily facilitated by sulfotransferases, plays a crucial role in the detoxification pathways of endogenous substances and xenobiotics, promoting metabolism and elimination. Traditionally, this bioconversion has been attributed to a family of human cytosolic sulfotransferases (hSULTs) known for their high sequence similarity and dependence on 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfo donor. However, recent studies have revealed the presence of PAPS-dependent sulfotransferases within gut commensals, indicating that the gut microbiome may harbor a diverse array of sulfotransferase enzymes and contribute to detoxification processes via sulfation. In this study, we investigated the prevalence of sulfotransferases in members of the human gut microbiome. Interestingly, we stumbled upon PAPS-independent sulfotransferases, known as aryl-sulfate sulfotransferases (ASSTs). Our bioinformatics analyses revealed that members of the gut microbial genus Sutterella harbor multiple asst genes, possibly encoding multiple ASST enzymes within its members. Fluctuations in the microbes of the genus Sutterella have been associated with various health conditions. For this reason, we characterized 17 different ASSTs from Sutterella wadsworthensis 3_1_45B. Our findings reveal that SwASSTs share similarities with E. coli ASST but also exhibit significant structural variations and sequence diversity. These differences might drive potential functional diversification and likely reflect an evolutionary divergence from their PAPS-dependent counterparts.

The Multienzyme Complex Nature of Dehydroepiandrosterone Sulfate Biosynthesis.

Dehydroepiandrosterone (DHEA), a precursor of steroid sex hormones, is synthesized by steroid 17-alpha-hydroxylase/17,20-lyase (CYP17A1) with the participation of microsomal cytochrome b5 (CYB5A) and cytochrome P450 reductase (CPR), followed by sulfation by two cytosolic sulfotransferases, SULT1E1 and SULT2A1, for storage and transport to tissues in which its synthesis is not available. The involvement of CYP17A1 and SULTs in these successive reactions led us to consider the possible interaction of SULTs with DHEA-producing CYP17A1 and its redox partners. Text mining analysis, protein-protein network analysis, and gene co-expression analysis were performed to determine the relationships between SULTs and microsomal CYP isoforms. For the first time, using surface plasmon resonance, we detected interactions between CYP17A1 and SULT2A1 or SULT1E1. SULTs also interacted with CYB5A and CPR. The interaction parameters of SULT2A1/CYP17A1 and SULT2A1/CYB5A complexes seemed to be modulated by 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Affinity purification, combined with mass spectrometry (AP-MS), allowed us to identify a spectrum of SULT1E1 potential protein partners, including CYB5A. We showed that the enzymatic activity of SULTs increased in the presence of only CYP17A1 or CYP17A1 and CYB5A mixture. The structures of CYP17A1/SULT1E1 and CYB5A/SULT1E1 complexes were predicted. Our data provide novel fundamental information about the organization of microsomal CYP-dependent macromolecular complexes.

Sulfation of chondroitin and bile acids converges to antagonize Wnt/β-catenin signaling and inhibit APC deficiency-induced gut tumorigenesis

Sulfation is a crucial and prevalent conjugation reaction involved in cellular processes and mammalian physiology. 3′-Phosphoadenosine 5′-phosphosulfate (PAPS) synthase 2 (PAPSS2) is the primary enzyme to generate the universal sulfonate donor PAPS. The involvement of PAPSS2-mediated sulfation in adenomatous polyposis coli (APC) mutation-promoted colonic carcinogenesis has not been reported. Here, we showed that the expression of PAPSS2 was decreased in human colon tumors along with cancer stages, and the lower expression of PAPSS2 was correlated with poor prognosis in advanced colon cancer. Gut epithelial-specific heterozygous Apc deficient and Papss2-knockout (ApcΔgut-HetPapss2Δgut) mice were created, and the phenotypes were compared to the spontaneous intestinal tumorigenesis of ApcΔgut-Het mice. ApcΔgut-HetPapss2Δgut mice were more sensitive to gut tumorigenesis, which was mechanistically accounted for by the activation of Wnt/β-catenin signaling pathway due to the suppression of chondroitin sulfation and inhibition of the farnesoid X receptor (FXR)-transducin-like enhancer of split 3 (TLE3) gene regulatory axis. Chondroitin sulfate supplementation in ApcΔgut-HetPapss2Δgut mice alleviated intestinal tumorigenesis. In summary, we have uncovered the protective role of PAPSS2-mediated chondroitin sulfation and bile acids–FXR–TLE3 activation in the prevention of gut carcinogenesis via the antagonization of Wnt/β-catenin signaling. Chondroitin sulfate may be explored as a therapeutic agent for Papss2 deficiency-associated colonic carcinogenesis.

Role of Conformational Dynamics of Sulfotransferases SULT1A1 and SULT1A3 in Substrate Specificity

Sulfotransferases (SULTs) are phase II metabolizing enzymes catalyzing the sulfoconjugation from the co-factor 3′-Phosphoadenosine 5′-Phosphosulfate (PAPS) to a wide variety of endogenous compounds, drugs and natural products. Although SULT1A1 and SULT1A3 share 93% identity, SULT1A1, the most abundant SULT isoform in humans, exhibits a broad substrate range with specificity for small phenolic compounds, while SULT1A3 displays a high affinity toward monoamine neurotransmitters like dopamine. To elucidate the factors determining the substrate specificity of the SULT1 isoenzymes, we studied the dynamic behavior and structural specificities of SULT1A1 and SULT1A3 by using molecular dynamics (MD) simulations and ensemble docking of common and specific substrates of the two isoforms. Our results demonstrated that while SULT1A1 exhibits a relatively rigid structure by showing lower conformational flexibility except for the lip (loop L1), the loop L2 and the cap (L3) of SULT1A3 are extremely flexible. We identified protein residues strongly involved in the recognition of different substrates for the two isoforms. Our analyses indicated that being more specific and highly flexible, the structure of SULT1A3 has particularities in the binding site, which are crucial for its substrate selectivity.

Engineering sulfonate group donor regeneration systems to boost biosynthesis of sulfated compounds

Sulfonation as one of the most important modification reactions in nature is essential for many biological macromolecules to function. Development of green sulfonate group donor regeneration systems to efficiently sulfonate compounds of interest is always attractive. Here, we design and engineer two different sulfonate group donor regeneration systems to boost the biosynthesis of sulfated compounds. First, we assemble three modules to construct a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) regeneration system and demonstrate its applicability for living cells. After discovering adenosine 5’-phosphosulfate (APS) as another active sulfonate group donor, we engineer a more simplified APS regeneration system that couples specific sulfotransferase. Next, we develop a rapid indicating system for characterizing the activity of APS-mediated sulfotransferase to rapidly screen sulfotransferase variants with increased activity towards APS. Eventually, the active sulfonate group equivalent values of the APS regeneration systems towards trehalose and p-coumaric acid reach 3.26 and 4.03, respectively. The present PAPS and APS regeneration systems are environmentally friendly and applicable for scaling up the biomanufacturing of sulfated products.

Analysis of evolutionary and functional features of the bullfrog SULT1 family

We identified the bullfrog Rana catesbeiana sulfotransferase 1 (SULT1) family from the BLAST search tool of the public databases based on the SULT1 families of Nanorana parkeri, Xenopus laevis, and Xenopus tropicalis as queries, revealing the characteristics of the anuran SULT1 family. The results showed that the anuran SULT1 family comprises six subfamilies, four of which were related to the mammalian SULT1 subfamily. Additionally, the bullfrog has two SULT1Cc subfamily members that are consistent with the characteristics of the expanded Xenopus SULT1C subfamily. Several members of the bullfrog SULT1 family were suggested to play important roles in sulfation during metamorphosis. Among these, cDNAs encoding SULT1Cc1 and SULT1Y1 were cloned, and the sulfation activity was analyzed using recombinant proteins. The affinity for 2-naphthol and 3′-phosphoadenosine 5′-phosphosulfate (PAPS) and the enzymatic reaction rate were higher in SULT1Cc1 than in SULT1Y1. Both the enzymes showed inhibitory effect of many thyroid hormones (THs) analogs on the sulfation of 2-naphthol. The potency of sulfation activities of SULT1Cc1 and SULT1Y1 against T4 indicated their possible role in the intracellular T4 clearance during metamorphosis.

ADY tripeptide is a minimum sequence for Tyrosylprotein sulfotransferases 1 and 2 substrate recognition

Tyrosylprotein sulfotransferases (TPSTs) catalyze the transfer of a sulphonate moiety from 3′-Phosphoadenosine 5′-Phosphosulfate (PAPS) to the hydroxyl group of a tyrosine residue in substrate proteins. The positively charged substrate binding region of TPST homodimer interacts with acidic residues located in N-terminal region from the sulfated tyrosine in substrates. However, the sequence pattern in TPST substrate recognition remains unclear. Therefore, we aimed to determine the minimum recognition chain length required for tyrosine sulfation. We prepared His-tagged polypeptide, His-TPST143-370 and His-TPST243-377, form 43–370 of TPST1 and 43–377 of TPST2. Next, we prepared a series of synthesized ADYAE peptides and used a combination of reverse phase high-performance liquid chromatography (RP-HPLC) and mass spectrometric analysis to show that the tripeptide amino acid sequence, ADY, was sulfated by TPST1 and TPST2. Furthermore, we found that the acidic residue, located two residues C-terminal region from the tyrosine residue, may be involved in the TPST-induced sulfation regulation. The results in our study propose that proteins with the ADY sequence may be useful for searching the novel TPST tyrosine sulfated substrates.

A tale of two switches: Redox regulation of adenosine-5′-phosphosulfate kinase in humans and plants

The sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is a near-universal component of sulfur metabolism. In a report by Zhang etal. in this issue of Structure, X-ray crystal structures of the APS kinase domains from human PAPS synthase reveal dynamic substrate recognition and a regulatory "redox switch" analogous to that previously described only in plant APS kinases.

Assimilatory sulfate reduction in the marine methanogen Methanothermococcus thermolithotrophicus

Methanothermococcus thermolithotrophicus is the only known methanogen that grows on sulfate as its sole sulfur source, uniquely uniting methanogenesis and sulfate reduction. Here we use physiological, biochemical and structural analyses to provide a snapshot of the complete sulfate reduction pathway of this methanogenic archaeon. We find that later steps in this pathway are catalysed by atypical enzymes. PAPS (3′-phosphoadenosine 5′-phosphosulfate) released by APS kinase is converted into sulfite and 3′-phosphoadenosine 5′-phosphate (PAP) by a PAPS reductase that is similar to the APS reductases of dissimilatory sulfate reduction. A non-canonical PAP phosphatase then hydrolyses PAP. Finally, the F420-dependent sulfite reductase converts sulfite to sulfide for cellular assimilation. While metagenomic and metatranscriptomic studies suggest that the sulfate reduction pathway is present in several methanogens, the sulfate assimilation pathway in M. thermolithotrophicus is distinct. We propose that this pathway was ‘mix-and-matched’ through the acquisition of assimilatory and dissimilatory enzymes from other microorganisms and then repurposed to fill a unique metabolic role.

Psychrobacter Phage Encoding an Antibiotics Resistance Gene Represents a Novel Caudoviral Family.

Psychrobacter is an important bacterial genus that is widespread in Antarctic and marine environments. However, to date, only two complete Psychrobacter phage sequences have been deposited in the NCBI database. Here, the novel Psychrobacter phage vB_PmaS_Y8A, infecting Psychrobacter HM08A, was isolated from sewage in the Qingdao area, China. The morphology of vB_PmaS_Y8A was characterized by transmission electron microscopy, revealing an icosahedral head and long tail. The genomic sequence of vB_PmaS_Y8A is linear, double-stranded DNA with a length of 40,226 bp and 44.1% G+C content, and encodes 69 putative open reading frames. Two auxiliary metabolic genes (AMGs) were identified, encoding phosphoadenosine phosphosulfate reductase and MarR protein. The first AMG uses thioredoxin as an electron donor for the reduction of phosphoadenosine phosphosulfate to phosphoadenosine phosphate. MarR regulates multiple antibiotic resistance mechanisms in Escherichia coli and is rarely found in viruses. No tRNA genes were identified and no lysogeny-related feature genes were detected. However, many similar open reading frames (ORFs) were found in the host genome, which may indicate that Y8A also has a lysogenic stage. Phylogenetic analysis based on the amino acid sequences of whole genomes and comparative genomic analysis indicate that vB_PmaS_Y8A contains a novel genomic architecture similar only to that of Psychrobacter phage pOW20-A, although at a low similarity. vB_PmaS_Y8A represents a new family-level virus cluster with 22 metagenomic assembled viral genomes, here named Minviridae. IMPORTANCE Although Psychrobacter is a well-known and important bacterial genus that is widespread in Antarctic and marine environments, genetic characterization of its phages is still rare. This study describes a novel Psychrobacter phage containing an uncharacterized antibiotic resistance gene and representing a new virus family, Minviridae. The characterization provided here will bolster current understanding of genomes, diversity, evolution, and phage-host interactions in Psychrobacter populations.

Protein engineering, cofactor engineering, and surface display engineering to achieve whole-cell catalytic production of chondroitin sulfate A.

Chondroitin sulfate A (CSA) is a valuable glycosaminoglycan that has great market demand. However, current synthetic methods are limited by requiring the expensive sulfate group donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and inefficient enzyme carbohydrate sulfotransferase 11 (CHST11). Herein, we report the design and integration of the PAPS synthesis and sulfotransferase pathways to realize whole-cell catalytic production of CSA. Using mechanism-based protein engineering, we improved the thermostability and catalytic efficiency of CHST11; its Tm and half-life increased by 6.9°C and 3.5 h, respectively, and its specific activity increased 2.1-fold. Via cofactor engineering, we designed a dual-cycle strategy of regenerating ATP and PAPS to increase the supply of PAPS. Through surface display engineering, we realized the outer membrane expression of CHST11 and constructed a whole-cell catalytic system of CSA production with an 89.5% conversion rate. This whole-cell catalytic process provides a promising method for the industrial production of CSA.

Enzymatic sulfation of coelenterazine by human cytosolic aryl sulfotransferase SULT1A1: identification of coelenterazine C2-benzyl monosulfate by LC/ESI-TOF-MS

Coelenterazine (CTZ) is known as a light-emitting source for the bioluminescence reaction in marine organisms. CTZ has two phenolic hydroxy groups at the C2-benzyl and C6-phenyl positions, and a keto-enol type hydroxy group at the C3-position in the core structure of imidazopyrazinone (= 3,7-dihydroimidazopyrazin-3-one). These hydroxy groups in CTZ could be sulfated by sulfotransferase(s), and the sulfates of Watasenia luciferin (CTZ disulfate at the C2- and C6-positions) and Renilla pre-luciferin (CTZ 3-enol sulfate) have been identified in marine organisms. To characterize the sulfation process of CTZ, human cytosolic aryl sulfotransferase SULT1A1 (SUTase) was used as a model enzyme. The sulfated products catalyzed by SUTase with 3′-phosphoadenosine 5′-phosphosulfate (PAPS) were analyzed by LC/ESI-TOF-MS. The product was the monosulfate of CTZ and identified as the C2-benzyl sulfate of CTZ (CTZ C2-benzyl monosulfate), but CTZ disulfate, CTZ 3-enol sulfate, and CTZ C6-phenyl monosulfate were not detected. The non-enzymatic oxidation products of dehydrocoelenterazine (dCTZ, dehydrogenated derivative of CTZ), coelenteramide (CTMD), and coelenteramine (CTM) from CTZ were also identified as their monosulfates.

Redox switching mechanism of the adenosine 5′-phosphosulfate kinase domain (APSK2) of human PAPS synthase 2

Adenosine 5'-phosphosulfate kinase (APSK) catalyzes the rate-limiting biosynthetic step of the universal sulfuryl donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS). In higher eukaryotes, the APSK and ATP sulfurylase (ATPS) domains are fused in a single chain. Humans have two bifunctional PAPS synthetase isoforms: PAPSS1 with the APSK1 domain and PAPSS2 containing the APSK2 domain. APSK2 displays a distinct higher activity for PAPSS2-mediated PAPS biosynthesis during tumorigenesis. How APSK2 achieves excess PAPS production has remained unclear. APSK1 and APSK2 lack the conventional redox-regulatory element present in plant PAPSS homologs. Here we elucidate the dynamic substrate recognition mechanism of APSK2. We discover that APSK1 contains a species-specific Cys-Cys redox-regulatory element that APSK2 lacks. The absence of this element in APSK2 enhances its enzymatic activity for excess PAPS production and promotes cancer development. Our results help to understand the roles of human PAPSSs during cell development and may facilitate PAPSS2-specific drug discovery.

Deciphering the biodesulfurization pathway employing marine mangrove Bacillus aryabhattai strain NM1-A2 according to whole genome sequencing and transcriptome analyses

In the biogeochemical cycle, sulfur oxidation plays a vital role and is typically referred to as the elemental sulfur or reductive sulfide oxidation process. This study aimed to characterize a subtropical mangrove-isolated bacterial strain using biochemical, whole-genome, and transcriptome sequencing analyses to enhance our understanding of sulfur metabolism and biodegradation from a molecular genetic perspective. Strain NM1-A2 was characterized as Gram-positive and found to have a close molecular phylogenetic relationship with Bacillus aryabhattai. NM1-A2 efficiently converted dibenzothiophene (DBT) into 2-hydroxybiphenyl (2-HBP) via a 4S pathway with 95% efficiency, using enzymes encoded by the dsz operon (dszA, dszB, and dszC), which determine monooxygenases (DszA & DszC) and desulfinase (DszB). The whole-genome sequence of NM1-A2 had a length of approximately 5,257,678 bp and included 16 sulfur metabolism-related genes, featuring the ABC transport system, small subunit (ssu) and cysteine (cys) gene families, and adenosine 5′-phosphosulfate (APS) and 3′-phosphoadenosine-5′-phosphosulfate (PAPS) biosynthesis-related genes. Transcriptomic analysis of NM1-A2 using three sulfur groups—magnesium sulfate (MS), sulfur powder (SP), and sodium thiosulfate (ST) resulted in a significant number of differentially expressed genes (1200, 2304, and 2001, respectively). This analysis revealed that intracellular cysteine concentration directly regulated the expression of cys and ssu genes. Sulfate did not directly affect cys gene expression but repressed ssu gene expression. The cys gene expression levels decreased during the conversion of sulfate to sulfide and cysteine. The transcriptomic data was validated by analyzing the expression patterns of NM1-A2 using real-time quantitative PCR validation analysis. The expression levels of cysl, mccB, and nrnA were significantly upregulated, while cysH, metB, and sat were downregulated in the SP, ST, and MS groups, respectively. This research contributes to our understanding of marine mangrove microorganisms' bacterial efficiency through characterization, whole-genome, and transcriptome sequencing-based molecular degradation of organic compounds in the mangrove ecosystem, which may enhance nutrient availability.

Construction of a Protein Crystalline Inclusion-Based Enzyme Immobilization System for Biosynthesis of PAPS from ATP and Sulfate.

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is the bioactive form of sulfate and is involved in all biological sulfation reactions. The enzymatic transformation method for PAPS is promising, but the low efficiency and high cost of enzyme purification and storage restrict its practical applications. Here, we reported PAPS biosynthesis with a protein crystalline inclusion (PCI)-based enzyme immobilization system. First, the in vivo crystalline inclusion protein CipA was identified as an efficient auto-assembly tag for immobilizing the bifunctional PAPS synthase (ASAK). After characterizing the pyrophosphokinase activity of a polyphosphate exonuclease PaPPX from Pseudomonas aeruginosa, and optimizing the linker fragment, auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA were constructed. Then, the auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA with high stability were co-expressed and immobilized for constructing a transformation system. The highest transformation rate of PAPS from ATP and sulfate reached 90%, and the immobilized enzyme can be reused 10 times. The present work provided a convenient, efficient, and easy to be enlarged auto-immobilization system for PAPS biosynthesis from ATP and sulfate. The immobilization system also represented a new approach to reduce the production cost of PAPS by facilitating the purification, storage, and reuse of related enzymes, and it would boost the studies on biotechnological production of glycosaminoglycans and sulfur-containing natural compounds.