Structural and biochemical basis of a marine bacterial glycoside hydrolase family 2 β-glycosidase with broad substrate specificity

Uronic acids are commonly found in marine polysaccharides and increase structural complexity sanand intrinsic recalcitrance to enzymatic attack. The glycoside hydrolase family 2 (GH2) include proteins that target sugar conjugates with hexuronates and are involved in the catabolism and cycling of marine polysaccharides. Here, we reported a novel GH2, Aq GalA from a marine algae-associated Bacteroidetes with broad-substrate specificity. Biochemical analyses revealed that Aq GalA exhibits hydrolyzing activities against β-galacturonide, β-glucuronide, and β-galactopyranoside via retaining mechanisms. We solved the Aq GalA crystal structure in complex with galacturonic acid (GalA) and showed (via mutagenesis) that charge characteristics at uronate-binding subsites controlled substrate selectivity for uronide hydrolysis. Additionally, conformational flexibility of the Aq GalA active site pocket was proposed as a key component for broad substrate enzyme selectivity. Our Aq GalA structural and functional data augments the current understanding of substrate recognition of GH2 enzymes and provided key insights into the bacterial use of uronic acid containing polysaccharides. IMPORTANCE The decomposition of algal glycans driven by marine bacterial communities represents one of the largest heterotrophic transformation of organic matter fueling marine food webs and global carbon cycling. However, our knowledge of the carbohydrate cycling is limited due to structural complexity of marine polysaccharides and the complicated enzymatic machinery of marine microbes. To degrade algal glycan, marine bacteria such as members of Bacteroidetes produce a complex repertoire of carbohydrate-active enzymes (CAZymes) matching the structural specificity of the different carbohydrates. In this study, we investigated an extracellular GH2 β-glycosidase, Aq GalA from a marine Bacteroidetes to identify the key components responsible for glycuronides recognition and hydrolysis. The broad substrate specificity of Aq GalA against glycosides with diverse stereochemical substitutions indicates its potential in processing complex marine polysaccharides. Our findings promote a better understanding of microbially-driven mechanisms of marine carbohydrate cycling.

P-Type ATPase Apt1 of the Fungal Pathogen Cryptococcus neoformans Is a Lipid Flippase of Broad Substrate Specificity

Lipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the P4-ATPase Apt1p is an important regulator of polysaccharide secretion and pathogenesis, but its biochemical characterization is lacking. Phylogenetic analysis revealed that Apt1p belongs to the subclade of P4A-ATPases characterized by the common requirement for a β-subunit. Using heterologous expression in S. cerevisiae, we demonstrate that Apt1p forms a heterodimeric complex with the C. neoformans Cdc50 protein. This association is required for both localization and activity of the transporter complex. Lipid flippase activity of the heterodimeric complex was assessed by complementation tests and uptake assays employing fluorescent lipids and revealed a broad substrate specificity, including several phospholipids, the alkylphospholipid miltefosine, and the glycolipids glucosyl- and galactosylceramide. Our results suggest that transbilayer lipid transport in C. neoformans is finely regulated to promote fungal virulence, which reinforces the potential of Apt1p as a target for antifungal drug development.

A new carboxypeptidase from Aspergillus niger with good thermostability, pH stability and broad substrate specificity

A new serine carboxypeptidase gene, capA, was identified in Aspergillus niger CBS 513.88 by reading genomic information and performing sequence alignment, and the gene was cloned and expressed in Pichia pastoris GS115. In a shake flask, the enzyme activity of the recombinant strain GS115 (pPIC9K-capA) reached 209.3 U mg−1. The optimal temperature and pH for enzyme activity were determined to be 45 °C and 6.0, respectively. After incubation at 40–50 °C or at pH 4.0–8.0 for 1 h, the enzyme retained more than 80% or 60% of its initial activity. The presence of 1–10 mmol L−1 Mg2+ enhanced the activity of CapA, whereas 1–10 mmol L−1 Cu2+, Fe2+, or Co2+, 10 mmol L−1 Mn2+, or 1–10 mmol L−1 phenylmethylsulfonyl fluoride (PMSF) significantly inhibited its activity. CapA had a broad substrate specificity and preferred the hydrophobic amino acids Leu and Lys at the C terminus of proteins, and N-benzyloxycarbonyl-l-phenylalanyl-l-leucine (Cbz-Phe-Leu) was the optimal substrate, for which CapA exhibited Km 0.063 mmol L−1 and kcat/Km 186.35 mmol L−1 s−1. The good thermostability, pH stability and hydrolysis characteristics of CapA provide a solid foundation for application in the food and biotechnology fields.

Polyphenol-Hydroxylating Tyrosinase Activity under Acidic pH Enables Efficient Synthesis of Plant Catechols and Gallols

Tyrosinase is generally known as a melanin-forming enzyme, facilitating monooxygenation of phenols, oxidation of catechols into quinones, and finally generating biological melanin. As a homologous form of tyrosinase in plants, plant polyphenol oxidases perform the same oxidation reactions specifically toward plant polyphenols. Recent studies reported synthetic strategies for large scale preparation of hydroxylated plant polyphenols, using bacterial tyrosinases rather than plant polyphenol oxidase or other monooxygenases, by leveraging its robust monophenolase activity and broad substrate specificity. Herein, we report a novel synthesis of functional plant polyphenols, especially quercetin and myricetin from kaempferol, using screened bacterial tyrosinases. The critical bottleneck of the biocatalysis was identified as instability of the catechol and gallol under neutral and basic conditions. To overcome such instability of the products, the tyrosinase reaction proceeded under acidic conditions. Under mild acidic conditions supplemented with reducing agents, a bacterial tyrosinase from Bacillus megaterium (BmTy) displayed efficient consecutive two-step monophenolase activities producing quercetin and myricetin from kaempferol. Furthermore, the broad substrate specificity of BmTy toward diverse polyphenols enabled us to achieve the first biosynthesis of tricetin and 3′-hydroxyeriodictyol from apigenin and naringenin, respectively. These results suggest that microbial tyrosinase is a useful biocatalyst to prepare plant polyphenolic catechols and gallols with high productivity, which were hardly achieved by using other monooxygenases such as cytochrome P450s.

Nudix proteins affecting microbial pathogenesis

Nudix proteins catalyse hydrolysis of pyrophosphate bonds in a variety of substrates and are ubiquitous in all domains of life. Their widespread presence and broad substrate specificity suggest that they have important cellular functions. In this review, we summarize the state of knowledge on microbial Nudix proteins involved in pathogenesis.