Long-term monitoring for short/branched-chain acyl-CoA dehydrogenase deficiency: A single-center 4-year experience and open issues.

Introduction: Short/branched-chain acyl-CoA dehydrogenase deficiency (SBCADD) is an inherited disorder of L-isoleucine metabolism due to mutations in the ACADSB gene. The role of current diagnostic biomarkers [i.e., blood 2-methylbutyrylcarnitine (C5) and urine 2-methylbutyrylglycine (2MBG)] in patient monitoring and the effects of proposed treatments remain uncertain as follow-data are lacking. This study presents first systematic longitudinal biochemical assessment in SBCADD patients. Methods: A retrospective, observational single-center study was conducted on newborns born between 2017 and 2020 and suspected with SBCADD. Biochemical, molecular, clinical and dietary data collected upon NBS recall and during the subsequent follow-up were recorded. Results: All enrolled subjects (n = 10) received adequate protein intake and L-carnitine supplementation. Nine subjects were diagnosed with SBCADD. During the follow-up [median: 20.5 (4-40) months] no patient developed symptoms related to SBCADD. No patient normalized serum C5 and urine 2MBG values. In 7/9 SBCADD patients mean serum C5 values decreased or stabilized compared to their first serum C5 value. A major increase in serum C5 values was observed in two patients after L-carnitine discontinuation and during intercurrent illness, respectively. Urine 2MBG values showed moderate intra-patient variability. Discussion: The relatively stable serum C5 values observed during L-carnitine supplementation together with C5 increase occurring upon L-carnitine discontinuation/intercurrent illness may support the value of serum C5 as a monitoring biomarker and the benefit of this treatment in SBCADD patients. The role of urine 2MBG in patient monitoring remains uncertain. As all patients were asymptomatic, no association between biochemical parameters and clinical phenotype could be investigated in this study.

A one-step procedure for immobilising the thermostable carbonic anhydrase (SspCA) on the surface membrane of Escherichia coli.

The carbonic anhydrase superfamily (CA, EC 4.2.1.1) of metalloenzymes is present in all three domains of life (Eubacteria, Archaea, and Eukarya), being an interesting example of convergent/divergent evolution, with its seven families (α-, ß-, γ-, δ-, ζ-, η-, and θ-CAs) described so far. CAs catalyse the simple, but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons. Recently, our groups characterised the α-CA from the thermophilic bacterium, Sulfurihydrogenibium yellowstonense finding a very high catalytic activity for the CO2 hydration reaction (kcat = 9.35 × 105 s-1 and kcat/Km = 1.1 × 108 M-1 s-1) which was maintained after heating the enzyme at 80 °C for 3 h. This highly thermostable SspCA was covalently immobilised within polyurethane foam and onto the surface of magnetic Fe3O4 nanoparticles. Here, we describe a one-step procedure for immobilising the thermostable SspCA directly on the surface membrane of Escherichia coli, using the INPN domain of Pseudomonas syringae. This strategy has clear advantages with respect to other methods, which require as the first step the production and the purification of the biocatalyst, and as the second step the immobilisation of the enzyme onto a specific support. Our results demonstrate that thermostable SspCA fused to the INPN domain of P. syringae ice nucleation protein (INP) was correctly expressed on the outer membrane of engineered E. coli cells, affording for an easy approach to design biotechnological applications for this highly effective thermostable catalyst.

Sequence Analysis, Kinetic Constants, and Anion Inhibition Profile of the Nacrein-Like Protein (CgiNAP2X1) from the Pacific Oyster Magallana gigas (Ex-Crassostrea gigas).

The carbonic anhydrase (CA, EC 4.2.1.1) superfamily of metalloenzymes catalyzes the hydration of carbon dioxide to bicarbonate and protons. The catalytically active form of these enzymes incorporates a metal hydroxide derivative, the formation of which is the rate-determining step of catalytic reaction, being affected by the transfer of a proton from a metal-coordinated water molecule to the environment. Here, we report the cloning, expression, and purification of a particular CA, i.e., nacrein-like protein encoded in the genome of the Pacific oyster Magallana gigas (previously known as Crassostrea gigas). Furthermore, the amino acid sequence, kinetic constants, and anion inhibition profile of the recombinant enzyme were investigated for the first time. The new protein, CgiNAP2X1, is highly effective as catalyst for the CO2 hydration reaction, based on the measured kinetic parameters, i.e., kcat = 1.0 × 106 s-1 and kcat/KM = 1.2 × 108 M-1·s-1. CgiNAP2X1 has a putative signal peptide, which probably allows an extracellular localization of the protein. The inhibition data demonstrated that the best anion inhibitors of CgiNAP2X1 were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, which showed a micromolar affinity for this enzyme, with KIs in the range of 76-87 µM. These studies may add new information on the physiological role of the molluskan CAs in the biocalcification processes.

Cloning, expression and purification of the α-carbonic anhydrase from the mantle of the Mediterranean mussel, Mytilus galloprovincialis.

We cloned, expressed, purified, and determined the kinetic constants of the recombinant α-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the α-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32 kDa. Moreover, the determined kinetic parameters for the CO2 hydration reaction were kcat = 4.2 × 105 s-1 and kcat/Km of 3.5 × 107 M-1 ×s-1. Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50 kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of α-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.

Production and covalent immobilisation of the recombinant bacterial carbonic anhydrase (SspCA) onto magnetic nanoparticles.

Carbonic anhydrases (CAs; EC 4.2.1.1) are metalloenzymes with a pivotal potential role in the biomimetic CO2 capture process (CCP) because these biocatalysts catalyse the simple but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons in all life kingdoms. The CAs are among the fastest known enzymes, with kcat values of up to 106 s-1 for some members of the superfamily, providing thus advantages when compared with other CCP methods, as they are specific for CO2. Thermostable CAs might be used in CCP technology because of their ability to perform catalysis in operatively hard conditions, typical of the industrial processes. Moreover, the improvement of the enzyme stability and its reuse are important for lowering the costs. These aspects can be overcome by immobilising the enzyme on a specific support. We report in this article that the recombinant thermostable SspCA (α-CA) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense can been heterologously produced by a high-density fermentation of Escherichia coli cultures, and covalently immobilised onto the surface of magnetic Fe3O4 nanoparticles (MNP) via carbodiimide activation reactions. Our results demonstrate that using a benchtop bioprocess station and strategies for optimising the bacterial growth, it is possible to produce at low cost a large amount SspCA. Furthermore, the enzyme stability and storage greatly increased through the immobilisation, as SspCA bound to MNP could be recovered from the reaction mixture by simply using a magnet or an electromagnetic field, due to the strong ferromagnetic properties of Fe3O4.

Biochemical characterization of the native α-carbonic anhydrase purified from the mantle of the Mediterranean mussel, Mytilus galloprovincialis.

A α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified and characterized biochemically from the mollusk Mytilus galloprovincialis. As in most mollusks, this α-CA is involved in the biomineralization processes leading to the precipitation of calcium carbonate in the mussel shell. The new enzyme had a molecular weight of 50 kDa, which is roughly two times higher than that of a monomeric α-class enzyme. Thus, Mytilus galloprovincialis α-CA is either a dimer, or similar to the Tridacna gigas CA described earlier, may have two different CA domains in its polypeptide chain. The Mytilus galloprovincialis α-CA sequence contained the three His residues acting as zinc ligands and the gate-keeper residues present in all α-CAs (Glu106-Thr199), but had a Lys in position 64 and not a His as proton shuttling residue, being thus similar to the human isoform hCA III. This probably explains the relatively low catalytic activity of Mytilus galloprovincialis α-CA, with the following kinetic parameters for the CO2 hydration reaction: kcat = 4.1 × 105 s-1 and kcat/Km of 3.6 × 107 M-1 × s-1. The enzyme activity was poorly inhibited by the sulfonamide acetazolamide, with a KI of 380 nM. This study is one of the few describing in detail the biochemical characterization of a molluskan CA and may be useful for understanding in detail the phylogeny of these enzymes, their role in biocalcification processes and their potential use in the biomimetic capture of the CO2.