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Biosynthesis of Starch
Published in Jean-Luc Wertz, Bénédicte Goffin, Starch in the Bioeconomy, 2020
Jean-Luc Wertz, Bénédicte Goffin
The α-subunit monomer of potato tuber AGPase is composed of an N-terminal catalytic domain and a C-terminal β-helix domain (Figure 3.6a).27 The overall fold of the catalytic domain shares a strong similarity with the three PPis whose structures have been elucidated, namely N-acetyl glucosamine-1-phosphate uridyltransferase (GlmU) and two G1P thymidylyltransferases (RmlA and RffH), although the primary sequences for the three distinct enzymes have very low similarities (Figure 3.6b). The catalytic domain is composed of a mostly parallel but mixed seven-stranded β-sheet covered by α-helices, a fold reminiscent of the dinucleotide-binding Rossmann fold. The catalytic domain makes strong hydrophobic interactions with the C-terminal β-helix domain via an α-helix that encompasses residues 285–297. The catalytic domain is connected to the C-terminal β-helix domain by a long loop containing residues 300–320. This loop makes numerous interactions with the equivalent region of another subunit.
Molecular Aspects of the Activity and Inhibition of the FAD-Containing Monoamine Oxidases
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
FAD, a redox cofactor important for catalysis in many enzymes, is derived from the vitamin riboflavin. As in most FAD-containing enzymes, the adenine part of the molecule binds to a Rossman fold in the protein. The isoalloxazine ring part of FAD is held in position by a covalently bond to a cysteine residue in MAO (Fig. 10.3). In all crystal structures of MAO B, the isoalloxazine ring is bent by about 30° (Binda et al., 2003) with about 0.3° difference between the oxidized and reduced forms. However, molecular dynamics has now enabled a more realistic picture of the shape of the flavin in the flexible protein active site. Two different studies have shown that it is almost planar in the oxidized form (FAD) (Vianello et al., 2012; Zapata-Torres et al., 2015), but when it is reduced either by a hydride ion from the substrate transferred to N5 in the first step of the catalytic mechanism or after the propargylamine adduct has formed at N5, the FADH2 is bent by almost 30 degrees (Borstnar et al., 2011) in agreement with the crystal structure.
Carbohydrate-Active Enzymes
Published in Jean-Luc Wertz, Magali Deleu, Séverine Coppée, Aurore Richel, Hemicelluloses and Lignin in Biorefineries, 2017
Jean-Luc Wertz, Magali Deleu, Séverine Coppée, Aurore Richel
As for other classes of carbohydrate-active enzymes, GTs are classified into families based on amino acid sequence similarities.12,13 The number of distinct GT families was 97 in June 2015. In striking contrast with glycosidases, which exhibit a wide variety of overall folds, nucleotide sugar-dependent (Leloir) GTs have been found to possess only two general folds termed GT-A and GT-B.11 This finding may indicate that the majority of GTs has evolved from a small number of progenitor sequences. However, it also reflects the requirement for at least one nucleotide-binding domain of the Rossmann fold14 type (structural motif found in proteins that bind nucleotides, especially the cofactor NAD). Other structural folds have been observed recently in non-Leloir GTs.
Retention of functional characteristics of glutathione-S-transferase and lactate dehydrogenase-A in fusion protein
Published in Preparative Biochemistry & Biotechnology, 2018
S. Lalitha Gavya, Neha Arora, Siddhartha Sankar Ghosh
To ascertain that both GST and LDHA are functionally active after fusion, their respective activity assays were performed. A Rossmann fold with a pair of β-α-β-α-β motifs forms the nucleotide binding site in LDHA, to which NADH binds to form a LDH/NADH substrate encounter complex suitable for the conversion of pyruvate to lactate.[45] The LDH activity against NADH and sodium pyruvate followed Michaelis–Menten kinetics. The kinetic parameters Km and Vmax were determined from the plot of activity over substrate concentration in GraphPad prism software (Figure 3a and 3b) using which, Kcat and Kcat/Km were calculated as illustrated in Table 1. The maximum velocity of the reaction for purified GST–hLDHA (0.667 mU µg−1 for sodium pyruvate and 0.6837 mU µg−1 for NADH) is lesser when compared to tetrameric LDH purified from breast tissues (1.7 mU µg−1 for pyruvate and 1.37 mU µg−1 for NADH).[39] This reduction in velocity can be attributed to the difference in number of catalytic subunits between the purified GST-hLDH and tetrameric LDH. However, the affinity for the substrates, sodium pyruvate and NADH, was higher in GST–hLDHA (Km 0.4988 mM for sodium pyruvate and 0.227 for NADH) in comparison with the tetrameric LDH (Km 0.63 mM for pyruvate and 0.33 mM for NADH).[39] Kinetic profile of LDH at temperature ranging from 4 to 50°C displayed an increase in activity with increase in temperature with maximum activity at 37°C above which, the activity decreases (Figure 3c). NADH and sodium pyruvate were dissolved in the corresponding buffers to maintain the desired pH. A plot of activity over pH showed maximum activity at pH 8 (Figure 3d). For both the assays, NADH and sodium pyruvate concentrations were maintained at 0.2 and 0.3 mM, respectively. Since pH 8 and 37°C are close to human physiological conditions, it leads to the inference that functionality of LDH in GST–hLDHA as well as its response to reaction conditions like temperature and pH is preserved.