Mation of abietadiene, neoabietadiene, palustradiene, and levopimaradiene, constant using the GC
Mation of abietadiene, neoabietadiene, palustradiene, and levopimaradiene, consistent with the GC S final results previously obtained for Pt DTPS LAS from P. taeda [31]. On the basis of such sequence similarity, Pnl DTPS1 could be predicted to be involved within the synthesis of abietane-type diterpene olefins. Interestingly, however, when aligned using the other group-1 DTPSs (Figure S7), Pnl DTPS1 from Calabrian pine revealed distinctive amino acids substitutions, namely D/G-515, G/E-565, and D/N-632, which could cause a adjust in the protein structure and hence in its solution(s) profile. The Pnl DTPS2 was found to become closely related to 4 PI3KC2β Compound mono-I DTPSs belonging to the phylogenetic group two (Figure 3), for which Hall et al. [22] observed no biochemical activity. All of these proteins, though really similar amongst every other (95 to 98 protein sequence identity), show a low identity both with all the above 5 putative bi-I/II DTPSs in the Pinus species (645 ), and with the other identified pine mono-I DTPSs (736 )Plants 2021, 10,8 of(Table S3). Though the 4 mono-DTPS from P. contorta and P. banksiana contain the class-I signature motif, and their homology modelling [33] predicts that they do possess a conserved -domain ErbB3/HER3 Synonyms folding pattern [22], the presence of unique structural characteristics close to their active websites, conserved also within the Pnl DTPS2 from Calabrian pine (Figure S8), could clarify their absence of function. In such a respect, it was proposed that, in these group-2 DTPSs, the side chains of F-592, positioned upstream with the class I motif, and likewise those of F-814 and H-817, can protrude into the active website cavity and might trigger a steric hindrance, possibly impeding catalytic activity [22]. It has been therefore speculated that these enzymes might have evolved from functional DTPSs into a trough of no function, from where they might evolve toward new DTPS activities or simply represent dead-end mutations of functional DTPSs [22]. Based on sequence similarity (Figure 3), and diverging from Pnl DTPS1, Pnl DTPS3 and Pnl DTPS4 were predicted to create pimarane-type olefins, namely pimaradiene, sandaracopimaradiene, and isopimaradiene. In particular, Pnl DTPS3 was identified to cluster in the phylogenetic group three, collectively with 1 protein from P. contorta (Pc DTPS mISO1) and a single from P. banksiana (Pb DTPS mISO1) (Figure 3), both of which had been identified to create isopimaradiene as the main product, with modest amounts of sandaracopimaradiene [22]. The members of such a group, displaying 96 to 99 protein sequence identity among each and every other, have been identified to become much more equivalent towards the mono-I DTPSs from the phylogenetic group 4 (790 ) than to these of phylogenetic group 2 (746 ; Table S3). On top of that, for the group-3 DTPS, as noted above for the group-1 ones, sequence alignment revealed amino acid substitutions exclusively present within the Pnl DTPS3 from Calabrian pine, namely K/N-642, D/N-748, and H/Y-749 (Figure S9), which could cause a transform inside the protein structure and hence in its item(s) profile. Likewise, Pnl DTPS4 was discovered to cluster inside the phylogenetic group four (Figure 3), with each other with two previously described mono-I DTPS, one from P. banksiana (Pb DTPS mPIM1) and a single from P. contorta (Computer DTPS mPIM1), each of which have been functionally characterized as forming pimaradiene as their major product [22]. Despite the pronounced sequence identity amongst the group-4 predicted proteins (about 94 ; Table S3), the higher quantity of amino acid substitutions identified in th.