Search

The improved production, recycling, and removal of plastic waste, such as polyethylene terephthalate (PET), are pressing environmental and economic issues for society. Biocatalytic (enzymatic) PET depolymerization is potentially a sustainable, low-energy solution to PET recycling, especially when compared with current disposal methods such as landfills, incineration, or gasification. IsPETase has been extensively studied for its use in PET depolymerization; however, its evolution from cutinases is not fully understood, and most engineering studies have neglected the majority of the available sequence space remote from the active site. In this study, ancestral protein reconstruction (ASR) has been used to trace the evolutionary trajectory from ancient serine hydrolases to IsPETase, while ASR and the related design approach, protein repair one-stop shop, were used to identify enzyme variants with improved activity and stability. Kinetic and structural characterization of these variants reveals new insights into the evolution of PETase activity and the role of second-shell mutations around the active site. Among the designed and reconstructed variants, we identified several with melting points 20 °C higher than that of IsPETase and two variants with significantly higher catalytic activity.

Cyanobacteria have evolved a suite of enzymes and inorganic carbon (Ci) transporters that improve photosynthetic performance by increasing the localized concentration of CO2around the primary CO2-fixating enzyme, Rubisco. This CO2-concentrating mechanism (CCM) is highly regulated, responds to illumination/darkness cycles, and allows cyanobacteria to thrive under limiting Ciconditions. While the transcriptional control of CCM activity is well understood, less is known about how regulatory proteins might allosterically regulate Citransporters in response to changing conditions. Cyanobacterial sodium-dependent bicarbonate transporters (SbtAs) are inhibited by PII-like regulatory proteins (SbtBs), with the inhibitory effect being modulated by adenylnucleotides. Here, we used isothermal titration calorimetry to show that SbtB from Cyanobiumsp. PCC7001 (SbtB7001) binds AMP, ADP, cAMP, and ATP with micromolar-range affinities. X-ray crystal structures of apo and nucleotide-bound SbtB7001 revealed that while AMP, ADP, and cAMP have little effect on the SbtB7001 structure, binding of ATP stabilizes the otherwise flexible T-loop, and that the flexible C-terminal C-loop adopts several distinct conformations. We also show that ATP binding affinity is increased 10-fold in the presence of Ca2+, and we present an X-ray crystal structure of Ca2+ATP:SbtB7001 that shows how this metal ion facilitates additional stabilizing interactions with the apex of the T-loop. We propose that the Ca2+ATP-induced conformational change observed in SbtB7001 is important for allosteric regulation of SbtA activity by SbtB and is consistent with changing adenylnucleotide levels in illumination/darkness cycles.