Photosystem I (PSI) is a very large membrane protein complex (∼1000 kDa) harboring P700*, the strongest reductant known in biological systems, which is responsible for driving NAD(P) + and ultimately for CO 2 reduction. Although PSI is one of the most important components in the photosynthesis machinery, it has remained difficult to enhance PSI functions through genetic engineering due to its enormous complexity. Inspired by PSI's ability to undergo multiple-step photo-induced electron hopping from P700* to iron−sulfur [Fe 4 S 4 ] clusters, we designed a 33 kDa miniature photocatalytic CO 2 -reducing enzyme (mPCE) harboring a chromophore (BpC) and two [Fe 4 S 4 ] clusters (Fe A /Fe B ). Through reduction potential fine-tuning, we optimized the multiple-step electron hopping from BpC to Fe A /Fe B , culminating in a CO 2 / HCOOH conversion quantum efficiency of 1.43%. As mPCE can be overexpressed with a high yield in Escherichia coli cells without requiring synthetic cofactors, further development along this route may result in rapid photo-enzyme quantum yield improvement and functional expansion through an efficient directed evolution process. KEYWORDS: photosystem I, rational design, photosensitizer protein, iron−sulfur (Fe 4 S 4 ) cluster, photocatalytic, CO 2 reductase
Humani ndoleamine 2,3-dioxygenase 1( IDO1) hasb ecome an increasingly valuable target for cancer immunotherapy because it promotes immune escape by tumor cells. To date, the functiono fp ost-translational modifications (PTMs) on IDO1 has not been fully elucidated. Among the many forms of PTMs, it has been identified that three tyrosine sites (Y15, Y345, and Y353) on IDO1a re nitrated and play important roles in catalytic function. Herein, by genetically encoding 3-nitro-ltyrosine into the tyrosine nitration sites of IDO1, the homogeneous and native nitrated IDO1 have been obtained. It is found that the nitration of differentt yrosine sites has different effects on the IDO1 structurea nd enzyme activity.N itration at positionY 15 has an egligible effect, but nitration at Y345 or Y353 decreases the enzyme activity,e specially Y353. Furthermore, these results demonstrate that the regulationo ft he catalytic function caused by tyrosine nitration is relatedt op erturbation of the protein structure and heme-binding disruption.Humani ndoleamine 2,3-dioxygenase 1( IDO1) is am etalloenzyme with heme as ac ofactor that catalyzest he metabolism of l-Trp to N-formylkynurenine (NFK), which is the first and rate-determining step of the kynurenine pathway. [1] In this pathway,I DO1 consumes l-Trp, causing al ack of tryptophan in the local microenvironment,a nd produces many immunosuppressives econdary metabolites, such as kynureninic acid (KA) and quinolinic acid (QA), whichare involved in several different immune signaling processes. [2] Consequently,t he cycle of Tlymphocytes is restricted to the G1 phase and proliferation is inhibited. Additionally,t hese signaling processes trigger the generation and activation of Tr egulatory cells. Finally,alocal immunosuppressivee nvironment is formed. [3] Therefore, the enzymea ctivity of IDO1 plays ac rucial role in IDO1-mediated immune tolerance.It has been confirmed that three tyrosine residues (Y15, Y345, and Y353) in IDO1 can undergo nitration modification by peroxynitrite, such as 3-(4-morpholinyl)sydnonimine. [4] As previously reported, the THP-1 and PBMC cells with induced IDO1 expression, or purified recombinant IDO1 protein treated with the peroxynitrite generator, can cause IDO1 tyrosine nitration modification at three sites, which results in ad ecrease in IDO1 enzyme activity. [4b] To clarify at which site tyrosine nitration has the most significant impact on IDO1 enzymea ctivity,t yrosine was mutated to phenylalanine, the peroxynitrite experiment was repeated, and it was found that nitration of the Y15 residue was the most important factor in the inactivation of IDO1. [4b] However,m utation of tyrosine to phenylalanine may directly change the conformation of IDO1, and thus, affect its enzymea ctivity.M oreover,t he in vitro reactiono fI DO1 protein with peroxynitrite may directly affect its enzymea ctivity due to nonspecific tyrosine oxidative modification. Hence, it might not be accurate to study the effects of tyrosine nitration based on the nonspecific reaction.Genet...
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