Structural basis for enzymatic photocatalysis in chlorophyll biosynthesis

Abstract

The enzyme protochlorophyllide oxidoreductase (POR) catalyses a lightdependent step in chlorophyll biosynthesis that is essential to photosynthesis and ultimately all life on Earth. 1-3 POR, which is one of three known light-dependent enzymes, 4,5 catalyzes reduction of the photosensitizer and substrate protochlorophyllide to form the pigment chlorophyllide. Despite its biological importance, a structural basis for POR photocatalysis has remained elusive. Here, we report crystal structures of cyanobacterial PORs from Thermosynechococcus elongatus and Synechocystis sp. in their free forms, and in complex with nicotinamide coenzyme. Our structural models and simulations of the ternary protochlorophyllide-NADPH-POR complex have identified multiple interactions in the POR active site that are important for protochlorophyllide binding, photosensitization and photochemical conversion to chlorophyllide. We demonstrate the importance of active-site architecture and protochlorophyllide structure in experiments using POR variants and protochlorophyllide analogues. These studies reveal how the POR active site facilitates light-driven reduction of protochlorophyllide by localized hydride transfer from NADPH and long-range proton transfer along structurally defined proton-transfer pathways. As the light-driven step in the chlorophyll biosynthetic pathway (Fig. 1), the POR reaction acts as the trigger for the germination of seedlings =in plants and provokes a marked change in the morphological development of the plant. 2,3 Given this crucial biological role, POR has been the focus of numerous mechanistic and biophysical investigations. A combination of time-resolved (at the femtosecond-to-second scale) and cryogenic spectroscopy methods have provided some understanding of the mechanism of POR photocatalysis in a range of photosynthetic organisms, including cyanobacteria and plants. Picosecond excited-state dynamics in the protochlorophyllide (Pchlide) molecule are thought to result in excited state interactions between the substrate and active-site residues that are necessary to trigger the subsequent reaction chemistry. 6-12 This involves sequential transfer of a hydride equivalent from NADPH and a proton transfer from either an active site residue or solvent. Proton transfer is reliant on solvent dynamics and an implied network of extended protein motions that occur on the microsecond timescale. 13-17 Hydride transfer from NADPH is not concerted, but occurs in a stepwise manner that involves

Biosynthesis of the modified tetrapyrroles—the pigments of life

“…Nature has discovered two completely different ways to achieve this reduction: in one, a lightdependent reaction is catalyzed by NADPH:protochlorophyllide oxidoreductase (LPOR; EC 1.3.1.33) (207,208); in the other, reduction of the C17-C18 double bond is catalyzed by a dark-operative protochlorophyllide reductase (DPOR), consisting of ChlL, ChlN and ChlB subunits that display similarity to the components of nitrogenase (209). The ability to trigger the catalytic cycle with short pulses of light has led to a number of kinetic studies (210)(211)(212), and recently the structure of LPOR has been reported (208). The phylogenetic distribution of LPOR and DPOR is interesting; anoxygenic photosynthetic bacteria contain only DPOR; cyanobacteria, green algae, mosses and most gymnosperms possess both LPOR and DPOR; and angiosperms (flowering plants) contain only LPOR.…”

Extending the Library of Light‐Dependent Protochlorophyllide Oxidoreductases and their Solvent Tolerance, Stability in Light and Cofactor Flexibility

“…Interestingly, these positions are not the amino acids responsible for direct NADPH binding as deduced from the recently published crystal structure from Synechocystis. [27] As selected enzymes, which are in nature not light-dependent, have been shown to possess promiscuous activity under the influence of light, [28][29] LPORs as NADPH-dependent natural photoenzymes may be targets for further studies of light dependent reactions.…”

“…When locating the corresponding amino acids identified above in the recently published crystal structures [27] of the LPORs from T. elongatus (PDB: 6R46) and Synechocytis (PDB: 6R48) it becomes clear that these three amino acids are not amino acids in close contact with the phosphate moiety of the NADPH or even the cofactor itself (Figure 7). The amino acids are in an α-helix next to the NADPH cofactor, but the distance between the amino acids and the NADPH are too long for an obvious interaction.…”

“…Amino acids like K197 and Y193 in the published paper interact with the ribose next to nicotinamide of the cofactor but they are also suggested to bind the substrate, [27] thus there needs to be a significant change of the structure for binding. Therefore, by careful comparison of sequences, a non-intuitive origin of cofactor flexibility was identified.…”

A novel cucumber albino mutant caused by chloroplast development deficiency

“…POR is a light dependent key enzyme required for chlorophyll biosynthesis by catalyzing protochlorophyllide to chlorophyllide (41,42). It has been reported that POR is crucial for plant growth and development since nonfunctional plants displayed reduced chlorophyll content and severe photoautotrophic growth defects (43,44). The expression level of POR is lower in albino mutant than in wild type, indicating that albino mutant might be incapable in chlorophyll biosynthesis.…”