Crystal structures of cyanobacterial light-dependent protochlorophyllide oxidoreductase

Abstract

The reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) is the penultimate step of chlorophyll biosynthesis. In oxygenic photosynthetic bacteria, algae, and plants, this reaction can be catalyzed by the light-dependent Pchlide oxidoreductase (LPOR), a member of the short-chain dehydrogenase superfamily sharing a conserved Rossmann fold for NAD(P)H binding and the catalytic activity. Whereas modeling and simulation approaches have been used to study the catalytic mechanism of this light-driven reaction, key details of the LPOR structure remain unclear. We determined the crystal structures of LPOR from two cyanobacteria, Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus Structural analysis defines the LPOR core fold, outlines the LPOR-NADPH interaction network, identifies the residues forming the substrate cavity and the proton-relay path, and reveals the role of the LPOR-specific loop. These findings provide a basis for understanding the structure-function relationships of the light-driven Pchlide reduction.

Keywords: NADPH; chlorophyll biosynthesis; crystal structure; photocatalysis; proton relay.

Fig. 1.

Ribbon representation of the overall structures of SyLPOR and TeLPOR. (A) Two side views of SyLPOR. The secondary structure elements are colored in blue except the antiparallel β8 in yellow. The loop region is in gray. The LPOR-specific insertion is colored in black. The NADPH-binding sequence is colored in green. Four cysteine residues are shown in sphere mode. The cofactor NADPH is shown in stick-and-ball mode. (B) Front view of SyLPOR (Left), TeLPOR (Right), and their superimposition (Middle). The secondary structure elements of TeLPOR are colored in deep green except β8 in magenta; the NADPH-binding sequence is colored in cyan. The α-helices are labeled alphabetically, and the β-strands are labeled numerically.

Fig. 2.

The NADPH-binding site. (A and B) NADPH interactions with (A) SyLPOR and (B) TeLPOR. The protein backbone is traced with thin lines. Residues directly interacting with NADPH are shown as sticks. The water molecules are shown as red spheres. The consensus sequence GASSGV/LG conserved for all LPORs is shown as thick lines. The color scheme is the same as in Fig. 1. The 2F_o − F_c maps are shown in gray mesh contoured at 1.0σ. The polar interactions are indicated by black dashed lines. (C) Detailed diagram showing NADPH interactions with SyLPOR/TeLPOR and water molecules. The residues that interact with NADPH by backbone atoms are shown in gray boxes, and the residues that interact with NADPH by their side-chain atoms are shown in white boxes. Water molecules are presented as light-blue circles. The black dashed lines indicate the interactions present in both SyLPOR and TeLPOR, the blue dashed lines indicate interactions only in SyLPOR, and the green dashed lines and green boxes indicate interactions only in TeLPOR.

Fig. 3.

The substrate-binding site and the LPOR-specific insertion. (A) The substrate cavity of SyLPOR. The two polypeptide chains in an asymmetric unit are superimposed and colored blue and cyan. For clarity, only one NADPH molecule is shown (yellow stick). The side chains of residues possibly participating in Pchlide binding are shown as sticks. The LPOR-specific insertion of one chain is colored black. The missing fragment is depicted as dashed lines. (B) The substrate cavity of TeLPOR. The polypeptide chains are colored deep green and green, and only one NADPH is shown (magenta). Other representations are same as in A. (C) The majority of the LPOR-specific insertion is folded with the αβα-core. The core-insertion interacting residues are shown as sticks. SyLPOR and TeLPOR, respectively, are colored blue and deep green except that the insertion sequence in SyLPOR is colored black. (D–F) Representation of the flexibility of A, B, and C. The backbone is shown as tubes whose radius corresponds to the temperature factor of the Cα-atoms.

Fig. 4.

Dimerization and structural comparison. (A) The SyLPOR dimer, (B) the TeLPOR dimer, and (C) their superimpositions with one monomer fixed in position. The gray box in the dashed lines indicates the dimeric interface. (D) Structural comparison of SyLPOR (Upper) and TeLPOR (Lower). SyLPOR chains A and B (Protein Data Bank [accession no. 6R48]) are in orange and light orange; the apo-TeLPOR (Protein Data Bank [accession no. 6RNV]) and NADPH-bound TeLPOR (Protein Data Bank [accession no. 6RNW]) structures are in pink and magenta. The gray sphere indicates the alternative conformation of αG not observed previously (44).

Fig. 5.

Proposed proton-relay path. (A) The hydrogen bond network bridging the Tyr193 ηO and a solvent water molecule within the SyLPOR and TeLPOR structures. The well-positioned water, shown in the red sphere, is fixed by the backbone oxygens of Ala91 and Asn115, and the ε-amino group of Lys197. The hydrogen bonds are shown in dashed lines and the bond lengths (Å) are in blue for SyLPOR and dark green for TeLPOR. (B) A proposed proton-relay path following the hydride transfer from NADPH to C17. The photon energy (hv) is represented by a yellow thunderbolt.