No abstract

During chlorophyll and bacteriochlorophyll biosynthesis in gymnosperms, algae, and photosynthetic bacteria, dark-operative protochlorophyllide oxidoreductase (DPOR) reduces ring D of aromatic protochlorophyllide stereospecifically to produce chlorophyllide. We describe the heterologous overproduction of DPOR subunits BchN, BchB, and BchL from Chlorobium tepidum in Escherichia coli allowing their purification to apparent homogeneity. The catalytic activity was found to be 3.15 nmol min ؊1 mg ؊1 with K m values of 6.1 M for protochlorophyllide, 13.5 M for ATP, and 52.7 M for the reductant dithionite. To identify residues important in DPOR function, 21 enzyme variants were generated by site-directed mutagenesis and investigated for their metal content, spectroscopic features, and catalytic activity.

Selenocysteine (Sec) is naturally incorporated into proteins by recoding the stop codon UGA. Sec is not hardwired to UGA, as we found the Sec insertion machinery to be able to site-specifically incorporate Sec directed by 58 of the 64 codons. For 15 sense codons, complete conversion of the codon meaning from canonical amino acid to Sec was observed along with a 10-fold increase in selenoprotein yield compared to Sec insertion at the three stop codons. This high-fidelity sense-codon recoding mechanism was demonstrated for Escherichia coli formate dehydrogenase and recombinant human thioredoxin reductase and confirmed by independent biochemical and biophysical methods. Although Sec insertion at UGA is known to compete against protein termination, it is surprising that the Sec machinery has the ability to outcompete abundant aminoacyl-tRNAs in decoding sense codons. The findings have implications for the process of translation and the information storage capacity of the biological cell.

The 21st amino acid, selenocysteine (Sec), is synthesized on its cognate transfer RNA (tRNASec). In bacteria, SelA synthesizes Sec from Ser-tRNASec, whereas in archaea and eukaryotes SepSecS forms Sec from phosphoserine (Sep) acylated to tRNASec. We determined the crystal structures of Aquifex aeolicus SelA complexes, which revealed a ring-shaped homodecamer that binds 10 tRNASec molecules, each interacting with four SelA subunits. The SelA N-terminal domain binds the tRNASec-specific D-arm structure, thereby discriminating Ser-tRNASec from Ser-tRNASer. A large cleft is created between two subunits and accommodates the 3′-terminal region of Ser-tRNASec. The SelA structures together with in vivo and in vitro enzyme assays show decamerization to be essential for SelA function. SelA catalyzes pyridoxal 5′-phosphate–dependent Sec formation involving Arg residues nonhomologous to those in SepSecS. Different protein architecture and substrate coordination of the bacterial enzyme provide structural evidence for independent evolution of the two Sec synthesis systems present in nature.

Photosynthesis represents the fundamental strategy of nature to convert solar radiation into biochemically accessible energy. Chlorophylls and bacteriochlorophylls constitute the pigments employed in both harvesting and utilizing photons of visible light. Biosynthesis of these complex tetrapyrroles, of which more than 6 billion tons are produced annually, utilizes a chain of enzymatic conversions, many of which delve deep into the biochemical treasure trove of early life on earth (1).One of the more unusual steps in (bacterio)chlorophyll biosynthesis involves the chemically challenging, stereospecific reduction of the C17ϭC18 double bond of ring D of the porphyrin protochlorophyllide (Pchlide) 2 to form the chlorin chlorophyllide (Chlide, Fig. 1A). Two unrelated pathways have evolved for this two-electron reduction (2-5). In angiosperms, a monomeric, light-dependent protochlorophyllide oxidoreductase (LPOR) (NADPH Pchlide oxidoreductase, EC 1.3.1.33) catalyzes the reaction. The bound substrate Pchlide needs to be activated by a photon to drive the NADPH-dependent reduction step (6 -10).Anoxygenic, photosynthetic bacteria, by contrast, make use of an ATP-dependent process catalyzed by the dark operative protochlorophyllide oxidoreductase (DPOR). Other photosynthetic organisms such as cyanobacteria, algae, or gymnosperms encode both LPOR and DPOR (4). DPOR consists of three subunits. In chlorophyll-synthesizing organisms, these are termed ChlN, ChlB, and ChlL (1, 11, 12); in bacteriochlorophyll synthesizers, they are BchN, BchB, and BchL (11, 13). ChlL (BchL) forms the homodimer ChlL 2 (BchL 2 ) that functions as an ATP-dependent electron shuttle carrying an intersubunit [4Fe-4S] cluster and two ATP binding sites (13)(14)(15). Recently, the crystal structure of the BchL 2 complex from Rhodobacter sphaeroides was solved (16). Subunits ChlN and ChlB instead constitute a heterotetrameric complex here denoted (ChlN/ChlB) 2 that bears two [4Fe-4S] clusters and two substrate binding sites (14).Some details of the catalytic mechanism of DPOR have been established biochemically. Upon binding of two molecules of ATP, ChlL 2 interacts with the catalytic, substrate binding (ChlN/ChlB) 2 complex. Ferredoxin provides a single electron to ChlL 2 (13), which in turn transfers an electron to (ChlN/ ChlB) 2 . Hydrolysis of the two ATP molecules results in the dissociation of ChlL 2 from reduced (ChlN/ChlB) 2 . Pchlide reduction is completed after two sequential catalytic redox cycles. Substrate recognition by (ChlN/ChlB) 2 essentially involves all functional groups of the substrate (14). Two ChlL 2 dimers simultaneously interact with the (ChlN/ChlB) 2 tetramer, giving