A Tale of Two Reductases: Extending the Bacteriochlorophyll Biosynthetic Pathway in <i>E. coli</i>

<div><p>The creation of a synthetic microbe that can harvest energy from sunlight to drive its metabolic processes is an attractive approach to the economically viable biosynthetic production of target compounds. Our aim is to design and engineer a genetically tractable non-photosynthetic microbe to produce light-harvesting molecules. Previously we created a modular, multienzyme system for the heterologous production of intermediates of the bacteriochlorophyll (BChl) pathway in <i>E. coli</i>. In this report we extend this pathway to include a substrate promiscuous 8-vinyl reductase that can accept multiple intermediates of BChl biosynthesis. We present an informative comparative analysis of homologues of 8-vinyl reductase from the model photosynthetic organisms <i>Rhodobacter sphaeroides</i> and <i>Chlorobaculum tepidum</i>. The first purification of the enzymes leads to their detailed biochemical and biophysical characterization. The data obtained reveal that the two 8-vinyl reductases are substrate promiscuous, capable of reducing the C8-vinyl group of Mg protoporphyrin IX, Mg protoporphyrin IX methylester, and divinyl protochlorophyllide. However, activity is dependent upon the presence of chelated Mg²⁺ in the porphyrin ring, with no activity against non-Mg²⁺ chelated intermediates observed. Additionally, CD analyses reveal that the two 8-vinyl reductases appear to bind the same substrate in a different fashion. Furthermore, we discover that the different rates of reaction of the two 8-vinyl reductases both <i>in vitro</i>, and <i>in vivo</i> as part of our engineered system, results in the suitability of only one of the homologues for our BChl pathway in <i>E. coli</i>. Our results offer the first insights into the different functionalities of homologous 8-vinyl reductases. This study also takes us one step closer to the creation of a nonphotosynthetic microbe that is capable of harvesting energy from sunlight for the biosynthesis of molecules of choice.</p></div

Substrate promiscuity of purified 8-vinyl reductases with BChl intermediates as determined by shifts in absorbance maxima.

<p>Conversion of a mixture of Bchl intermediates (MgP^IX, MgP^IXME, P^IXME) was analyzed by HPLC at a single wavelength (412 nm) to detect all porphyrins present in the reaction mixtures after 18 hours. Reactions with enzyme (dotted traces) and control reactions (solid traces) are shown. Wavelengths displayed above arrows (pointing to peak shoulder or peak maximum) indicate the absorbance maximum measured at that time point, and illustrate the 5 nm absorbance shift which occurs after the reduction of the C-8 vinyl group. (<b>A</b>) Purified <i>RS</i>BciA partially reduces the C8-vinyl group of MgP^IX and MgP^IXME to generate a peak shoulder for each substrate at which the absorbance maximum is shifted from 415 nm to 410 nm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Chew2" target="_blank">[27]</a>. Non-Mg chelated compounds are not reduced. Note that the shift in retention time observed for P^IXME in the enzyme and control reaction is the results from an aberrance in column running conditions as both compounds retain the absorbance maximum of the P^IXME substrate. (<b>B</b>) Purified <i>CT</i>BciA reduces the C8-vinyl group on MgP^IX and MgP^IXME, as indicated by a complete shift in compound peak absorbance maxima from 415 nm to 410 nm. No activity and correspondingly, no shift in absorbance maximum is observed against non-Mg chelated compounds P^IX and P^IXME. For abbreviations of substrate names see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone-0089734-t001" target="_blank">Table 1</a>.</p

Amino acid sequence alignment of <i>Chlorobaculum tepidum CT</i>BciA and <i>Rhodobacter sphaeroides RS</i>BciA.

<p>The two divinyl reductases share 53% sequence identity. Conserved residues are highlighted in blue. The conserved GxxGxxG motif, required for NAD(P)H binding <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Rescigno1" target="_blank">[48]</a>, is marked with asterisks.</p

Bacteriochlorophyll pathway intermediates produced by <i>E. coli</i> cells expressing various combinations of genes from the Heme and BChl pathways as detected by HPLC.

<p><i>E. coli</i> cells expressing HemA-F and the magnesium chelatase complex BchSID produce P^IX and MgP^IX. Addition of the methyl transferase BchM results in production of both P^IX ME and MgP^IX ME. Expression with the divinyl reductase <i>CT</i>BciA in the presence and absence of BchM leads to the production of mono-vinyl forms of pathway intermediates. <i>RS</i>BciA is not active in our <i>in vivo</i> system. Abbreviations: P^IX - protoporphyrin IX, MgP^IX - Mg-protoporphyrin IX, P^IXME - protoporphyrin IX methylester, MgP^IXME - Mg-protoporphyrin IX methylester, mvP^IX - mono-vinyl protoporphyrin IX, mvMgPIX - mono-vinyl Mg-protoporphyrin IX, mvP^IXME - mono-vinyl protoporphyrin IX methylester, mvMgP^IXME - mono-vinyl Mg-protoporphyrin IX methylester, ND – none detected.</p

Engineered pathway design for the heterologous production of BChl in the non-photosynthetic host <i>E. coli</i>.

<p>Using succinyl-CoA and glycine as precursor molecules, expression of the heme pathway enzymes HemA-F in <i>E. coli</i> results in production of P^IX as the common intermediate of the heme and BChl biosynthetic pathways. Addition of the BChl enzymes magnesium chelatase (BchHID) and methyltransferase (BchM) yields MgP^IX and MgP^IXME in <i>E. coli </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Kwon1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Johnson3" target="_blank">[15]</a>. Subsequent steps have not yet been functionally assembled in a heterologous system and depending on the enzymes substrate specificities, the order in which the enzymes operate may differ from the depicted pathway. Briefly, formation of the characteristic fifth E ring of chlorophylls is catalyzed by two unrelated and yet to be biochemically characterized cyclases AcsF (aerobic) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Tang1" target="_blank">[20]</a> or BchE (anaerobic) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-BoldarevaNuianzina1" target="_blank">[19]</a>. The D pyrrole ring is reduced either by a light-dependent, nitrogenase-like (LPOR, three-subunit enzyme BchLNB) or a light-independent (DPOR) protochlorophyllide reductase; both enzymes have been biochemically characterized <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Brocker1" target="_blank">[23]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Paddock1" target="_blank">[26]</a>. Reduction of the C8-vinyl group of BChl intermediates is catalyzed by the NADPH-dependent reductase BciA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Chew2" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Canniffe1" target="_blank">[30]</a> investigated in this study. Seven additional enzymatic steps are required for production of Bchl <i>a </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Willows1" target="_blank">[14]</a>.</p

Circular dichroism analysis of the secondary structure of 8-vinyl reductase and the Soret band of MgP^IX reveals a difference in binding mode.

<p>CD spectra of purified protein in the far UV region show that (<b>A</b>) <i>CT</i>BciA and (<b>B</b>) <i>RS</i>BciA display the characteristic double minima at 222 nm and 208 nm associated with α-helical content <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Greenfield1" target="_blank">[57]</a> (solid line). Upon addition of MgP^IX to the protein, a shift is observed in the CD spectrum of <i>CT</i>BciA, but not <i>RS</i>BciA (dotted line). (<b>C</b>) and (<b>D</b>) Analysis in the Soret region of MgP^IX (dotted line) shows no spectra. Upon addition of purified protein (<b>C</b>) <i>CT</i>BciA and (<b>D</b>) <i>RS</i>BciA a change is observed in the Soret band of the porphyrin ring (solid line). The differences in peak and inflection wavelengths may represent MgP^IX interactions with different amino acid isomers in the two different proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Huang1" target="_blank">[58]</a>.</p

Reaction efficiency as a measure of percent conversion of divinyl-protochlorophyllide to mono-vinyl protochlorophyllide by 8-vinyl reductase.

<p>Purified <i>CT</i>BciA reduces greater than 85% DVP to mono-vinyl form in 1.5 hours (black bar). Purified <i>RS</i>BciA acts more slowly, reaching 100% conversion of divinyl to mono-vinyl in 18 hours (hashed bars). Attempts to improve reaction efficiency of <i>RS</i>BciA by addition of crude cell lysate to the reaction vessel actually reduced the rate of reaction as well as the overall conversion to less than 80% in 18 hours (white bars). Error bars are calculated from reactions carried out in duplicate.</p

Hydrolysis of X-gal by <i>E. coli</i> co-expressing EutC^1–19-β-galactosidase and recombinant Eut shell proteins.

<p><i>E. coli</i> C2566 cells with constructs for constitutive expression of β-galactosidase (β-gal) or EutC^1–19-β-gal and different combinations of Eut shell proteins were grown with the β-gal substrate X-gal. Intracellular accumulation of the insoluble X-gal cleavage product was observed by Differential Interference Contrast (DIC) microscopy. Arrows point to intracellular indole deposits.</p

Coenzyme-B₁₂-dependent ethanolamine utilization (<i>eut</i>) genes of <i>Salmonella enterica.</i>

<p>(<b>A</b>) <i>eut</i> operon in <i>S. enterica</i>. <i>eutS</i>, <i>eutM</i>, <i>eutN</i>, <i>eutL</i> and <i>eutK</i> encode BMC shell proteins that are proposed to form the Eut microcompartment (yellow and orange) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342-Kofoid1" target="_blank">[20]</a>. Asterisks indicate genes that encode for enzymes with predicted N-terminal signal sequences that target them to the BMC interior <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342-Fan1" target="_blank">[19]</a>. Transcription is induced from the P_I promoter in the presence of both ethanolamine and vitamin B₁₂, while the promoter P_II regulates weak constitutive expression of the transcription factor EutR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342-Roof1" target="_blank">[49]</a>. (<b>B</b>) Model for catabolism of ethanolamine by the Eut BMC. Ethanolamine enters the microcompartment and is metabolized to ethanol, acetyl-phosphate and acetyl-CoA, which can enter the tricarboxylic acid cycle <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342-Kerfeld1" target="_blank">[7]</a>. Eut BMC prevents dissipation of acetaldehyde, a volatile and toxic reaction intermediate (red) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342-Penrod1" target="_blank">[21]</a>. Enzymes assumed to reside in the BMC lumen include coenzyme-B₁₂-dependent ethanolamine ammonia lyase (EAL, EutBC), EAL reactivase (EutA), alcohol dehydrogenase (EutG), aldehyde dehydrogenase (EutE), and phosphotransacetylase (EutD).</p

Purification of Eut compartments.

<p>(<b>A</b>) Silver stained SDS-PAGE gel showing purification of (lane 1) Eut BMCs from <i>S. enterica</i> cells harboring EutC^1–19-EGFP, (lane 2) recombinant EutSMNLK BMCs, and (lane 3) recombinant EutS BMCs from <i>E. coli</i> C2566 cells co-expressing EutC^1–19-EGFP. Calculated protein sizes are as follows: EutS (11.6 kDa), EutM (9.8 kDa), EutN (10.4 kDa), EutL (22.7 kDa), EutK (17.5 kDa), EutC^1–19-EGFP (29.1 kDa). (<b>B</b>) Transmission electron micrographs of isolated native and recombinant Eut compartments. From left to right: Eut BMCs from <i>S. enterica</i>, EutSMNLK shells from <i>E. coli</i> C2566, EutS shells from <i>E. coli</i> C2566. (Scale bar: 100 nm).</p