Acrylyl-coenzyme A reductase, an enzyme involved in the assimilation of 3-hydroxypropionate by Rhodobacter sphaeroides

Abstract

The anoxygenic phototroph Rhodobacter sphaeroides uses 3-hydroxypropionate as a sole carbon source for growth. Previously, we showed that the gene (RSP_1434) known as acuI, which encodes a protein of the medium-chain dehydrogenase/reductase (MDR) superfamily, was involved in 3-hydroxypropionate assimilation via the reductive conversion to propionyl-coenzyme A (CoA). Based on these results, we speculated that acuI encoded acrylyl-CoA reductase. In this work, we characterize the in vitro enzyme activity of purified, recombinant AcuI using a coupled spectrophotometric assay. AcuI from R. sphaeroides catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA. Two other members of the MDR012 family within the MDR superfamily, the products of SPO_1914 from Ruegeria pomeroyi and yhdH from Escherichia coli, were shown to also be part of this new class of NADPH-dependent acrylyl-CoA reductases. The activities of the three enzymes were characterized by an extremely low Km for acrylyl-CoA (<3 μM) and turnover numbers of 45 to 80 s(-1). These homodimeric enzymes were highly specific for NADPH (Km = 18 to 33 μM), with catalytic efficiencies of more than 10-fold higher for NADPH than for NADH. The introduction of codon-optimized SPO_1914 or yhdH into a ΔacuI::kan mutant of R. sphaeroides on a plasmid complemented 3-hydroxypropionate-dependent growth. However, in their native hosts, SPO_1914 and yhdH are believed to function in the metabolism of substrates other than 3-hydroxypropionate, where acrylyl-CoA is an intermediate. Complementation of the ΔacuI::kan mutant phenotype by crotonyl-CoA carboxylase/reductase from R. sphaeroides was attributed to the fact that the enzyme also uses acrylyl-CoA as a substrate.

Fig 1

Reactions of the proposed reductive pathway for 3-hydroxypropionate assimilation by Rhodobacter sphaeroides 2.4.1. Acrylyl-CoA reductase (AcuI) is encoded by RSP_1434 (shown in the box). The reactions indicated by the dotted arrows are hypothetical; however, at least part of these reactions should exist, based on the fact that the overall conversion of 3-hydroxypropionate or acrylate to propionyl-CoA can be measured in cell extracts as described previously (14). In the case of R. sphaeroides, propionyl-CoA is assimilated via the methylmalonyl-CoA pathway and enters the citric acid cycle at succinyl-CoA. The methylmalonyl-CoA pathway consists of the following enzymes: propionyl-CoA carboxylase (alpha and beta subunits encoded by RSP_2191 and RSP_2189, respectively), ethylmalonyl-CoA/methylmalonyl-CoA epimerase (encoded by RSP_0812), and (2R)-methylmalonyl–CoA mutase (encoded by RSP_2192).

Fig 2

Purification of recombinant acrylyl-CoA reductases heterologously produced by E. coli Rosetta2 (DE3). Various fractions obtained during purification were separated on a 10% SDS-PAGE gel and stained with Coomassie blue. (A) Acrylyl-CoA reductase (AcuI) from Rhodobacter sphaeroides 2.4.1. Lane 1, whole cells before induction; lane 2, cell extract after 17 h of induction (20 μg); lane 3, recombinant AcuI eluted with E. coli GroEL (arrow) from the Ni⁺ affinity column (10 μg); lane 4, purified recombinant AcuI from the Superose 12 column (2 μg); lane 5, molecular mass standards. (B) Acrylyl-CoA reductase (product of SPO_1914) from Ruegeria pomeroyi DSS-3. Lane 1, whole cells before induction; lane 2, cell extract after 17 h of induction (20 μg); lane 3, recombinant product of SPO_1914 from the Ni⁺ affinity column (4 μg); lane 4, purified recombinant product of SPO_1914 from the Superose 12 column (4 μg); lane 5, molecular mass standards. (C) Acrylyl-CoA reductase (YhdH) from Escherichia coli K-12 substrain MG1655. Lane 1, molecular mass standards; lane 2, whole cells before induction; lane 3, cell extract after 18 h of induction (20 μg); lane 4, recombinant YhdH from the Ni⁺ affinity columns (4 μg); lane 5, purified recombinant YhdH from the Superose 12 column (2 μg).

Fig 3

HPLC analysis of products formed by 3-hydroxypropionyl–CoA synthetase and acrylyl-CoA reductase (AcuI). Acrylyl-CoA was produced in situ by recombinant Sulfolobus tokodaii 3-hydroxypropionyl–CoA synthetase (A to C). (A) The reaction mixture contained 100 mM Tris-Cl (pH 8.0), 10 mM MgCl₂, 3 mM ATP, recombinant S. tokodaii 3-hydroxypropionyl–CoA synthetase, and 0.1 mM CoA; (B) 1 min after the addition of 20 mM acrylate; (C) after 2 additional minutes of incubation; (D) 1 min after the addition of 1 mM NADPH; (E) 1 min after the addition of 0.3 μg of purified recombinant AcuI; (F) after 3 additional minutes of incubation. In panel C, the peaks denoted by the dotted arrows were considered the CoA adduct of acrylyl-CoA (30), which were not used as the substrates by AcuI. Free CoA and propionyl-CoA were identified by coelution with standard compounds (not shown).

Fig 4

Photoheterotrophic (anoxic/light) growth of Rhodobacter sphaeroides 2.4.1. R. sphaeroides wild-type, ΔacuI::kan mutant, ΔacuI::kan mutant complemented with constitutively expressing acuI, SPO_1914, yhdH, or ccr from the plasmid (indicated in parentheses), and ΔacuI::kan mutant carrying the null vector pBBRsm2MCS5 were grown in the minimal medium containing 3-hydroxypropionate (10 mM) (A) or succinate (10 mM) (B) as a sole carbon source. See Materials and Methods for the names of the plasmids used in the study.