Function and Phylogeny of Bacterial Butyryl Coenzyme A:Acetate Transferases and Their Diversity in the Proximal Colon of Swine.

Studying the host-associated butyrate-producing bacterial community is important, because butyrate is essential for colonic homeostasis and gut health. Previous research has identified the butyryl coenzyme A (CoA):acetate-CoA transferase (EC 2.3.8.3) as a gene of primary importance for butyrate production in intestinal ecosystems; however, this gene family (but) remains poorly defined. We developed tools for the analysis of butyrate-producing bacteria based on 12 putative but genes identified in the genomes of nine butyrate-producing bacteria obtained from the swine intestinal tract. Functional analyses revealed that eight of these genes had strong But enzyme activity. When but paralogues were found within a genome, only one gene per genome encoded strong activity, with the exception of one strain in which no gene encoded strong But activity. Degenerate primers were designed to amplify the functional but genes and were tested by amplifying environmental but sequences from DNA and RNA extracted from swine colonic contents. The results show diverse but sequences from swine-associated butyrate-producing bacteria, most of which clustered near functionally confirmed sequences. Here, we describe tools and a framework that allow the bacterial butyrate-producing community to be profiled in the context of animal health and disease. IMPORTANCE: Butyrate is a compound produced by the microbiota in the intestinal tracts of animals. This compound is of critical importance for intestinal health, and yet studying its production by diverse intestinal bacteria is technically challenging. Here, we present an additional way to study the butyrate-producing community of bacteria using one degenerate primer set that selectively targets genes experimentally demonstrated to encode butyrate production. This work will enable researchers to more easily study this very important bacterial function that has implications for host health and resistance to disease.

Characterization of hydroxycinnamoyltransferase from rice and its application for biological synthesis of hydroxycinnamoyl glycerols.

Hydroxycinnamoyltransferases (HCTs) catalyze the transfer of the cinnamoyl moiety from hydroxycinnamoyl-CoA to various acceptors such as shikimic acid, quinic acid, hydroxylated acid, and glycerol. Four rice HCT homologues (OsHCT1-4) to tobacco HST were cloned, and OsHCT4 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. Using the purified recombinant protein and biotransformation techniques, whether OsHCT4 shows hydroxycinnamoyltransferase activity with a variety of acyl group acceptors was investigated. The results of high performance liquid chromatography (HPLC) and mass spectrometry (MS) established that OsHCT4 mediated the trans-esterification of glycerol as well as shikimic acid in the presence of hydroxycinnamoyl-CoA. The structure of the reaction product was determined using nuclear magnetic resonance spectroscopy (NMR). E. coli cells co-expressing 4CL (4-coumarate:coenzyme A ligase) and OsHCT4 converted p-coumaric acid, ferulic acid, and caffeic acid into the corresponding glycerides. While this conversion is very efficient in vitro, the physiological significant in rice is currently unknown.

Characterization of (R)-2-hydroxyisocaproate dehydrogenase and a family III coenzyme A transferase involved in reduction of L-leucine to isocaproate by Clostridium difficile.

The strictly anaerobic pathogenic bacterium Clostridium difficile occurs in the human gut and is able to thrive from fermentation of leucine. Thereby the amino acid is both oxidized to isovalerate plus CO(2) and reduced to isocaproate. In the reductive branch of this pathway, the dehydration of (R)-2-hydroxyisocaproyl-coenzyme A (CoA) to (E)-2-isocaprenoyl-CoA is probably catalyzed via radical intermediates. The dehydratase requires activation by an ATP-dependent one-electron transfer (J. Kim, D. Darley, and W. Buckel, FEBS J. 272:550-561, 2005). Prior to the dehydration, a dehydrogenase and a CoA transferase are supposed to be involved in the formation of (R)-2-hydroxyisocaproyl-CoA. Deduced amino acid sequences of ldhA and hadA from the genome of C. difficile showed high identities to d-lactate dehydrogenase and family III CoA transferase, respectively. Both putative genes encoding the dehydrogenase and CoA transferase were cloned and overexpressed in Escherichia coli; the recombinant Strep tag II fusion proteins were purified to homogeneity and characterized. The substrate specificity of the monomeric LdhA (36.5 kDa) indicated that 2-oxoisocaproate (K(m) = 68 muM, k(cat) = 31 s(-1)) and NADH were the native substrates. For the reverse reaction, the enzyme accepted (R)- but not (S)-2-hydroxyisocaproate and therefore was named (R)-2-hydroxyisocaproate dehydrogenase. HadA showed CoA transferase activity with (R)-2-hydroxyisocaproyl-CoA as a donor and isocaproate or (E)-2-isocaprenoate as an acceptor. By site-directed mutagenesis, the conserved D171 was identified as an essential catalytic residue probably involved in the formation of a mixed anhydride with the acyl group of the thioester substrate. However, neither hydroxylamine nor sodium borohydride, both of which are inactivators of the CoA transferase, modified this residue. The dehydrogenase and the CoA transferase fit well into the proposed pathway of leucine reduction to isocaproate.

Crystal structure of CaiB, a type-III CoA transferase in carnitine metabolism.

Carnitine is an important molecule in human metabolism, mainly because of its role in the transport of long-chain fatty acids across the inner mitochondrial membrane. Escherichia coli uses carnitine as a terminal electron acceptor during anaerobic metabolism. Bacteria present in our large intestine break down carnitine that is not absorbed in the small intestine. One part of this catabolic pathway is reversible and can be utilized for bioproduction of large amounts of stereochemically pure L-carnitine, which is used medically for the treatment of a variety of human diseases. Here, we present the crystal structure of the E. coli protein CaiB, which is a member of the recently identified type-III coenzyme A (CoA) transferase family and catalyzes the transfer of the CoA moiety between gamma-butyrobetaine-CoA and carnitine forming carnityl-CoA and gamma-butyrobetaine. This is the first protein from the carnitine metabolic pathway to be structurally characterized. The structure of CaiB reveals a spectacular fold where two monomers are interlaced to form an interlocked dimer. A molecule of the crystallization buffer bis-(2-hydroxyethyl)imino-tris(hydroxymethyl)methane (bis-tris) is bound in a large pocket located primarily in the small domain, and we propose that this pocket constitutes the binding site for both substrate moieties participating in the CaiB transfer reaction. The binding of CoA to CaiB induces a domain movement that closes the active site of the protein. This is the first observation of a domain movement in the type-III CoA transferase family and can play an important role in coupling substrate binding to initiation of the catalytic reaction.

A new family of CoA-transferases.

CoA-transferases are found in organisms from all lines of descent. Most of these enzymes belong to two well-known enzyme families, but recent work on unusual biochemical pathways of anaerobic bacteria has revealed the existence of a third family of CoA-transferases. The members of this enzyme family differ in sequence and reaction mechanism from CoA-transferases of the other families. Currently known enzymes of the new family are a formyl-CoA: oxalate CoA-transferase, a succinyl-CoA: (R)-benzylsuccinate CoA-transferase, an (E)-cinnamoyl-CoA: (R)-phenyllactate CoA-transferase, and a butyrobetainyl-CoA: (R)-carnitine CoA-transferase. In addition, a large number of proteins of unknown or differently annotated function from Bacteria, Archaea and Eukarya apparently belong to this enzyme family. Properties and reaction mechanisms of the CoA-transferases of family III are described and compared to those of the previously known CoA-transferases.

Glutaconate CoA-transferase from Acidaminococcus fermentans: the crystal structure reveals homology with other CoA-transferases.

BACKGROUND: Coenzyme A-transferases are a family of enzymes with a diverse substrate specificity and subunit composition. Members of this group of enzymes are found in anaerobic fermenting bacteria, aerobic bacteria and in the mitochondria of humans and other mammals, but so far none have been crystallized. A defect in the human gene encoding succinyl-CoA: 3-oxoacid CoA-transferase causes a metabolic disease which leads to severe ketoacidosis, thus reflecting the importance of this family of enzymes. All CoA-transferases share a common mechanism in which the CoA moiety is transferred from a donor (e.g. acetyl CoA) to an acceptor, (R)-2-hydroxyglutarate, whereby acetate is formed. The transfer has been described by a ping-pong mechanism in which CoA is bound to the active-site residue of the enzyme as a covalent thiol ester intermediate. We describe here the crystal structure of glutaconate CoA-transferase (GCT) from the strictly anaerobic bacterium Acidaminococcus fermentans. This enzyme activates (R)-2-hydroxyglutarate to (R)-2-hydroxyglutaryl-CoA in the pathway of glutamate fermentation. We initiated this project to gain further insight into the function of this enzyme and the structural basis for the characteristics of CoA-transferases. RESULTS: The crystal structure of GCT was solved by multiple isomorphous replacement to 2.55 A resolution. The enzyme is a heterooctamer and its overall arrangement of subunits can be regarded as an (AB)4tetramer obeying 222 symmetry. Both subunits A and B belong to the open alpha/beta-protein class and can be described as a four-layered alpha/alpha/beta/alpha type with a novel composition and connectivity of the secondary structure elements. The core of subunit A consists of seven alpha/beta repeats resulting in an all parallel central beta sheet, against which helices pack from both sides. In contrast, the centre of subunit B is formed by a ninefold mixed beta sheet. In both subunits the helical C terminus is folded back onto the N-terminal domain to form the third layer of helices. CONCLUSIONS: The active site of GCT is located at the interface of subunits A and B and is formed by loops of both subunits. The funnel-shaped opening to the active site has a depth and diameter of about 20 A with the catalytic residue, Glu54 of subunit B, at the bottom. The active-site glutamate residue is stabilized by hydrogen bonds. Despite very low amino acid sequence similarity, subunits A and B reveal a similar overall fold. Large parts of their structures can be spatially superimposed, suggesting that both subunits have evolved from a common ancestor.