The ANL superfamily of adenylating enzymes contains acyl- and aryl-CoA synthetases, firefly luciferase, and the adenylation domains of the modular Non-Ribosomal Peptide Synthetases (NRPSs). Members of this family catalyze two partial reactions, the initial adenylation of a carboxylate to form an acyl-AMP intermediate, followed by a second partial reaction, most commonly, the formation of a thioester. Recent biochemical and structural evidence has been presented that supports the use by this enzyme family of a remarkable catalytic strategy for the two catalytic steps. The enzymes use a 140° domain rotation to present opposing faces of the dynamic C-terminal domain to the active site for the different partial reactions. Support for this Domain Alternation strategy is presented along with an explanation of the advantage of this catalytic strategy on the reaction catalyzed by the ANL enzymes. Finally, the ramifications of this domain rotation in the catalytic cycle of the modular NRPS enzymes are discussed.
The acyl-AMP forming family of adenylating enzymes catalyze two-step reactions to activate a carboxylate with the chemical energy derived from ATP hydrolysis. X-ray crystal structures have been determined for multiple members of this family and, together with biochemical studies, provide insights into the active site and catalytic mechanisms used by these enzymes. These studies have shown that the enzymes use a domain rotation of 140° to reconfigure a single active site to catalyze the two partial reactions. We present here the crystal structure of a new medium chain acyl-CoA synthetase from Methanosarcina acetivorans. The binding pocket for the three substrates is analyzed, with many conserved residues present in the AMP binding pocket. The CoA binding pocket is compared to the pockets of both acetyl-CoA synthetase and 4-chlorobenzoate:CoA ligase. Most interestingly, the acyl binding pocket of the new structure is compared with other acyl- and aryl-CoA synthetases. A comparison of the acyl-binding pocket of the acyl-CoA synthetase from M. acetivorans with other structures identifies a shallow pocket that is used to bind the medium chain carboxylates. These insights emphasize the high sequence and structural diversity among this family in the area of the acyl binding pocket.
Adenylate-forming enzyme; substrate specificity; X-ray crystallography
The adenylate-forming enzymes, including acyl-CoA synthetases, the adenylation domains of non-ribosomal peptide synthetases (NRPS), and firefly luciferase, perform two half-reactions in a ping-pong mechanism. We have proposed a Domain Alternation mechanism for these enzymes whereby, upon completion of the initial adenylation reaction, the C-terminal domain of these enzymes undergoes a 140° rotation to perform the second thioester-forming half reaction. Structural and kinetic data of mutant enzymes support this hypothesis. We present here mutations to Salmonella enterica Acetyl-CoA Synthetase (Acs) and test the ability of the enzymes to catalyze the complete reaction and the adenylation half-reaction. Substitution of Lys609 with alanine results in an enzyme that is unable to catalyze the adenylate reaction, while the Gly524 to leucine substitution is unable to catalyze the complete reaction yet catalyzes the adenylation half-reaction with activity comparable to the wild-type enzyme. The positions of these two residues, which are located on the mobile C-terminal domain, strongly support the domain alternation hypothesis. We also present steady-state kinetic data of putative substrate-binding residues and demonstrate that no single residue plays a dominant role in dictating CoA binding. We have also created two mutations in the active site to alter the acyl substrate specificity. Finally, the crystallographic structures of wild-type Acs and mutants R194A, R584A, R584E, K609A, and V386A are presented to support the biochemical analysis.
Adenylate-forming Enzymes; Non-Ribosomal Peptide Synthetase; Domain Alteration; X-ray Crystallography; CoA-ligase
The fungal neurotoxin α-cyclopiazonic acid (CPA), a nanomolar inhibitor of Ca2+-ATPase, has a pentacyclic indole tetramic acid scaffold that arises from one molecule of tryptophan, acetyl-CoA, malonyl-CoA and dimethylallyl pyrophosphate by consecutive action of three enzymes CpaS, D, O. CpaS is a hybrid, two module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) that makes and releases cyclo-acetoacetyl-L-tryptophan (cAATrp), the tetramic acid that serves as substrate for subsequent prenylation and oxidative cyclization to the five ring CPA scaffold. The NRPS module in CpaS has a predicted four domain organization of Condensation, Adenylation, Thiolation, Reductase* (C-A-T-R*) where R* lacks the critical Ser-Tyr-Lys catalytic triad of the short chain dehydrogenase/reductase (SDR) super family. By heterologous overproduction in E. coli of the 56 kDa Aspergillus flavus CpaS TR* didomain, and the single T and R* domains, we demonstrate that CpaS catalyzes a Dieckmann type cyclization on the N-acetoacetyl-Trp intermediate bound in thioester linkage to the phosphopantetheinyl arm of the T domain to form and release cAATrp. This occurs without any participation of NAD(P)H, so R* does not function as a canonical SDR family member. Use of the T and R* domains in in trans assays enabled multiple turnovers and evaluation of specific mutants. Mutation of the D3803 residue in the R* domain, conserved in other fungal tetramate synthetases, abolished activity both in in trans and in cis (TR*) activity assays. It is likely that cyclization of β-ketoacyl-aminoacyl-S-pantetheinyl intermediates to released tetramates represents a default cyclization/release route for redox-incompetent R* domains embedded in NRPS assembly lines.
Beetle luciferases catalyze a two-step reaction that includes the initial adenylation of the luciferin substrate, followed by an oxidative decarboxylation that ultimately produces light. Evidence for homologous acyl-CoA synthetases supports a domain alternation catalytic mechanism in which these enzymes’ C-terminal domain rotates by ~140° to adopt two conformations that are used to catalyze the two partial reactions. While many structures exist of acyl-CoA synthetases in both conformations, to date only biochemical evidence supports domain alternation with luciferase. We have determined the structure of a cross-linked luciferase enzyme that is trapped in the second conformation. This new structure supports the role of the second catalytic conformation and provides insights into the biochemical mechanism of the luciferase oxidative step.
Acyl-CoA synthetase enzymes are essential for de novo lipid synthesis, fatty acid catabolism, and remodeling of membranes. Activation of fatty acids requires a two-step reaction catalyzed by these enzymes. In the first step, an acyl-AMP intermediate is formed from ATP. AMP is then exchanged with CoA to produce the activated acyl-CoA. The release of AMP in this reaction defines the superfamily of AMP-forming enzymes. The length of the carbon chain of the fatty acid species defines the substrate specificity for the different acyl-CoA synthetases (ACS). On this basis, five sub-families of ACS have been characterized. The purpose of this review is to report on the large family of mammalian long-chain acyl-CoA synthetases (ACSL), which activate fatty acids with chain lengths of 12 to 20 carbon atoms. Five genes and several isoforms generated by alternative splicing have been identified and limited information is available on their localization. The structure of these membrane proteins has not been solved for the mammalian ACSLs but homology to a bacterial form, whose structure has been determined, points at specific structural features that are important for these enzymes across species. The bacterial form acts as a dimer and has a conserved short motif, called the fatty acid Gate domain, that seems to determine substrate specificity. We will discuss the characterization and identification of the different spliced isoforms, draw attention to the inconsistencies and errors in their annotations, and their cellular localizations. These membrane proteins act on membrane-bound substrates probably as homo- and as heterodimer complexes but have often been expressed as single recombinant isoforms, apparently purified as monomers and tested in Triton X-100 micelles. We will argue that such studies have failed to provide an accurate assessment of the activity and of the distinct function of these enzymes in mammalian cells.
Activation of fatty acids by acyl-CoA synthetase enzymes is required for de novo lipid synthesis, fatty acid catabolism, and remodeling of biological membranes. Human long-chain acyl-CoA synthetase member 6, ASCL6, is a form present in the plasma membrane of cells. Splicing events affecting the amino-terminus and alternative motifs near the ATP-binding site generate different isoforms of ACSL6.
Isoforms with different fatty acid Gate-domain motifs have different activity and the form lacking this domain, isoform 3, showed no detectable activity. Enzymes truncated of the first 40 residues generate acyl-CoAs at a faster rate than the full-length protein. The gating residue, which prevents entry of the fatty acid substrate unless one molecule of ATP has already accessed the catalytic site, was identified as a tyrosine for isoform 1 and a phenylalanine for isoform 2 at position 319. All isoforms, with or without a fatty acid Gate-domain, as well as recombinant protein truncated of the N-terminus, can interact to form enzymatic complexes with identical or different isoforms.
The alternative fatty acid Gate-domain motifs are essential determinants for the activity of the human ACSL6 isoforms, which appear to act as homodimeric enzyme as well as in complex with other spliced forms. These findings provide evidence that the diversity of these enzyme species could produce the variety of acyl-CoA synthetase activities that are necessary to generate and repair the hundreds of lipid species present in membranes.
Members of the adenylate-forming family of enzymes play a role in the metabolism of halogenated aromatics and of short, medium, and long chain fatty acids, as well as in the biosynthesis of menaquinone, peptide antibiotics, and peptide siderophores. This family includes a subfamily of acyl- and aryl-CoA ligases that catalyze thioester synthesis through two half-reactions. A carboxylate substrate first reacts with ATP to form an acyl-adenylate. Subsequent to the release of the product PPi, the enzyme binds CoA, which attacks the activated acyl group to displace AMP. Structural and functional studies on different family members suggest that these enzymes alternate between two conformations during catalysis of the two half-reactions. Specifically, after the initial adenylation step, the C-terminal domain rotates by ~140° to adopt a second conformation for thioester formation. Previously, we determined the structure of 4-chlorobenzoate:CoA ligase (CBL) in the adenylate forming conformation bound to 4-chlorobenzoate. We have determined two new crystal structures. We have determined the structure of CBL in the original adenylate-forming conformation, bound to the adenylate intermediate. Additionally, we have used a novel product analog, 4-chlorophenacyl-CoA, to trap the enzyme in the thioester-forming conformation and determined this structure in a new crystal form. This work identifies a novel binding pocket for the CoA nucleotide. The structures presented herein provide the foundation for biochemical analyses presented in the accompanying manuscript (Wu et al.). The complete characterization of this enzyme allows us to provide an explanation for the use of the domain alternation strategy by these enzymes.
4-chorobenzoate: CoA ligase; 4-chlorobenzoate; coenzyme A; adenylate-forming enzyme superfamily; acyl-adenylate; X-ray structure; Domain Alternation; enzyme conformational changes
By complementation of a previously described non-phosphinothricin tripeptide (PTT)-producing mutant, NTG1, which is blocked in nonribosomal synthesis of the peptide, a DNA fragment including the putative peptide synthetase gene phsA was isolated (W. Wohlleben, R. Alijah, J. Dorendorf, D. Hillemann, B. Nussbaumer, and S. Pelzer, Gene 115:127-132, 1992). Sequence analysis of phsA revealed that it encodes a protein of 622 amino acids with regions which are highly similar to core motifs characteristic for peptide synthetases. PhsA represents one functional domain of a peptide synthetase which is necessary for activation and condensation of one amino acid, probably N-acetyl-demethyl-phosphinothricin. With regard to the arrangement of the flanking genes, phsA is the first peptide synthetase gene which is not in the direct neighborhood of additional peptide synthetase genes involved in the formation of peptide antibiotics. Gene disruption mutants with internal fragments of phsA subcloned in temperature-sensitive pGM vectors were generated. Integration occurred either into the chromosomal copy of phsA or into a gene outside the known phsA locus, resulting in two classes of non-PTT-producing mutants. In cofeeding experiments the former phsA mutants showed the same phenotype as did NTG1, which confirmed participation of phsA in nonribosomal synthesis of PTT. A truncated phsA gene was overexpressed in Escherichia coli, and the resulting protein of 593 amino acids was purified for raising antibodies. By performing immunoblotting experiments, the expression of phsA could be detected in Streptomyces viridochromogenes Tü494 in the stationary-growth phase after 4 days of incubation.
The formation of acyl-CoA by the action of acyl-CoA synthetases plays a crucial role in membrane lipid turnover, including the plasma membrane of erythrocytes. In human, five Acyl-CoA Synthetase Long-chain (ACSL) genes have been identified with as many as 3 different transcript variants for each.
Acyl-CoA Synthetase Long-chain member 6 (ACSL6) is responsible for activation of long-chain fatty acids in erythrocytes. Two additional transcript variants were also isolated from brain and testis. We report the expression in reticulocytes of two new variants and of the one isolated from brain. All three represented different spliced variants of a mutually exclusive exon pair. They encode a slightly different short motif which contains a conserved structural domain, the fatty acid Gate domain. The motifs differ in the presence of either the aromatic residue phenylalanine (Phe) or tyrosine (Tyr). Based on homology, two new isoforms for the closely related ACSL1 were predicted and characterized. One represented a switch of the Phe- to the Tyr-Gate domain motif, the other resulted from the exclusion of both. Swapping of this motif also appears to be common in all mammalian ACSL member 1 and 6 homologs.
We propose that a Phe to Tyr substitution or deletion of the Gate domain, is the structural reason for the conserved alternative splicing that affects these motifs. Our findings support our hypothesis that this region is structurally important to define the activity of these enzymes.
Short- and medium-chain acyl coenzyme A (acyl-CoA) synthetases catalyze the formation of acyl-CoA from an acyl substrate, ATP, and CoA. These enzymes catalyze mechanistically similar two-step reactions that proceed through an enzyme-bound acyl-AMP intermediate. Here we describe the characterization of a member of this enzyme family from the methane-producing archaeon Methanosarcina acetivorans. This enzyme, a medium-chain acyl-CoA synthetase designated MacsMa, utilizes 2-methylbutyrate as its preferred substrate for acyl-CoA synthesis but cannot utilize acetate and thus cannot catalyze the first step of acetoclastic methanogenesis in M. acetivorans. When propionate or other less favorable acyl substrates, such as butyrate, 2-methylpropionate, or 2-methylvalerate, were utilized, the acyl-CoA was not produced or was produced at reduced levels. Instead, acyl-AMP and PPi were released in the absence of CoA, whereas in the presence of CoA, the intermediate was broken down into AMP and the acyl substrate, which were released along with PPi. These results suggest that although acyl-CoA synthetases may have the ability to utilize a broad range of substrates for the acyl-adenylate-forming first step of the reaction, the intermediate may not be suitable for the thioester-forming second step. The MacsMa structure has revealed the putative acyl substrate- and CoA-binding pockets. Six residues proposed to form the acyl substrate-binding pocket, Lys256, Cys298, Gly351, Trp259, Trp237, and Trp254, were targeted for alteration. Characterization of the enzyme variants indicates that these six residues are critical in acyl substrate binding and catalysis, and even conservative alterations significantly reduced the catalytic ability of the enzyme.
In Methanothrix soehngenii, acetate is activated to acetyl-coenzyme A (acetyl-CoA) by an acetyl-CoA synthetase. Cell extracts contained high activities of adenylate kinase and pyrophosphatase, but no activities of a pyrophosphate:AMP and pyrophosphate:ADP phosphotransferase, indicating that the activation of 1 acetate in Methanothrix requires 2 ATP. Acetyl-CoA synthetase was purified 22-fold in four steps to apparent homogeneity. The native molecular mass of the enzyme from M. soehngenii estimated by gel filtration was 148 kilodaltons (kDa). The enzyme was composed of two subunits with a molecular mass of 73 kDa in an alpha 2 oligomeric structure. The acetyl-CoA synthetase constituted up to 4% of the soluble cell protein. At the optimum pH of 8.5, the Vmax was 55 mumol of acetyl-CoA formed per min per mg of protein. Analysis of enzyme kinetic properties revealed a Km of 0.86 mM for acetate and 48 microM for coenzyme A. With varying amounts of ATP, weak sigmoidal kinetic was observed. The Hill plot gave a slope of 1.58 +/- 0.12, suggesting two interacting substrate sites for the ATP. The kinetic properties of the acetyl-CoA synthetase can explain the high affinity for acetate of Methanothrix soehngenii.
A conserved catalytic core of the ATP-dependent DNA ligases is composed of an N-terminal domain (domain 1, containing nucleotidyl transferase motifs I, III, IIIa and IV) and a C-terminal domain (domain 2, containing motif VI) with an intervening cleft. Motif V links the two structural domains. Deletion analysis of the 298 amino acid Chlorella virus DNA ligase indicates that motif VI plays a critical role in the reaction of ligase with ATP to form ligase-adenylate, but is dispensable for the two subsequent steps in the ligation pathway; DNA-adenylate formation and strand closure. We find that formation of a phosphodiester at a pre-adenylated nick is subject to a rate limiting step that does not apply during the sealing of nicked DNA by ligase-adenylate. This step, presumably conformational, is accelerated or circumvented by deleting five amino acids of motif VI. The motif I lysine nucleophile (Lys27) is not required for strand closure by wild-type ligase, but this residue enhances the closure rate by a factor of 16 when motif VI is truncated. We find that a more extensively truncated ligase consisting of only N-terminal domain 1 and motif V is inert in ligase--adenylate formation, but competent to catalyze strand closure at a pre-adenylated nick. These results suggest that different enzymic catalysts facilitate the three steps of the DNA ligase reaction.
Crystal structures of histidyl-tRNA synthetase from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme, and reveal differences from bacterial homologs. Histidyl-tRNA synthetases in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active site rearrangement upon histidine binding, but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme’s first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human histidyl-tRNA synthetases. The essentiality of the single histidyl-tRNA synthetase gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference.
aminoacyl-tRNA synthetase; protozoa; drug target; tropical disease; RNAi
In Salmonella enterica, the protein acetyltransferase (Pat) enzyme is part of the sirtuin-dependent acylation/deacylation system (SDPADS) that modulates the activity of several proteins via the acylation of lysine residues critical to their activities. Pat is a ~98 kDa protein that, with two distinct domains, an N-terminal acyl-CoA synthetase (NDP-forming) domain (~700 aa) and a C-terminal acetyltransferase domain (~160 aa), with homology to proteins of the Gcn5-related N-acetyltransferase (GNAT) superfamily. Although the role of the GNAT-like domain is likely responsible for the catalytic activity of Pat, the role of the N-terminal domain remains unclear. Here we report the use of positive selection for identification of residues critical for Pat enzyme activity. This approach revealed seven residues that, when changed, resulted in drastic loss of Pat activity in vitro which caused a discernable loss-of-function phenotype. Five of the seven residues were located in the N-terminal region of Pat and two were located in the GNAT-like domain. Each single-amino-acid variant had a circular dichroism spectrum that differed from that of the wild-type Pat protein, suggesting that loss of enzymatic activity in the mutant proteins was likely due to an inability to acquire its biologically active fold.
Post-translational modification; Protein acetylation; Lysine acylation; Protein acetyltransferases; Metabolic regulation; Acetate metabolism
Biosynthesis of coenzyme A (CoA) requires a five-step process using pantothenate and cysteine in the fission yeast Schizosaccharomyces pombe. CoA contains a thiol (SH) group, which reacts with carboxylic acid to form thioesters, giving rise to acyl-activated CoAs such as acetyl-CoA. Acetyl-CoA is essential for energy metabolism and protein acetylation, and, in higher eukaryotes, for the production of neurotransmitters. We isolated a novel S. pombe temperature-sensitive strain ppc1-537 mutated in the catalytic region of phosphopantothenoylcysteine synthetase (designated Ppc1), which is essential for CoA synthesis. The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation. Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence. Ppc1 is thus the product of a super-housekeeping gene. The ppc1-537 mutant showed combined synthetic lethal defects with five of six histone deacetylase mutants, whereas sir2 deletion exceptionally rescued the ppc1-537 phenotype. In synchronous cultures, ppc1-537 cells can proceed to the S phase, but lose viability during mitosis failing in sister centromere/kinetochore segregation and nuclear division. Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation. The CoA-supported metabolism thus controls the state of chromosome DNA.
acetyl-CoA; pantothenate; phosphopantothenoylcysteine synthetase; lipid droplet; centromere/kinetochore
Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS;
acetate:CoA ligase (AMP-forming), EC 22.214.171.124) is a key enzyme for
conversion of acetate to acetyl-CoA, an essential intermediate at the
junction of anabolic and catabolic pathways. Phylogenetic analysis of
putative short and medium chain acyl-CoA synthetase sequences
indicates that the ACSs form a distinct clade from other acyl-CoA
synthetases. Within this clade, the archaeal ACSs are not monophyletic
and fall into three groups composed of both bacterial and archaeal
sequences. Kinetic analysis of two archaeal enzymes, an ACS from
thermautotrophicus (designated as MT-ACS1) and an ACS
from Archaeoglobus fulgidus
(designated as AF-ACS2), revealed that these enzymes have very
different properties. MT-ACS1 has nearly 11-fold higher affinity and
14-fold higher catalytic efficiency with acetate than with propionate,
a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold
higher affinity and catalytic efficiency with acetate than with
propionate. This enzyme has an affinity for propionate that is almost
identical to that of MT-ACS1 for acetate and nearly tenfold higher
than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is
limited to acetate and propionate as acyl substrates, whereas AF-ACS2
can also utilize longer straight and branched chain acyl substrates.
Phylogenetic analysis, sequence alignment and structural modeling
suggest a molecular basis for the altered substrate preference and
expanded substrate range of AF-ACS2 versus MT-ACS1.
acetate; Archaeoglobus fulgidus; Methanothermobacter thermautotrophicus
NAGS catalyzes the conversion of glutamate and acetyl-CoA to N-acetylglutamate (NAG) the essential allosteric activator of carbamyl phosphate synthetase I, the first urea cycle enzyme in mammals. A 17-year-old female with recurrent hyperammonemia attacks, the cause of which remained undiagnosed for 8 years in spite of multiple molecular and biochemical investigations, showed markedly enhanced ureagenesis (measured by isotope incorporation) in response to N-carbamylglutamate (NCG). This led to sequencing of the regulatory regions of the NAG synthase (NAGS) gene and identification of a deleterious single-base substitution in the upstream enhancer.
The homozygous mutation (-3063C>A), affecting a highly conserved nucleotide within the Hepatic Nuclear Factor 1 (HNF-1) binding site, was not found in SNP databases and in a screen of 1086 alleles from a diverse population. Functional assays demonstrated that this mutation decreases transcription and binding of HNF-1 to the NAGS gene, while a consensus HNF-1 binding sequence enhances binding to HNF-1 and increases transcription. Oral daily NCG therapy appears to have restored ureagenesis in this patient, normalizing her biochemical markers, and allowing discontinuation of alternate pathway therapy and normalization of her diet with no recurrence of hyperammonemia.
N-acetyl-L-glutamate; acetylglutamate; hyperammonemia; bio-informatics; urea cycle; stable isotope; gene expression regulation
tRNAs are transcribed as precursors and processed in a series of required reactions leading to aminoacylation and translation. The 3′ end trailer can be removed by the pre-tRNA processing endonuclease tRNase Z, an ancient, conserved member of the β-lactamase superfamily of metal-dependent hydrolases. The signature sequence of this family, the His domain (HxHxDH, Motif II), and histidines in Motifs III and V and aspartate in Motif IV contribute seven side chains for coordination of two divalent metal ions. We previously investigated the effects on catalysis of substitutions in Motif II, and in the PxKxRN loop and Motif I on the amino side of Motif II. Herein we present the effects of substitutions on the carboxy side of Motif II within Motifs III, IV, the HEAT and HST loops and Motif V. Substitution of the Motif IV aspartate reduces catalytic efficiency more than 10,000-fold. Histidines in Motif III, V and the HST loop are also functionally important. Strikingly, replacement of Glu in the HEAT loop with Ala reducesefficiency by ~1000-fold. Proximity and orientation of this Glu side chain relative to His in the HST loop and the importance of both residues for catalysis suggest that they function as a duo in a proton transfer at the final stage of reaction, characteristic of the tRNase Z class of RNA endonucleases.
Long-chain acyl-coenzyme A (CoA) compounds (palmityl, stearyl, and oleyl) were found to be potent inhibitors of acetyl-CoA synthetase (ACS) of Saccharomyces cerevisiae strain LK2G12 from aerobic, but not from nonaerobic, cells. The effectiveness of the inhibitors of the aerobic enzyme was in the following order: palmityl-CoA < stearyl-CoA < oleyl-CoA. Short-chain acyl-CoA compounds (propionyl, butyryl, and valeryl) and long-chain fatty acids had no effect on ACS from either source. The inhibition by oleyl-CoA was found to be dependent on enzyme concentration, whereas the inhibition by palmityl- and stearyl-CoA was independent of ACS concentration. Inhibition by palmityl-CoA was noncompetitive with respect to both acetate and CoA, and with increasing concentration of inhibitor the pattern was sigmoidal, with a Hill value of 3.24. At maximally inhibitory concentrations of palmityl-CoA, a small amount of enzyme activity remained. This noninhibitable enzyme in aerobic cells was shown not to be of nonaerobic origin.
Anabaenopeptins (AP) are bioactive cyclic hexapeptides synthesized nonribosomally in cyanobacteria. APs are characterized by several conserved motifs, including the ureido bond, N-methylation in position 5, and d-Lys in position 2. All other positions of the AP molecule are variable, resulting in numerous structural variants. We have identified a nonribosomal peptide synthetase (NRPS) operon from Planktothrix agardhii strain CYA126/8 consisting of five genes (apnA to apnE) encoding six NRPS modules and have confirmed its role in AP synthesis by the generation of a mutant via insertional inactivation of apnC. In order to correlate the genetic diversity among adenylation domains (A domains) with AP structure variation, we sequenced the A domains of all six NRPS modules from seven Planktothrix strains differing in the production of AP congeners. It is remarkable that single strains coproduce APs bearing either of the chemically divergent amino acids Arg and Tyr in exocyclic position 1. Since the A domain of the initiation module (the ApnA A1 domain) has been proposed to activate the amino acid incorporated into exocyclic position 1, we decided to analyze this domain both biochemically and phylogenetically. Only ApnA A1 enzymes from strains producing AP molecules containing Arg or Tyr in position 1 were found to activate these two chemically divergent amino acids in vitro. Phylogenetic analysis of apn A domain sequences revealed that strains with a promiscuous ApnA A1 domain are derived from an ancestor that activates only Arg. Surprisingly, positive selection appears to affect only three codons within the apnA A1 gene, suggesting that this remarkable promiscuity has evolved from point mutations only.
Trichoderma virens (synonym, Gliocladium virens), a deuteromycete fungus, suppresses soilborne plant diseases caused by a number of fungi and is used as a biocontrol agent. Several traits that may contribute to the antagonistic interactions of T. virens with disease-causing fungi involve the production of peptide metabolites (e.g., the antibiotic gliotoxin and siderophores used for iron acquisition). We cloned a 5,056-bp partial cDNA encoding a putative peptide synthetase (Psy1) from T. virens using conserved motifs found within the adenylate domain of peptide synthetases. Sequence similarities with conserved motifs of the adenylation domain, acyl transfer, and two condensation domains support identification of the Psy1 gene as a gene that encodes a peptide synthetase. Disruption of the native Psy1 gene through gene replacement was used to identify the function of this gene. Psy1 disruptants produced normal amounts of gliotoxin but grew poorly under low-iron conditions, suggesting that Psy1 plays a role in siderophore production. Psy1 disruptants cannot produce the major T. virens siderophore dimerum acid, a dipetide of acylated Nδ-hydroxyornithine. Biocontrol activity against damping-off diseases caused by Pythium ultimum and Rhizoctonia solani was not reduced by the Psy1 disruption, suggesting that iron competition through dimerum acid production does not contribute significantly to disease suppression activity under the conditions used.
Biotin is a water-soluble vitamin required by all organisms, but only synthesized by plants and some bacterial and fungal species. As a cofactor, biotin is responsible for carbon dioxide transfer in all biotin-dependent carboxylases, including acetyl-CoA carboxylase, methylcrotonyl-CoA carboxylase, and pyruvate carboxylase. Adding biotin to carboxylases is catalyzed by the enzyme holocarboxylase synthetase (HCS). Biotin is also involved in gene regulation, and there is some indication that histones can be biotinylated in humans. Histone proteins and most histone modifications are highly conserved among eukaryotes. HCS1 is the only functional biotin ligase in Arabidopsis and has a high homology with human HCS. Therefore, we hypothesized that HCS1 also biotinylates histone proteins in Arabidopsis. A comparison of the catalytic domain of HCS proteins was performed among eukaryotes, prokaryotes, and archaea, and this domain is highly conserved across the selected organisms. Biotinylated histones could not be identified in vivo by using avidin precipitation or two-dimensional gel analysis. However, HCS1 physically interacts with Arabidopsis histone H3 in vitro, indicating the possibility of the role of this enzyme in the regulation of gene expression.
Aeruginosins are bioactive oligopeptides that are produced in high structural diversity by strains of the bloom-forming cyanobacterial genera Microcystis and Planktothrix. A hallmark of aeruginosins is the unusual Choi moiety central to the tetrapeptides, while other positions are occupied by variable moieties in individual congeners. Here we report on three aeruginosin synthetase gene clusters (aer) of Microcystis aeruginosa (strains PCC 7806, NIES-98, and NIES-843). The analysis and comparison the aer gene clusters provide the first insight into the molecular basis of biosynthetic and structural plasticity in aeruginosin pathways. Major parts of the aer gene clusters are highly similar in all strains, particularly the genes coding for the first three nonribosomal peptide synthetase (NRPS) modules except for the region coding for the second adenylation domain. However, the gene clusters differ largely in genes coding for tailoring enzymes such as halogenases and sulfotransferases, reflecting structural peculiarities in aeruginosin congeners produced by the individual strains. Significant deviations were further observed in the C-terminal NRPS modules, suggesting two distinct release mechanisms. The architecture of the gene clusters is in agreement with the particular aeruginosin variants that are produced by individual strains, the structures of two of which (aeruginosins 686 A and 686 B) were elucidated. The aer gene clusters of Microcystis and Planktothrix are proposed to originate from a common ancestor and to have evolved to their present-day diversity largely through horizontal gene transfer and recombination events.
Siderophores are known virulence factors and their biosynthesis is a target for new antibacterial agents. A nonribosomal peptide synthetase independent siderophore (NIS) biosynthetic pathway in Dickeya dadantii is responsible for production of the siderophore achromobactin. The D. dadantii AcsD enzyme has been shown to enantioselectively esterify citric acid with L-serine in the first committed step of achromobactin biosynthesis. The reaction occurs in two steps: stereospecific activation of citric acid by adenylation, followed by attack of the enzyme bound citryl adenylate by L-serine to give the homochiral ester. We now report a detailed characterization of the substrate profile and mechanism of the second (acyl transfer) step of AcsD enzyme. We demonstrate that the enzyme catalyzes formation of not only esters, but also amides from the citryl-adenylate intermediate. We have rationalized the substrate utilization profile for the acylation reaction by determining the first X-ray crystal structure of a product complex for this enzyme class. We have identified the residues that are important for both recognition of L-serine and catalysis of ester formation. Our hypotheses were tested by biochemical analysis of various mutants, one of which shows a reversal of specificity from the wild type with respect to non-natural substrates. This change can be rationalized on the basis of our structural data. That this change in specificity is accompanied by no loss in activity suggests that AcsD and other members of the NIS synthetase superfamily may have biotransformation potential.