AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1) 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 Methanothermobacter 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.     

Crystal structure of yeast acetyl-coenzyme A synthetase in complex with AMP

Acetyl-coenzyme A synthetase (ACS) belongs to the family of AMP-forming enzymes that also includes acyl-CoA synthetases, firefly luciferase, and nonribosomal peptide synthetases. ACS catalyzes the two-step activation of acetate to acetyl-CoA: formation of an acetyl-AMP intermediate from acetate and ATP and the transfer of the acetyl group to CoA. In mammals, the acetyl-CoA product is used for biosynthesis of long chain fatty acids as well as energy production. We have determined the crystal structure of yeast ACS in a binary complex with AMP at 2.3 A resolution. The structure contains a large, N-terminal domain and a small, C-terminal domain. AMP is bound at the interface between the two domains. This structure represents a new conformation for the ACS enzyme, which may be competent for catalyzing the first step of the reaction. A Lys residue that is critical for this step is located in the active site. A rotation of 140 degrees in the small domain is needed for the binding of CoA and the catalysis of the second step. In contrast to the monomeric bacterial enzyme, yeast ACS is a stable trimer.     

Regulation of acetate and acetyl-CoA converting enzymes during growth on acetate and/or glucose in the halophilic archaeon Haloarcula marismortui

Haloarcula marismortui formed acetate during aerobic growth on glucose and utilized acetate as growth substrate. On glucose/acetate mixtures diauxic growth was observed with glucose as the preferred substrate. Regulation of enzyme activities, related to glucose and acetate metabolism was analyzed. It was found that both glucose dehydrogenase (GDH) and ADP-forming acetyl-CoA synthetase (ACD) were upregulated during periods of glucose consumption and acetate formation, whereas both AMP-forming acetyl-CoA synthetase (ACS) and malate synthase (MS) were downregulated. Conversely, upregulation of ACS and MS and downregulation of ACD and GDH were observed during periods of acetate consumption. MS was also upregulated during growth on peptides in the absence of acetate. From the data we conclude that a glucose-inducible ACD catalyzes acetate formation whereas acetate activation is catalyzed by an acetate-inducible ACS; both ACS and MS are apparently induced by acetate and repressed by glucose.     

The 2.1 Å crystal structure of an acyl‐CoA synthetase from Methanosarcina acetivorans reveals an alternate acyl‐binding pocket for small branched acyl substrates

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. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

A novel octameric AMP-forming acetyl-CoA synthetase from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum

AMP-forming acetyl-CoA synthetases (ACSs) are ubiquitous in all three domains of life. Here, we report the first characterization of an ACS from a hyperthermophilic organism, from the archaeon Pyrobaculum aerophilum. The recombinant ACS, the gene product of ORF PAE2867, showed extremely high thermostability and thermoactivity at temperatures around 100 degrees C. In contrast to known monomeric or homodimeric mesophilic ACSs, the P. aerophilum ACS was a 610 kDa homooctameric protein, with a significant lower content of thermolabile (Cys, Asn, and Gln) and higher content of charged (Glu, Lys, and Arg) amino acids. Kinetic analyses revealed an unusual broad substrate spectrum for organic acids and an extremely high affinity for acetate (K(m) 3 microM).     

Short-chain fatty acid activation by acyl-coenzyme A synthetases requires SIR2 protein function in Salmonella enterica and Saccharomyces cerevisiae

SIR2 proteins have NAD(+)-dependent histone deacetylase activity, but no metabolic role has been assigned to any of these proteins. In Salmonella enterica, SIR2 function was required for activity of the acetyl-CoA synthetase (Acs) enzyme. A greater than two orders of magnitude increase in the specific activity of Acs enzyme synthesized by a sirtuin-deficient strain was measured after treatment with homogeneous S. enterica SIR2 protein. Human SIR2A and yeast SIR2 proteins restored growth of SIR2-deficient S. enterica on acetate and propionate, suggesting that eukaryotic cells may also use SIR2 proteins to control the synthesis of acetyl-CoA by the level of acetylation of acetyl-CoA synthetases. Consistent with this idea, growth of a quintuple sir2 hst1 hst2 hst3 hst4 mutant strain of the yeast Saccharomyces cerevisiae on acetate or propionate was severely impaired. The data suggest that the Hst3 and Hst4 proteins are the most important for allowing growth on these short-chain fatty acids.     

The 1.75 A crystal structure of acetyl-CoA synthetase bound to adenosine-5'-propylphosphate and coenzyme A

Acetyl-coenzyme A synthetase catalyzes the two-step synthesis of acetyl-CoA from acetate, ATP, and CoA and belongs to a family of adenylate-forming enzymes that generate an acyl-AMP intermediate. This family includes other acyl- and aryl-CoA synthetases, firefly luciferase, and the adenylation domains of the modular nonribosomal peptide synthetases. We have determined the X-ray crystal structure of acetyl-CoA synthetase complexed with adenosine-5'-propylphosphate and CoA. The structure identifies the CoA binding pocket as well as a new conformation for members of this enzyme family in which the approximately 110-residue C-terminal domain exhibits a large rotation compared to structures of peptide synthetase adenylation domains. This domain movement presents a new set of residues to the active site and removes a conserved lysine residue that was previously shown to be important for catalysis of the adenylation half-reaction. Comparison of our structure with kinetic and structural data of closely related enzymes suggests that the members of the adenylate-forming family of enzymes may adopt two different orientations to catalyze the two half-reactions. Additionally, we provide a structural explanation for the recently shown control of enzyme activity by acetylation of an active site lysine.     

Structural and mutational studies on an aldo‐keto reductase AKR5C3 from Gluconobacter oxydans

An aldo‐keto reductase AKR5C3 from Gluconobacter oxydans (designated as Gox0644) is a useful enzyme with various substrates, including aldehydes, diacetyl, keto esters, and α‐ketocarbonyl compounds. The crystal structures of AKR5C3 in apoform in complex with NADPH and the D53A mutant (AKR5C3^‐D53A) in complex with NADPH are presented herein. Structure comparison and site‐directed mutagenesis combined with biochemical kinetics analysis reveal that the conserved Asp53 in the AKR5C3 catalytic tetrad has a crucial role in securing active pocket conformation. The gain‐of‐function Asp53 to Ala mutation triggers conformational changes on the Trp30 and Trp191 side chains, improving NADPH affinity to AKR5C3, which helps increase catalytic efficiency. The highly conserved Trp30 and Trp191 residues interact with the nicotinamide moiety of NADPH and help form the NADPH‐binding pocket. The AKR5C3^‐W30A and AKR5C3^‐W191Y mutants show decreased activities, confirming that both residues facilitate catalysis. Residue Trp191 is in the loop structure, and the AKR5C3^‐W191Y mutant does not react with benzaldehyde, which might also determine substrate recognition. Arg192, which is involved in the substrate binding, is another important residue. The introduction of R192G increases substrate‐binding affinity by improving hydrophobicity in the substrate‐binding pocket. These results not only supplement the AKRs superfamily with crystal structures but also provide useful information for understanding the catalytic properties of AKR5C3 and guiding further engineering of this enzyme.     

Genome analysis and heterologous expression of acetate-activating enzymes in the anammox bacterium Kuenenia stuttgartiensis

Anaerobic ammonium-oxidizing bacteria were recently shown to use short-chain organic acids as additional energy source. The AMP-forming acetyl-CoA synthetase gene (acs) of Kuenenia stuttgartiensis, encoding an important enzyme involved in the conversion of these organic acids, was identified and heterologously expressed in Escherichia coli to investigate the activation of several substrates, that is, acetate, propionate and butyrate. The heterologously expressed ACS enzyme could complement an E. coli triple mutant deficient in all pathways of acetate activation. Activity was observed toward several short-chain organic acids, but was highest with acetate. These properties are in line with a mixotrophic growth of anammox bacteria. In addition to acs, the genome of K. stuttgartiensis contained the essential genes of an acetyl-CoA synthase/CO dehydrogenase complex and genes putatively encoding two isoenzymes of archaeal-like ADP-forming acetyl-CoA synthetase underlining the importance of acetyl-CoA as intermediate in the carbon assimilation metabolism of anammox bacteria.     

System-wide studies of N-lysine acetylation in Rhodopseudomonas palustris reveal substrate specificity of protein acetyltransferases

N-lysine acetylation is a posttranslational modification that has been well studied in eukaryotes and is likely widespread in prokaryotes as well. The central metabolic enzyme acetyl-CoA synthetase is regulated in both bacteria and eukaryotes by acetylation of a conserved lysine residue in the active site. In the purple photosynthetic α-proteobacterium Rhodopseudomonas palustris, two protein acetyltransferases (RpPat and the newly identified RpKatA) and two deacetylases (RpLdaA and RpSrtN) regulate the activities of AMP-forming acyl-CoA synthetases. In this work, we used LC/MS/MS to identify other proteins regulated by the N-lysine acetylation/deacetylation system of this bacterium. Of the 24 putative acetylated proteins identified, 14 were identified more often in a strain lacking both deacetylases. Nine of these proteins were members of the AMP-forming acyl-CoA synthetase family. RpPat acetylated all nine of the acyl-CoA synthetases identified by this work, and RpLdaA deacetylated eight of them. In all cases, acetylation occurred at the conserved lysine residue in the active site, and acetylation decreased activity of the enzymes by >70%. Our results show that many different AMP-forming acyl-CoA synthetases are regulated by N-lysine acetylation. Five non-acyl-CoA synthetases were identified as possibly acetylated, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Rpa1177, a putative 4-oxalocrotonate tautomerase. Neither RpPat nor RpKatA acetylated either of these proteins in vitro. It has been reported that Salmonella enterica Pat (SePat) can acetylate a number of metabolic enzymes, including GAPDH, but we were unable to confirm this claim, suggesting that the substrate range of SePat is not as broad as suggested previously.     

Biochemical and crystallographic analysis of substrate binding and conformational changes in acetyl-CoA synthetase

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 degrees 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.     

Characterization of an Archaeal Medium-Chain Acyl Coenzyme A Synthetase from Methanosarcina acetivorans

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 Macs_Ma, 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 PP_i 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 PP_i. 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 Macs_Ma 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.AMP-forming acetyl coenzyme A (acetyl-CoA) synthetase (Acs; acetate:CoA ligase [AMP forming], EC 6.2.1.1), which catalyzes the activation of acetate to acetyl-CoA, is a member of the acyl-adenylate-forming enzyme superfamily (8), which consists of acyl- and aryl-CoA ligases, nonribosomal peptide synthetases that mediate the synthesis of peptide and polyketide secondary metabolites, such as gramicidin and tyrocidine, and the enzymes firefly luciferase and α-aminoadipate reductase. Although these enzymes share the property of forming an acyl-adenylate intermediate and are structurally related, they share limited sequence homology and catalyze unrelated reactions in which the intermediate serves different functions for different members of this enzyme family.A two-step mechanism for Acs (equations 1 and 2) in which the reaction proceeds through an acetyl-AMP intermediate has been proposed based on evidence including detection of an enzyme-bound acetyl-AMP (2-4, 38): (1) (2)In the CoA-dependent first step of the reaction, an enzyme-bound acetyl-AMP intermediate is formed from acetate and ATP, and inorganic pyrophosphate (PP_i) is released. In the second step, the acetyl group is transferred to the sulfhydryl group of CoA and AMP is released. Other short- (Sacs) and medium-chain acyl-CoA synthetases (Macs) follow a similar reaction mechanism using acyl substrates other than acetate (8, 15).In the 2.3-Å structure of trimeric Saccharomyces cerevisiae Acs1 in a binary complex with AMP (19), the C-terminal domain is positioned away from the N-terminal domain in a conformation for catalysis of the first step of the reaction (equation 1). The 1.75-Å structure of the monomeric Salmonella enterica Acs (Acs_Se) (13) in complex with both CoA and adenosine-5′-propylphosphate, an inhibitor of the related propionyl-CoA synthetase (12, 15), which mimics the acetyl-adenylate intermediate, reveals that the C-terminal domain of Acs is rotated approximately 140° toward the N-terminal domain to form the complete active site for catalysis of the second half-reaction (equation 2). In this orientation, the CoA thiol is properly positioned for nucleophilic attack on the acetyl group. In structure/function studies of 4-chlorobenzoate:CoA ligase (CBAL), a distant member of the acyl- and aryl-CoA synthetase subfamily of the acyl-adenylate-forming enzyme superfamily, Wu et al. (39) and Reger et al. (28) provide evidence that PP_i produced in the first step of the reaction dissociates from the enzyme before the switch from the first conformation to the second conformation required for CoA binding and catalysis of the second step of the reaction.Acs and Sacs/Macs are widespread in all three domains of life and play a key role in archaea, as suggested by the finding that several thermophilic archaea have multiple open reading frames (ORFs) (up to seven) that encode putative Sacs or Macs (33). The chemolithoautotrophic methanoarchaeon Methanothermobacter thermautotrophicus has two ORFs with high identity to Acs and a third ORF that is likely to encode a Macs. M. thermautotrophicus Acs1 (Acs1_Mt) has been biochemically and kinetically characterized, has been shown to have a strong preference for acetate as the acyl substrate, and can also utilize propionate but not butyrate (16, 17).Methanosarcina and Methanosaeta are the only two methanoarchaea isolated that are able to utilize acetate as substrate for methane production. Unlike Methanosaeta species, which utilize Acs for catalyzing the first step of methanogenesis (18, 34), Methanosarcina species employ the acetate kinase-phosphotransacetylase pathway for activation of acetate to acetyl-CoA, and an Acs activity has not been observed in Methanosarcina (1, 23, 30, 32). Surprisingly, an Acs-related sequence was identified in the Methanosarcina acetivorans genome. Here we describe the kinetic characterization this enzyme, designated Macs_Ma, and show that it utilizes longer acyl substrates than Acs. The preferred acyl substrate was shown to be 2-methylbutyrate, and 2-methylbutyryl-CoA, AMP, and PP_i were the products of the reaction, as expected. However, when propionate was used as the acyl substrate, propionyl-CoA was not produced. Instead, in the absence of CoA, propionyl-AMP and PP_i were released, whereas in the presence of CoA, the propionyl-AMP intermediate was broken down into AMP and propionate and released along with PP_i. Intermediate results were obtained with other acyl substrates, with both acyl-CoA and acyl-AMP production observed.The 2.1-Å crystal structure of Macs_Ma (31), determined in the absence of any substrate, revealed the enzyme to be in a conformation similar to that of the S. enterica Acs (13) with respect to the position of the C-terminal domain. Through inspection of the Macs_Ma structure and alignment of Acs, Sacs, and Macs sequences, we identified six residues that form the putative acyl substrate-binding pocket. Individual alterations at these residues dramatically diminished enzyme activity and indicate that the acyl substrate-binding pocket of Macs_Ma has a very precise architecture that cannot be perturbed.     

Reaction mechanism and structural model of ADP-forming Acetyl-CoA synthetase from the hyperthermophilic archaeon Pyrococcus furiosus: evidence for a second active site histidine residue

In Archaea, acetate formation and ATP synthesis from acetyl-CoA is catalyzed by an unusual ADP-forming acetyl-CoA synthetase (ACD) (acetyl-CoA + ADP + P(i) acetate + ATP + HS-CoA) catalyzing the formation of acetate from acetyl-CoA and concomitant ATP synthesis by the mechanism of substrate level phosphorylation. ACD belongs to the protein superfamily of nucleoside diphosphate-forming acyl-CoA synthetases, which also include succinyl-CoA synthetases (SCSs). ACD differs from SCS in domain organization of subunits and in the presence of a second highly conserved histidine residue in the beta-subunit, which is absent in SCS. The influence of these differences on structure and reaction mechanism of ACD was studied with heterotetrameric ACD (alpha(2)beta(2)) from the hyperthermophilic archaeon Pyrococcus furiosus in comparison with heterotetrameric SCS. A structural model of P. furiosus ACD was constructed suggesting a novel spatial arrangement of the subunits different from SCS, however, maintaining a similar catalytic site. Furthermore, kinetic and molecular properties and enzyme phosphorylation as well as the ability to catalyze arsenolysis of acetyl-CoA were studied in wild type ACD and several mutant enzymes. The data indicate that the formation of enzyme-bound acetyl phosphate and enzyme phosphorylation at His-257alpha, respectively, proceed in analogy to SCS. In contrast to SCS, in ACD the phosphoryl group is transferred from the His-257alpha to ADP via transient phosphorylation of a second conserved histidine residue in the beta-subunit, His-71beta. It is proposed that ACD reaction follows a novel four-step mechanism including transient phosphorylation of two active site histidine residues:     

Crystal structure of auxin-binding protein 1 in complex with auxin

The structure of auxin-binding protein 1 (ABP1) from maize has been determined at 1.9 A resolution, revealing its auxin-binding site. The structure confirms that ABP1 belongs to the ancient and functionally diverse germin/seed storage 7S protein superfamily. The binding pocket of ABP1 is predominantly hydrophobic with a metal ion deep inside the pocket coordinated by three histidines and a glutamate. Auxin binds within this pocket, with its carboxylate binding the zinc and its aromatic ring binding hydrophobic residues including Trp151. There is a single disulfide between Cys2 and Cys155. No conformational rearrangement of ABP1 was observed when auxin bound to the protein in the crystal, but examination of the structure reveals a possible mechanism of signal transduction.     

The Acetylation Motif in AMP-Forming Acyl Coenzyme A Synthetases Contains Residues Critical for Acetylation and Recognition by the Protein Acetyltransferase Pat of Rhodopseudomonas palustris

The AMP-forming acyl coenzyme A (acyl-CoA) synthetases are a large class of enzymes found in both anabolic and catabolic pathways that activate fatty acids to acyl-CoA molecules. The protein acetyltransferase (Pat) from Rhodopseudomonas palustris (RpPat) inactivates AMP-forming acyl-CoA synthetases by acetylating the ε-amino group of a conserved, catalytic lysine residue. In all of the previously described RpPat substrates, this lysine residue is located within a PX₄GK motif that has been proposed to be a recognition motif for RpPat. Here, we report five new substrates for RpPat, all of which are also AMP-forming acyl-CoA synthetases. This finding supports the idea that Pat enzymes may have evolved to control the activity of this family of enzymes. Notably, RpPat did not acetylate the wild-type long-chain acyl-CoA synthetase B (RpLcsB; formerly Rpa2714) enzyme of this bacterium. However, a single amino acid change two residues upstream of the acetylation site was sufficient to convert RpLcsB into an RpPat substrate. The results of mutational and functional analyses of RpLcsB and RpPimA variants led us to propose PK/RTXS/T/V/NGKX₂K/R as a substrate recognition motif. The underlined positions within this motif are particularly important for acetylation by RpPat. The first residue, threonine, is located 4 amino acids upstream of the acetylation site. The second residue can be S/T/V/N and is located two positions upstream of the acetylation site. Analysis of published crystal structures suggests that the side chains of these two residues are very close to the acetylated lysine residue, indicating that they may directly interact with RpPat.     

Functional analyses of two acetyl coenzyme A synthetases in the ascomycete Gibberella zeae

Acetyl coenzyme A (acetyl-CoA) is a crucial metabolite for energy metabolism and biosynthetic pathways and is produced in various cellular compartments with spatial and temporal precision. Our previous study on ATP citrate lyase (ACL) in Gibberella zeae revealed that ACL-dependent acetyl-CoA production is important for histone acetylation, especially in sexual development, but is not involved in lipid synthesis. In this study, we deleted additional acetyl-CoA synthetic genes, the acetyl-CoA synthetases (ACS genes ACS1 and ACS2), to identify alternative acetyl-CoA production mechanisms for ACL. The ACS1 deletion resulted in a defect in sexual development that was mainly due to a reduction in 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol production, which is required for perithecium development and maturation. Another ACS coding gene, ACS2, has accessorial functions for ACS1 and has compensatory functions for ACL as a nuclear acetyl-CoA producer. This study showed that acetate is readily generated during the entire life cycle of G. zeae and has a pivotal role in fungal metabolism. Because ACSs are components of the pyruvate-acetaldehyde-acetate pathway, this fermentation process might have crucial roles in various physiological processes for filamentous fungi.     

Structures of human N-Acetylglucosamine kinase in two complexes with N-Acetylglucosamine and with ADP/glucose: insights into substrate specificity and regulation

N-Acetylglucosamine (GlcNAc), a major component of complex carbohydrates, is synthesized de novo or salvaged from lysosomally degraded glycoconjugates and from nutritional sources. The salvage pathway requires that GlcNAc kinase converts GlcNAc to GlcNAc-6-phosphate, a component utilized in UDP-GlcNAc biosynthesis or energy metabolism. GlcNAc kinase belongs to the sugar kinase/Hsp70/actin superfamily that catalyze phosphoryl transfer from ATP to their respective substrates, and in most cases catalysis is associated with a large conformational change in which the N-terminal small and C-terminal large domains enclose the substrates. Here we report two crystal structures of homodimeric human GlcNAc kinase, one in complex with GlcNAc and the other in complex with ADP and glucose. The active site of GlcNAc kinase is located in a deep cleft between the two domains of the V-shaped monomer. The enzyme adopts a "closed" configuration in the GlcNAc-bound complex and GlcNAc interacts with residues of both domains. In addition, the N-acetyl methyl group contacts residues of the other monomer in the homodimer, a unique feature compared to other members of the sugar kinase/Hsp70/actin superfamily. This contrasts an "open" configuration in the ADP/glucose-bound structure, where glucose cannot form these interactions, explaining its low binding affinity for GlcNAc kinase. Our results support functional implications derived from apo crystal structures of GlcNAc kinases from Chromobacter violaceum and Porphyromonas gingivalis and show that Tyr205, which is phosphorylated in thrombin-activated platelets, lines the GlcNAc binding pocket. This suggests that phosphorylation of Tyr205 may modulate GlcNAc kinase activity and/or specificity.     

Mammalian long-chain acyl-CoA synthetases

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.     

In vivo and in vitro analyses of single-amino acid variants of the Salmonella enterica phosphotransacetylase enzyme provide insights into the function of its N-terminal domain

The function of the N-terminal domain ( approximately 350 residues) of the Pta (phosphotransacetylase) enzyme of Salmonella enterica is unclear. Results from in vivo genetic and in vitro studies suggest that the N-terminal domain of Pta is a sensor for NADH and pyruvate. We isolated 10 single-amino acid variants of Pta that, unlike the wild-type protein, supported growth of a strain of S. enterica devoid of Acs (acetyl-CoA synthetase; AMP-forming) activity on 10 mm acetate. All mutations were mapped within the N-terminal domain of the protein. Kinetic analyses of the wild type and three variant Pta proteins showed that two of the variant proteins were faster enzymes (k(cat) 2.5-3-fold > k(cat) Pta(WT). Results from sedimentation equilibrium experiments are consistent with Pta(WT) being a trimer. Pta variants formed more hexamer than the Pta(WT) protein. NADH inhibited Pta(WT) activity by inducing a conformational change detectable by limited trypsin proteolysis; NADH did not inhibit variant protein Pta(R252H). Pyruvate stimulated Pta(WT) activity, and its effect was potentiated in the variants, being most pronounced on Pta(R252H).     

AMP-forming acetyl coenzyme A synthetase in the outermost membrane of the hyperthermophilic crenarchaeon Ignicoccus hospitalis

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic crenarchaeon was found to possess a new CO(2) fixation pathway, the dicarboxylate/4-hydroxybutyrate cycle. The primary acceptor molecule for this pathway is acetyl coenzyme A (acetyl-CoA), which is regenerated in the cycle via the characteristic intermediate 4-hydroxybutyrate. In the presence of acetate, acetyl-CoA can alternatively be formed in a one-step mechanism via an AMP-forming acetyl-CoA synthetase (ACS). This enzyme was identified after membrane preparation by two-dimensional native PAGE/SDS-PAGE, followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry and N-terminal sequencing. The ACS of I. hospitalis exhibits a molecular mass of ～690 kDa with a monomeric molecular mass of 77 kDa. Activity tests on isolated membranes and bioinformatic analyses indicated that the ACS is a constitutive membrane-associated (but not an integral) protein complex. Unexpectedly, immunolabeling on cells of I. hospitalis and other described Ignicoccus species revealed that the ACS is localized at the outermost membrane. This perfectly coincides with recent results that the ATP synthase and the H(2):sulfur oxidoreductase complexes are also located in the outermost membrane of I. hospitalis. These results imply that the intermembrane compartment of I. hospitalis is not only the site of ATP synthesis but may also be involved in the primary steps of CO(2) fixation.