Most gram-negative organisms require lipopolysaccharide and its membrane anchor, lipid A, for growth and survival. Also known as endotoxin, lipid A is synthesized via a nine-step enzymatic process, culminating in a conserved hexa-acylated, bis-phosphorylated disaccharide of glucosamine. This framework is often altered by condition- or species-specific lipid A modifications, which change the biochemical properties of the molecule in response to and to defend against environmental stress signals. Here, we expound on two stories in different gram-negative organisms, both involving novel or unanticipated biochemistry that impacts lipid A structure. First, the missing acyltransferase in the Epsilonproteobacterium Helicobacter pylori lipid A biosynthesis pathway is identified. This enzyme transfers a secondary acyl chain to the 3'-linked primary acyl chain of lipid A like E. coli LpxM, but shares almost no sequence similarity with the E. coli acyltransferase. It is reannotated as LpxJ and demonstrated to possess an unprecedented ability to act before the 2'-secondary acyltransferase, LpxL, as well as the 3-deoxy-D-manno-octulosonic acid transferase, KdtA. LpxJ is one member of a large class of acyltransferases found in a diverse range of organisms that lack an E. coli LpxM homolog, suggesting that LpxJ participates in lipid A biosynthesis in place of an LpxM homolog. The second story focuses on regulation of modifications to endotoxin structure that occur after the conserved biosynthesis pathway. E. coli pmrD is shown to be required for PmrAB-dependent lipid A modifications in conditions that exclusively activate PhoPQ; this result proves that PmrD connects PhoPQ and PmrAB despite previous reports that it is an inactive connector in this organism. Further, RNA sequencing and polymyxin B survival assays solidify the role of E. coli pmrD in influencing expression of pmrA and its target genes and promoting survival during exposure to cationic antimicrobial peptides. Notably, the presence of an unknown factor or system capable of activating pmrD to promote lipid A modification in the absence of the PhoPQ system is also revealed. In all, the findings presented here expand our understanding of alternative approaches to lipid A biosynthesis and the complex systems that regulate modifications of this dynamic molecule.

The vast variety of long-chain fatty acid amides identified in biological systems is intriguing. The general structure of a fatty acid amide is R-CO-NH-X, where R is an alkyl group and X is derived from an immense variety of biogenic amines. Although structurally simple, the bioactivities of these molecules as signaling lipids are very diverse and have just recently begun to emerge in the literature. Interest in the long-chain fatty acid amides dramatically increased following the identification and characterization of one specific N-acylethanolamine, N-arachidonoylethanolamine (anandamide), as the endogenous ligand for the cannabinoid receptors in the mammalian brain. Since this discovery, the details of N-acylethanolamine metabolism have been elucidated. However, a lesser extent of progress has been made in the last twenty years to identify and study the non-N-acylethanolamine long-chain fatty acid amides. The focus of this dissertation is the elucidation of the biosynthetic pathways for long-chain fatty acid amides, including N-acylglycines, primary fatty acid amides, N-acylarylalkylamides, and N-acylethanolamines. The details of long-chain fatty acid amide metabolism will lead to the determination of possible therapeutic targets. We identified mammalian glycine N-acyltransferase like 3 as the enzyme that catalyzes the formation of long-chain N-acylglycines in mouse N18TG2 neuoblastoma cells, identified and quantified a panel of long-chain fatty acid amides in Drosophila melanogaster extracts by LC/QTOF-MS, established Drosophila melanogaster as a model system to study long-chain fatty acid amide metabolism, and identified arylalkylamine N-acyltransferase like 2 as the enzyme that catalyzes the formation of long-chain N-acylserotonins and N-acyldopamines in Drosophila melanogaster.

Glycine-N-acyltransferase (GLYAT, EC 2.3.1.13) has been characterised in a number of species including: humans, chimpanzees, rhesus monkeys and bovines. The characterisation of GLYAT from various species contributes to a better understanding of the diversity of the enzyme which in turn might help improve the current understanding of detoxification in mammals. The GLYAT enzyme of both the chacma baboon and vervet monkey has not been characterised. In this project, tissue samples were obtained from a chacma baboon (Papio ursinus) and a vervet monkey (Chlorocebus pygerythrus) to determine the nucleic acid sequence that encodes GLYAT in these two species to broaden our current understanding on the diversity of GLYAT in primates. A liver of a chacma baboon was used to extract total RNA. Complementary DNA (cDNA) was synthesised using an oligo (dT) primer. An open reading frame (ORF) encoding GLYAT of the chacma baboon was amplified with a PCR (polymerase chain reaction) using primers designed from a human GLYAT transcript. The PCR product containing an ORF encoding GLYAT of the chacma baboon was cloned, sequenced and expressed. The recombinant GLYAT of the chacma baboon expressed well in bacteria, but was insoluble and did not have enzyme activity. A crude cytoplasmic extract was prepared from the liver of a chacma baboon. The objective was to compare enzyme activity between the native and recombinant GLYAT. The prepared liver extract from the chacma baboon was assayed for enzyme activity and compared to the activity in a liver extract from bovine, previously prepared by Ms M Snyders. Both the chacma baboon and bovine liver extracts had GLYAT enzyme activity. To obtain sequence information on vervet monkey GLYAT, leukocytes were isolated from blood obtained from a living vervet monkey. A human GLYAT gene sequence was used as a reference DNA sequence in the design of PCR primers that were used to amplify the exons of GLYAT of the vervet monkey. All six GLYAT exons were individually amplified and PCR products were sequenced. The sequences were combined to reconstruct an ORF encoding GLYAT of the vervet monkey. The ORFs coding the GLYAT of both chacma baboon and vervet monkey were found to be 888 bp long (excluding stop codon) and encoded a protein of 296 amino acids. A fragment of 1256 bp of the chacma baboon GLYAT transcript was sequenced. The two GLYAT ORF sequences were translated to amino acid sequences and aligned to that of GLYAT of primates obtained from the Ensembl sequence database. The GLYAT amino acid sequences of the chacma baboon, vervet monkey and rhesus monkey formed a related group, distinct from other primates. The chacma baboon and vervet monkey sequences were 99 % identical to the rhesus monkey sequence and 92.6 % identical to the human sequence. There were 4 new variations introduced by GLYAT amino acid sequences from the chacma baboon and the vervet monkey. The vervet monkey introduced an isoleucine in place of a valine at position 32 and an arginine in place of a histidine or glutamine at position 224. The chacma baboon introduced a tyrosine in place of isoleucine at position 201 and an arginine in place of histidine or glutamine at position 240. The knowledge generated in this project will broaden the understanding of GLYAT diversity relating to GLYAT in primates. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2005" Includes bibliographical references.

Les proanthocyanidines (PA) du raisin jouent un rôle majeur dans les propriétés organoleptiques du vin notamment dans l'astringence et la stabilité de la couleur. Elles sont accumulées principalement dans la pellicule et les pépins pendant les premiers stades du développement de la baie. La biosynthèse des PA commence à être bien décrite, cependant certaines étapes restent à élucider.Une étude transcriptomic avait permis d'indentifier de nouveaux gènes potentiellemnt impliqués dans la biosynthèse des PA (Terrier et al., 2009). Parmi ces gènes figuraient 3 glucosyltransférases et 2 glucose- acyltransférases.Les trois glucosyltransférases VvgGT1, VvgGT2 et VvgGT3 présentent de fortes homologies entres elles et avec d'autres glucosyltransférases capables de catalyser la formation de glucose ester. Les transcrits sont exprimés durant les premiers stades de développement de la baie principalement dans les pépins et la pellicule mais aussi dans la pulpe pour VvgGT2. Les propriétés de ces 3 enzymes (Km, Vm, spécificité de substrat, effet du pH) ont été étudiées in vitro après production de protéines recombinantes. Les 3 VvgGTs sont capables de catalyser la synthèse d'esters de glucose en présence de dérivés d'acide hydroxybenzoique ou d'acide hydroxycinnamique.Deux acyltransférases VvGAT1 et VvGAT2 ont été isolées. Elle présente de fortes similarités avec des acyltransférases glucose-ester dépendantes de type serine carboxypeptidase like. Les transcrits sont exprimés surtout avant la véraison, dans les pépins et la pellicule pour VvGAT1 et dans la pulpe et les pépins pour la VvGAT2. L'expression hétérologue dans différents hôtes n'a pas permis de détecter d'activité enzymatique. Les analyses en microscopie confocale suggèrent que VvGAT1 fusionnée à une GFP est localisée dans des vésicules cytoplasmiques.L'implication successive des VvgGTs et des VvGATs dans la galloylation des PA et dans la synthèse d'esters d'acides hydroxcinnamiques est discutée. / Grape proanthocyanidins (PA) play a major role in organoleptic properties of wine, being involved in astringency and color stability. They are accumulated mainly in skin and seeds during early stages of berry development. Numerous structural genes involved in PA biosynthesis have already been identified even in grape, but some steps are still not documented.A previous transcriptomic study led to identify new genes putatively involved in the PA pathway (Terrier et al., 2009). Among them, 3 glucosyltransferases and 2 glucose acyl transferases were identified. The objective of this work is to clarify the function of these genes in the phenylpropanoid pathway.The three glucosyltransferases called VvgGT1, VvgGT2, VvgGT3 displayed high sequence similarities between them and with other plant glucosyltransferases able to catalyze the formation of glucose esters. The transcripts are expressed in the early stages of grape berry development, mainly in skins and seeds and also in the pulp for VvgGT2. The properties of these 3 enzymes (Km, Vm, substrate specificity, pH sensitivity) were studied in vitro after production of recombinant proteins. The three of them are able to catalyse the synthesis of glucose ester with derivates of hydroxybenzoic acids and hydroxycinnamic acids as substrates and with similar kinetic properties.Two glucose-acyltransferases called VvGAT1 and VvGAT2 were isolated. They displayed high sequence similarity with other Serine carboxypeptidase like acyltransferases-glucose dependent. The transcripts are expressed in early stages of grape berry development mainly in skins and seeds for VvGAT1 and in pulp and seeds for VvGAT2. Heterologous expression of the proteins in different hosts were unsuccessful. The confocal microscopy data suggest that VvGAT1 fused to GFP are localized in cytosolic vesicles.The successive involvement of those VvgGT and VvGAT in the galloylation of PAs and in the synthesis of hydroxycinnamic esters is discussed.

Master of Science / Department of Biochemistry / Rachel Zufferey / Glycerolipids are the most abundant lipids and are important constituents of various virulence factors in the protozoan parasite Leishmania. The dihydroxyacetone phosphate acyltransferase LmDAT catalyzes the first step of the ether, and possibly ester glycerolipid biosynthetic pathway. A L. major null mutant of LmDAT grew slowly, died rapidly during the stationary phase of growth, and more importantly, was attenuated in virulence in mice. The goal of this study was to determine the molecular basis responsible for the attenuated virulence. Western blot analysis revealed that the ∆lmdat/∆lmdat null mutant synthesized altered versions of the virulence factor lipophosphoglycans that were not released in the media, suggesting that its lipid anchor structure was altered. The ∆lmdat/∆lmdat strain differentiated into virulent metacyclics, but with lower efficiency compared to the wild type. Using the autophagosomal marker ATG8-GFP, the ∆lmdat/∆lmdat line produced twice as many autophagosomes as the wild type, suggesting that it is either defective in degradation of autophagosomes or that autophagy is simply induced. In conclusion, the attenuated virulence of ∆lmdat/∆lmdat may be explained by i) its inability to synthesize and release normal forms of lipophosphoglycan, ii) its inability to fully differentiate into virulent metacyclics, and iii) altered autophagy.

Parmi les métabolites secondaires impliqués dans la qualité du raisin et du vin, les tanins condensés ou proanthocyanidines (PAs) jouent un rôle majeur, en particulier dans l'astringence et la stabilité de la couleur du vin. Ces molécules sont également impliquées dans la défense des plantes contre des stress biotiques et abiotiques. En outre, les effets bénéfiques des PAs pour la santé humaine sont bien documentés. Les PAs de la vigne ont la particularité d’être estérifiées avec de l’acide gallique. Une réaction d’acylation appelée galloylation est responsable de cette modification. Les études montrent que la galloylation influence les propriétés œnologiques et pharmacologiques des PAs. Dans la baie de raisin, les PAs sont synthétisés dans les premiers stades de développement, principalement dans les pellicules et les pépins. Un nombre relativement faible d'étapes enzymatiques sont nécessaires pour la biosynthèse de la structure de base de ces métabolites et les gènes correspondants sont aujourd'hui largement connus chez les plantes modèles, y compris chez la vigne. Cependant, les mécanismes moléculaires impliqués dans les étapes finales, y compris la galloylation, ne sont encore que partiellement connus. Des résultats antérieurs obtenus après la recherche de QTL influençant la composition du raisin, et en particulier le taux de galloylation des PAs, et des études transcriptomiques après surexpression de facteurs de transcription régulant la biosynthèse de la voie des PAs, ont permis l'identification de gènes potentiellement impliqués dans ces étapes. Des gènes de shikimate déshydrogénase (SDH) ont été identifiés. Ces gènes interviendraient en amont, pour la biosynthèse de l'acide gallique. Trois glucosyltransférases ainsi identifiées et déjà caractérisées au laboratoire sont impliquées dans la biosynthèse de l'ester de glucose de l'acide gallique (β-glucogalline), qui servirait d'intermédiaire pour la galloylation des PAs. Ces méthodes de criblage ont également permis d’identifier 2 acyltransférases de type sérine carboxypeptidase, nommées glucose acyltransférases (GATs) qui seraient capables de catalyser la dernière étape de galloylation: le transfert de l'acide gallique depuis la β-glucogalline sur les PAs. Le premier objectif de cette thèse a été de déterminer la fonction des SDHs codées par les gènes de vigne. Certaines SDHs recombinantes produites de façon hétérologue chez E.coli ont la capacité à produire de l'acide gallique in vitro. Leur niveau d’expression au cours du développement et dans différents tissus de la baie a également été établi. Les résultats obtenus in vitro sont étayés par le profil métabolique (acide gallique, β-glucogalline et PAs) de hairy-roots de vigne transformées avec un gène de SDH. Le second objectif de cette thèse a été de valider la fonction des GATs par expression transitoire dans des feuilles de tabac et des tests enzymatiques in vitro. La transformation transitoire de feuilles de vigne avec les GATs a permis de moduler la concentration d’esters phénoliques et nomment des flavan-3-ols galloylés in planta. L’étude de ces gènes a été étendue aux plantes vasculaires par des analyses phylogénétiques et a permis d’identifier des motifs peptidiques potentiellement impliqués dans les mécanismes étudiés et reflétant la sub-fonctionnalisation de certains gènes. Ce travail a fourni des informations sur les bases génétiques et les mécanismes moléculaires impliqués dans la biosynthèse de l'acide gallique et son transfert en deux étapes sur les flavan-3-ols (galloylation). De nouvelles hypothèses sur l'intervention de différents transporteurs et la nature des molécules transportées pourront être formulées. / Among the secondary metabolites involved in grape berry and wine quality, condensed tannins or proanthocyanidins (PAs) play a major role, especially in astringency and color stability of wine. These molecules are also involved in plant defence against biotic and abiotic stresses. Furthermore, the beneficial effects of PAs to human health are well documented. In grapevine, PAs have the distinctive feature of being esterified with gallic acid. An acylation reaction called galloylation is responsible for this modification. Studies show that the galloylation influences oenological and pharmacological properties of PAs. In the grape berry, PAs are synthesized in the early stages of development, mainly in skin and seeds. A relatively small number of enzymatic steps are required for the biosynthesis of the basic structure of these metabolites and the corresponding genes are now widely known in model plants, including in grapevine. However, the molecular mechanisms involved in the final steps, including galloylation, are only partially known. Earlier results obtained after the search of QTL influencing the composition of the grape berry, especially the galloylation ratio of PAs, and transcriptomic studies after overexpression of transcription factors that regulate PAs biosynthesis pathway, have allowed the identification of genes potentially involved in these steps. Shikimate dehydrogenase (SDH) genes were identified. These genes would intervene upstream, for the biosynthesis of gallic acid. Three identified glucosyltransferases, already characterized in the laboratory, are involved in the biosynthesis of glucose ester of gallic acid (β-glucogalline), which could serve as an intermediary for PAs galloylation. These screening methods have also helped to identify 2 serine carboxypeptidase-like acyltransferases, called glucose acyltransferases (GATs) which are capable of catalyzing the last step of galloylation: the transfer of gallic acid from β-glucogalline to PAs. The first objective of this thesis was to determine the function of the SDHs encoded by grapevine genes. Recombinant SDHs, produced heterologously in E. coli, have the capacity to generate gallic acid in vitro. Their level of expression during development and in different tissues of the berry was also established. In vitro results are supported by the metabolic profile (gallic acid, β-glucogallin and PAs) of grapevine hairy -roots transformed with a SDH gene. The second objective of this thesis was to validate the function of the GATs by transient expression in tobacco leaves and in vitro enzyme assays. The transient transformation of grapevine leaves with GATs allowed to modulate the concentration of phenolic esters and notably galloylated flavan-3-ols in planta. The study of these genes was extended to vascular plants by phylogenetic analyses which allowed to identify peptide motifs potentially involved in the studied mechanisms and reflecting the sub-functionalization of certain genes. This work has provided informations on the genetic basis and molecular mechanisms involved in the biosynthesis of gallic acid and its two-step transfer on flavan-3-ols (galloylation). New hypotheses on the intervention of different carriers and nature of transported molecules can be proposed.

Afin de s'adapter à son environnement chimique, l'organisme a développé au cours de l'évolution des systèmes enzymatiques capables de transformer de nombreuses molécules étrangères ou xénobiotiques (médicaments, composés toxiques, carcinogènes...), le plus souvent de nature hydrophobe, en métabolites suffisamment hydrophiles pour être plus facilement excrétés par voie urinaire et/ou biliaire. Certaines de ces enzymes sont également impliquées dans des processus cataboliques ou de biosynthèses de composés endogènes (acides gras, rétinoïdes, stéroïdes, prostaglandines…). Ces enzymes jouent ainsi un rôle fondamental à la fois dans la défense de l'organisme face à son environnement chimique et dans des processus physiologiques essentiels. On comprend dès lors que s'il existe, chez certains individus, des anomalies de séquence ou de structure des gènes codant pour ces enzymes, une partie de la population présentera une susceptibilité particulière à certaines molécules de l'environnement, voire des dysfonctionnements de certaines réactions biologiques indispensables. Les travaux de cette thèse s'inscrivent dans cette démarche. Dans un premier temps, ils ont consisté à évaluer la nature et l'étendue de la variabilité de la séquence nucléotidique de trois gènes codants pour les enzymes CYP4A11, CYP4A22 et la Glycine N-acyltransférase (GLYAT). Dans un deuxième temps, les analyses fonctionnelles des variations de séquence identifiées ont été abordées par des approches in silico et in vitro. Les cytochromes P450 CYP4A11 et CYP4A22, participent à la biotransformation de composés endogènes et sont impliqués plus particulièrement dans la voie d’activation de l’acide arachidonique. Des travaux récents suggèrent que des anomalies génétiques de ces enzymes constituent des facteurs de susceptibilité à l’hypertension artérielle chez l’homme. Nous avons ainsi analysé les variations de séquence du gène CYP4A11 et CYP4A22 dans des échantillons d'ADN provenant de volontaires sains. Au total, 26 polymorphismes ont été identifiés et 5 nouveaux CYP4A* allèles ont été caractérisés pour chaque isoforme CYP4A. Les structures 3D des protéines CYP4A ont été construites et validées pour l’analyse de l’impact des mutations identifiées. Bien que des travaux supplémentaires soient nécessaires pour confirmer le lien entre le polymorphisme génétique du CYP4A11 et du CYP4A22 et l’hypertension artérielle, ce travail représente la première description et caractérisation du polymorphisme génétique des isoformes CYP4A dans une population Française. De plus, nous avons mise en évidence une variabilité interethnique de ce polymorphisme génétique dans différentes populations testées. La glycine N-acyltransférase ou GLYAT est une enzyme impliquée dans la détoxication de xénobiotiques contenant un groupement carboxylique par conjugaison d’un résidu de glycine. Sept variations de séquence de la GLYAT ont été identifiées et quatre nouveaux GLYAT* allèles ont été caractérisés. La localisation des certaines mutations dans des structures secondaires très conservées de la protéine suggère un impact sur l’activité catalytique de cette enzyme. Bien que les conséquences cliniques potentielles de ces variations restent encore à étudier, ces résultats seront utiles pour de futures études d’association de ce polymorphisme génétique de la GLYAT avec les altérations de détoxications de xénobiotiques contenant un groupement carboxylique comme l’aspirine, certains pesticides ou le toluène. / Through evolution, in order to adapt to its chemical environment, the human organism has developed enzymatic systems that can transform exogenous molecules or xenobiotic (drugs, toxins, carcinogens…), generally of hydrophobic nature, in metabolites more easily excretable via urinary or biliary tract. Some of these enzymes are also involved in catabolic processes or in the biosynthesis of endogenous compounds (fatty acids, retinoids, steroids, prostaglandins…). These enzymes thus play a major role in the protective response of the body toward chemicals and in essential physiological processes. The existence of anomalies in the sequence or structure of the genes encoding these enzymes can expose carriers of these anomalies to particular susceptibility toward xenobiotics or to impairment of essential biological reactions. In a first step, we investigated the nature and extent of the sequence variability of three genes coding for the enzymes CYP4A11, CYP4A22 and Glycine N-acyltransferase (GLYAT). In a second step, functional analyses of sequence variations were carried out, by in silico and in vitro experiments. The CYP4A11 and CYP4A22 genes are the only members of the human CYP4A subfamily. The activity of the recently identified CYP4A22 isoform is still unknown, but the CYP4A11 isoform is know as a ω-hydroxylase of the arachidonic acid, which converted into 20-hydroxyeicosatetraenoic acid (20-HETE). Several studies have shown that genetic anomalies of CYP4A are likely to contribute for susceptibility to hypertension in humans. We analyzed the sequence variations of the CYP4A11 and CYP4A22 genes in genomic DNA samples of healthy volunteers. A total of 26 polymorphisms were identified and 5 novel CYP4A* alleles were characterized for each CYP4A gene. The CYP4A 3D models were built and validated to analyse the potential impact of sequence variations identified. This work represents the first description and characterisation of genetic polymorphism of the human CYP4A genes in a French population. The glycine N-acyltranferase or GLYAT plays an important role in the detoxification of xenobiotics containing a carboxylic group via conjugation with a glycine residue. Seven sequence variations of the GLYAT gene were identified and four novel GLYAT* alleles were characterized. Localisation of missense mutations in predicted secondary structures suggest that these variants might have a potential role on the GLYAT protein activity. These results could be helpful in investigating the potential association of GLYAT variants with an incidence of reduced efficiency in xenobiotic carboxylic acids detoxification in humans, such as acetylsalicylic acid, pesticides, and solvents (Toluene).

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24795号 / 医博第4987号 / 新制||医||1066(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小林 恭, 教授 波多野 悦朗, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The kinetics of the reaction catalyzed by acyl-CoA: sn-glycerol-3-phosphate O-acyltransferase solubilized from Euglena gracilis microsomes were examined. For myristoyl-, palmitoyl-, stearoyl-, and oleoyl-CoAs, the initial reaction rates rose with increasing substrate concentration up to an optimal concentration that varied from 18.5 to 25 μ M, well above the respective critical micelle concentrations. At higher substrate concentrations, reaction was progressively inhibited. Arachidoyl-CoA was a relatively poor substrate for the acyltransferase, and substrate inhibition was not seen with it. Km values for acyl-CoAs ranged from 13 to 20 μ M while the corresponding V values varied almost 40-fold. Bovine serum albumin, among other effects, caused a change in the kinetic pattern of the reaction acyl-CoA dependency. Both acyl-CoA micelles and albumin-bound acyl-CoA were substrates. The binding of palmitoyl- and oleoyl-CoA was 2.7 and 1.5 mol, respectively, per mol of albumin. The critical micelle concentration of palmitoyl-CoA under the reaction conditions was shown by low angle light scattering photometry to be 7.1 p.M. The sn-glycerol 3-phosphate concentration dependency of the acyltransferase initial velocity exhibited Michaelis-Menten kinetics with Km values of 1.3 and 2.9 mM in the presence of 12.5 and 25 μM palmitoyl-CoA, respectively. The substrate analogues sn-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate inhibited the reaction.