Structural and Biochemical Studies of Substrate Selectivity in Ascaris suum Thiolases.

Thiolases are a class of carbon-carbon bond forming enzymes with important applications in biotechnology and metabolic engineering as they provide a general method for the condensation of two acyl coenzyme A (CoA) substrates. As such, developing a greater understanding of their substrate selectivity would expand our ability to engineer the enzymatic or microbial production of a broad range of small-molecule targets. Here, we report the crystal structures and biochemical characterization of Acat2 and Acat5, two biosynthetic thiolases from Ascaris suum with varying selectivity toward branched compared to linear compounds. The structure of the Acat2-C91S mutant bound to propionyl-CoA shows that the terminal methyl group of the substrate, representing the α-branch point, is directed toward the conserved Phe 288 and Met 158 residues. In Acat5, the Phe ring is rotated to accommodate a hydroxyl-π interaction with an adjacent Thr side chain, decreasing space in the binding pocket and possibly accounting for its strong preference for linear substrates compared to Acat2. Comparison of the different Acat thiolase structures shows that Met 158 is flexible, adopting alternate conformations with the side chain rotated toward or away from a covering loop at the back of the active site. Mutagenesis of residues in the covering loop in Acat5 with the corresponding residues from Acat2 allows for highly increased accommodation of branched substrates, whereas the converse mutations do not significantly affect Acat2 substrate selectivity. Our results suggest an important contribution of second-shell residues to thiolase substrate selectivity and offer insights into engineering this enzyme class.

The promiscuous enzyme medium-chain 3-keto-acyl-CoA thiolase triggers a vicious cycle in fatty-acid beta-oxidation.

Mitochondrial fatty-acid beta-oxidation (mFAO) plays a central role in mammalian energy metabolism. Multiple severe diseases are associated with defects in this pathway. Its kinetic structure is characterized by a complex wiring of which the functional implications have hardly been explored. Repetitive cycles of reversible reactions, each cycle shortening the fatty acid by two carbon atoms, evoke competition between intermediates of different chain lengths for a common set of 'promiscuous' enzymes (enzymes with activity towards multiple substrates). In our validated kinetic model of the pathway, substrate overload causes a steep and detrimental flux decline. Here, we unravel the underlying mechanism and the role of enzyme promiscuity in it. Comparison of alternative model versions elucidated the role of promiscuity of individual enzymes. Promiscuity of the last enzyme of the pathway, medium-chain ketoacyl-CoA thiolase (MCKAT), was both necessary and sufficient to elicit the flux decline. Subsequently, Metabolic Control Analysis revealed that MCKAT had insufficient capacity to cope with high substrate influx. Next, we quantified the internal metabolic regulation, revealing a vicious cycle around MCKAT. Upon substrate overload, MCKAT's ketoacyl-CoA substrates started to accumulate. The unfavourable equilibrium constant of the preceding enzyme, medium/short-chain hydroxyacyl-CoA dehydrogenase, worked as an amplifier, leading to accumulation of upstream CoA esters, including acyl-CoA esters. These acyl-CoA esters are at the same time products of MCKAT and inhibited its already low activity further. Finally, the accumulation of CoA esters led to a sequestration of free CoA. CoA being a cofactor for MCKAT, its sequestration limited the MCKAT activity even further, thus completing the vicious cycle. Since CoA is also a substrate for distant enzymes, it efficiently communicated the 'traffic jam' at MCKAT to the entire pathway. This novel mechanism provides a basis to explore the role of mFAO in disease and elucidate similar principles in other pathways of lipid metabolism.

[Disorder of cholesterol metabolism: regulation of intracellular cholesterol and membrane trafficking].

It has been clarified that several transcription factors and functioning proteins play important roles regulating intracellular cholesterol levels. They bind to the ER membrane and sense changes in cholesterol levels in the membrane through SSD. An important membrane-binding transcription factor, SREBP, is retained in the ER membrane, forming an SREBP/SCAP/INSIG trimer when cellular cholesterol levels are abundant. This complex blocks the transport of SREBPs to the Golgi apparatus, thus preventing subsequent transcriptional activation. When cellular cholesterol levels are low, the ER cholesterol concentration is below a threshold value ( <5 mol %). Under these conditions, SCAP escorts SREBPs from the ER to Golgi apparatus by binding to a component of the CopII protein coat. Once in the Golgi apparatus, the SREBPs are proteolytically processed to generate their nuclear form, the bHLH leucine zipper, that activates genes for cholesterol synthesis and uptake. HMG-CoA reductase is also post-transcriptionally regulated by sterol, with INSIG binding of the protein leading to its proteosomal degradation. We demonstrated that Tangier disease and Niemann-Pick disease type B and type C are metabolic disorders of membrane cholesterol. These diseases are not so common in clinical medicine; however, it is very important to understand membrane lipid metabolism, especially in the ER. It will be clarified in the near future disorders of membrane cholesterol trafficking contribute to the pathogeneses of many kinds of disease affecting through ER functioning.

Overexpression of a 3-ketoacyl-CoA thiolase in Leptosphaeria maculans causes reduced pathogenicity on Brassica napus.

Agrobacterium tumefaciens-mediated random mutagenesis was used to generate insertional mutants of the fungus Leptosphaeria maculans. Of 91 transformants screened, only one (A3) produced lesions of reduced size on cotyledons of canola (Brassica napus). Genes flanking the T-DNA insertion had the best matches to an alcohol dehydrogenase class 4 (ADH4)-like gene (Adh4L) and a 3-ketoacyl-CoA thiolase gene (Thiol) and were expressed in mutant A3 in vitro and in planta at significantly higher levels than in the wild type. This is the first report of a T-DNA insertion in fungi causing increased gene expression. Transformants of the wild-type isolate expressing both Adh4L and Thiol under the control of a heterologous promoter had similar pathogenicity to mutant A3. Ectopic expression of only thiolase resulted in loss of pathogenicity, suggesting that thiolase overexpression was primarily responsible for the reduced pathogenicity of the A3 isolate. The thiolase gene encoded a functional protein, as shown by assays in which a nontoxic substrate (2, 4 dichlorophenoxybutyric acid) was converted to a toxic product. The use of a translational fusion with a reporter gene showed thiolase expressed in organelles that are most likely peroxisomes.

Non-conventional yeasts as producers of polyhydroxyalkanoates--genetic engineering of Arxula adeninivorans.

The non-conventional yeast Arxula adeninivorans was equipped with the genes phbA, phbB and phbC of the polyhydroxyalkanoate (PHA) biosynthetic pathway of Ralstonia eutropha, which encode beta-ketothiolase, NADPH-linked acetoacetyl-CoA reductase and PHA synthase, respectively. Arxula strains transformed solely with the PHA synthase gene (phbC) were able to produce PHA. However, the maximum content of the polymer detected in these strains was just 0.003% poly-3-hydroxybutyrate (PHB) and 0.112% poly-3-hydroxyvalerate (PHV). The expression of all three genes (phbA, phbB, phbC) resulted in small increases in the PHA content of the transgenic Arxula cells. However, under controlled cultivation conditions with minimal medium and ethanol as the carbon source, the recombinant yeast was able to accumulate up to 2.2% PHV and 0.019% PHB. Possible reasons for these differences are discussed.

The role of the peroxisome proliferator-activated receptor alpha (PPAR alpha) in the control of cardiac lipid metabolism.

The postnatal mammalian heart uses mitochondrial fatty acid oxidation (FAO) as the chief source of energy to meet the high energy demands necessary for pump function. Flux through the cardiac FAO pathway is tightly controlled in accordance with energy demands dictated by diverse physiologic and dietary conditions. In this report, we demonstrate that the lipid-activated nuclear receptor, peroxisome proliferator-activated receptor alpha (PPARalpha), regulates the expression of several key enzymes involved in cardiac mitochondrial FAO. In response to the metabolic stress imposed by pharmacologic inhibition of mitochondrial long-chain fatty acid import with etomoxir, PPARa serves as a molecular 'lipostat' factor by inducing the expression of target genes involved in fatty acid utilization including enzymes involved in mitochondrial and peroxisomal beta-oxidation pathways. In mice lacking PPARalpha (PPARalpha-/- mice), etomoxir precipitates a cardiac phenotype characterized by myocyte lipid accumulation. Surprisingly, this metabolic regulatory response is influenced by gender as demonstrated by the observation that male PPARalpha-/- mice are more susceptible to the metabolic stress compared to female animals. These results identify an important role for PPARalpha in the control of cardiac lipid metabolism.

How proteins get into microbodies (peroxisomes, glyoxysomes, glycosomes).

All microbody proteins studies, including one microbody membrane protein, are made on free polysomes and imported post-translationally. This holds for animal tissues, plants, and fungi. The majority of microbody protein sub-units are synthesized in a form not detectably different from mature sub-units. In five cases a larger precursor protein has been found. The position of the extra piece in this precursor is not known. In two of the five cases, processing of the precursor is not coupled to import; in the other three this remains to be determined. It is not even known whether information in the prepiece contributes to topogenesis, or serves other purposes. Microbody preparations from Neurospora, plant tissue and rat liver can take up some newly synthesized microbody proteins in vitro. In most cases uptake is inefficient. No special requirements for uptake have been established and whether a receptor is involved is not yet known. Several examples have been reported of peroxisomal enzymes with a counterpart in another cell compartment. With the exception of catalase, no direct evidence is available in any of these cases for two isoenzymes specified by the same gene. In the Zellweger syndrome, a lethal hereditary disease of man, characterized by a lack of peroxisomes, the levels of several enzymes of lipid metabolism are strongly decreased. In contrast, D-amino-acid oxidase, L-alpha-hydroxyacid oxidase and catalase levels are normal. The catalase resides in the cytosol. Since there is no separate gene for cytosolic catalase, the normal catalase levels in Zellweger cells show that some peroxisomal enzymes can mature and survive stably in the cytosol. It is possible that maturation of the peroxisomal enzyme in the cytoplasm can account for the finding of cytosolic catalase in some normal mammalian cells. The glycosomes of trypanosomes are microbodies that contain a glycolytic system. Comparison of the glycosomal phosphoglycerate kinase with its cytosolic counterpart has shown that these isoenzymes are 93% homologous in amino-acid sequence, but less than 50% homologous to the corresponding enzymes of yeast and mammals. This implies that few alterations are required to direct a protein into microbodies. This interpretation is supported by the evidence for homology between some microbody and mitochondrial isoenzymes in other organisms mentioned under point 4. The major changes of the glycosomal phosphoglycerate kinase relative to the cytosolic enzyme are a large increase in positive charge and a C-terminal extension of 20 amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)