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Mitochondrial fatty acid synthesis (FAS) generates the octanoyl-group that is required for the synthesis of lipoic acid and is linked to mitochondrial RNA metabolism. All of the human enzymes involved in mitochondrial FAS have been characterized except for β-ketoacyl thioester reductase (HsKAR), which catalyzes the second step in the pathway. We report here the unexpected finding that a heterotetramer composed of human 17β-hydroxysteroid dehydrogenase type 8 (Hs17β-HSD8) and human carbonyl reductase type 4 (HsCBR4) forms the long-sought HsKAR. Both proteins share sequence similarities to the yeast 3-oxoacyl-(acyl carrier protein) reductase (Oarlp) and the bacterial FabG, although HsKAR is NADH dependent, whereas FabG and Oarlp are NADPH dependent. Hs17β-HSD8 and HsCBR4 show a strong genetic interaction in vivo in yeast, where, only if they are expressed together, they rescue the respiratory deficiency and restore the lipoic acid content of oarlΔ cells. Moreover, these two proteins display a stable physical interaction and form an active heterotetramer. Both Hsl7β-HSD8 and HsCBR4 are targeted to mitochondria in vivo in cultured HeLa cells. Notably, 17β-HSD8 was previously classified as a steroid-metabolizing enzyme, but our data suggest that 17β-HSD8 is primarily involved in mitochondrial FAS. [ABSTRACT FROM AUTHOR]

Diaminopropionate (DAP) ammonia lyase (a PLP-dependent enzyme; EC4.3.1.15) catalyzes the α, β-elimination reaction of both L- and D-α, β-diaminopropionate to form pyruvate and ammonia; Escherichia coli DAP ammonia lyase gene was cloned and over expressed in E. coli and the protein was purified to homogeneity and crystallized using the hanging-drop vapour diffusion technique. Crystals of two different morphologies were obtained, one of which belonged to the tetragonal space group P4[SUB1]2[SUB1]2(orP4[SUB3]2[SUB1]2), with unit-cell parameters a = b = 86.01, c = 209.56Å, and the other to the monoclinic space group P2[SUB1], with unit-cell parameters a = 87.78, b = 94.35, c = 96.02Å, β = 109.73°. The tetragonal crystals diffracted X-rays to 3.0Å resolution, while diffraction from the monoclinic form extended to 2.5Å. Complete X-ray diffraction data sets have been collected for both crystal forms. [ABSTRACT FROM AUTHOR]