Rodrigue A, Batia N, Müller M, Fayet O, Böhm R, Mandrand-Berthelot M A, Wu L F
Laboratoire de Génétique Moleculaire des Microorganismes et des Interactions Cellulaires, Unité Mixte de Recherche Centre National de la Recherche Scientifique, Villeurbanne, France.
J Bacteriol. 1996 Aug;178(15):4453-60. doi: 10.1128/jb.178.15.4453-4460.1996.
We analyzed the involvement of chaperonins GroES and GroEL in the biosynthesis of the three hydrogenase isoenzymes, HYD1, HYD2, and HYD3, of Escherichia coli. These hydrogenases are NiFe-containing, membrane-bound enzymes composed of small and large subunits, each of which is proteolytically processed during biosynthesis. Total hydrogenase activity was found to be reduced by up to 60% in groES and groEL thermosensitive mutant strains. This effect was specific because it was not seen for another oligomeric, membrane-bound metalloenzyme, i.e., nitrate reductase. Analyses of the single hydrogenase isoenzymes revealed that a temperature shift during the growth of groE mutants led to an absence of HYD1 activity and to an accumulation of the precursor of the large subunit of HYD3, whereas only marginal effects on the processing of HYD2 and its activity were observed under these conditions. A decrease in total hydrogenase activity, together with accumulation of the precursors of the large subunits of HYD2 and HYD3, was also found to occur in a nickel uptake mutant (nik). The phenotype of this nik mutant was suppressed by supplementation of the growth medium with nickel ions. On the contrary, Ni2+ no longer restored hydrogenase activity and processing of the large subunit of HYD3 when the nik and groE mutations were combined in one strain. This finding suggests the involvement of these chaperonins in the biosynthesis of a functional HYD3 isoenzyme via the incorporation of nickel. In agreement with these in vivo results, we demonstrated a specific binding of GroEL to the precursor of the large subunit of HYD3 in vitro. Collectively, our results are consistent with chaperonin-dependent incorporation of nickel into the precursor of the large subunit of HYD3 as a prerequisite of its proteolytic processing and the acquisition of enzymatic activity.
我们分析了伴侣蛋白GroES和GroEL在大肠杆菌三种氢化酶同工酶HYD1、HYD2和HYD3生物合成中的作用。这些氢化酶是含镍铁的膜结合酶，由小亚基和大亚基组成，每个亚基在生物合成过程中都经过蛋白水解加工。在groES和groEL温度敏感突变株中，总氢化酶活性降低了多达60%。这种效应是特异性的，因为对于另一种寡聚膜结合金属酶即硝酸还原酶未观察到这种效应。对单一氢化酶同工酶的分析表明，groE突变体生长过程中的温度变化导致HYD1活性缺失以及HYD3大亚基前体的积累，而在这些条件下，仅观察到对HYD2加工及其活性的轻微影响。在镍摄取突变体（nik）中也发现总氢化酶活性降低，同时伴有HYD2和HYD3大亚基前体的积累。通过在生长培养基中补充镍离子，该nik突变体的表型得到了抑制。相反，当nik和groE突变在一个菌株中同时存在时，Ni2+不再恢复氢化酶活性和HYD3大亚基的加工。这一发现表明这些伴侣蛋白通过镍的掺入参与功能性HYD3同工酶的生物合成。与这些体内结果一致，我们在体外证明了GroEL与HYD3大亚基前体的特异性结合。总体而言，我们的结果与伴侣蛋白依赖性地将镍掺入HYD3大亚基前体一致，这是其蛋白水解加工和获得酶活性的前提条件。
