Erlandsen Heidi, Kim Joo Y, Patch Marianne G, Han Andrew, Volner Alon, Abu-Omar Mahdi M, Stevens Raymond C
The Scripps Research Institute, Department of Molecular Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
J Mol Biol. 2002 Jul 12;320(3):645-61. doi: 10.1016/s0022-2836(02)00496-5.
Structure determination of bacterial homologues of human disease-related proteins provides an efficient path to understanding the three-dimensional fold of proteins that are associated with human diseases. However, the precise locations of active-site residues are often quite different between bacterial and human versions of an enzyme, creating significant differences in the biological understanding of enzyme homologs. To study this hypothesis, phenylalanine hydroxylase from a bacterial source has been structurally characterized at high resolution and comparison is made to the human analog. The enzyme phenylalanine hydroxylase (PheOH) catalyzes the hydroxylation of l-phenylalanine into l-tyrosine utilizing the cofactors (6R)-l-erythro-5,6,7,8 tetrahydrobiopterin (BH(4)) and molecular oxygen. Previously determined X-ray structures of human and rat PheOH, with a sequence identity of more than 93%, show that these two structures are practically identical. It is thus of interest to compare the structure of the divergent Chromobacterium violaceum phenylalanine hydroxylase (CvPheOH) ( approximately 24% sequence identity overall) to the related human and rat PheOH structures. We have determined crystal structures of CvPheOH to high resolution in the apo-form (no Fe-added), Fe(III)-bound form, and 7,8-dihydro-l-biopterin (7,8-BH(2)) plus Fe(III)-bound form. The bacterial enzyme displays higher activity and thermal melting temperature, and structurally, differences are observed in the N and C termini, and in a loop close to the active-site iron atom.
确定与人类疾病相关蛋白质的细菌同源物的结构，为理解与人类疾病相关蛋白质的三维折叠提供了一条有效途径。然而，一种酶的细菌版本和人类版本中活性位点残基的精确位置往往有很大差异，这在对酶同源物的生物学理解上造成了显著不同。为了研究这一假设，已对来源于细菌的苯丙氨酸羟化酶进行了高分辨率结构表征，并与人类同源物进行了比较。苯丙氨酸羟化酶（PheOH）利用辅因子（6R）-l-赤藓糖型-5,6,7,8-四氢生物蝶呤（BH(4)）和分子氧，催化l-苯丙氨酸羟基化为l-酪氨酸。先前测定的人类和大鼠PheOH的X射线结构，序列同一性超过93%，表明这两种结构几乎相同。因此，将差异较大的紫色色杆菌苯丙氨酸羟化酶（CvPheOH）（总体序列同一性约为24%）的结构与相关的人类和大鼠PheOH结构进行比较很有意义。我们已经确定了CvPheOH在无辅因子（未添加铁）形式、铁（III）结合形式以及7,8-二氢-l-生物蝶呤（7,8-BH(2)）加铁（III）结合形式下的高分辨率晶体结构。该细菌酶表现出更高的活性和热解链温度，在结构上，在N和C末端以及靠近活性位点铁原子的一个环中观察到差异。
