Structure of the β₂ homodimer of bacterial luciferase from vibrio harveyi: X‐ray analysis of a kinetic protein folding trap

Abstract

Luciferase, as isolated from Vibrio harveyi, is an ap heterodimer. When allowed to fold in the absence of the a subunit, either in vitro or in vivo, the p subunit of the enzyme will form a kinetically stable homodimer that does not unfold even after prolonged incubation in 5 M urea at pH 7.0 and 18 "C. This form of the p subunit, arising via kinetic partitioning on the folding pathway, appears to constitute a kinetically trapped alternative to the heterodimeric enzyme (Sinclair JF, Ziegler MM, Baldwin TO. 1994. Kinetic partitioning during protein folding yields multiple native states. Nature Struct Biol I : 320-326). Here we describe the X-ray crystal structure of the p2 homodimer of luciferase from V harveyi determined and refined at 1.95 A resolution. Crystals employed in the investigation belonged to the orthorhombic space group P212121 with unit cell dimensions of a = 58.8 A, b = 62.0 A, and c = 218.2 A and contained one dimer per asymmetric unit. Like that observed in the functional luciferase ap heterodimer, the major tertiary structural motif of each p subunit consists of an (a@)* barrel (Fisher AJ, Raushel FM, Baldwin TO, Rayment I. 1995. Three-dimensional structure of bacterial luciferase from Vibrio harveyi at 2.4 8, resolution. Biochemistry 3 4 6581-6586). The root-meansquare deviation of the a-carbon coordinates between the p subunits of the hetero-and homodimers is 0.7 A. This high resolution X-ray analysis demonstrates that "domain" or ''loop'' swapping has not occurred upon formation of the p2 homodimer and thus the stability of the p2 species to denaturation cannot be explained in such simple terms. In fact, the subunitmbunit interfaces observed in both the p2 homodimer and ap heterodimer are remarkably similar in hydrogenbonding patterns and buried surface areas.

GroE modulates kinetic partitioning of folding intermediates between alternative states to maximize the yield of biologically active protein

“…The high-resolution structure of the enzyme (Fisher et al, 1995(Fisher et al, , 1996 and of the b 2 homodimer is known (Thoden et al, 1997), and low-resolution structural information regarding various intermediates formed during refolding is available (Sinclair et al, , 1994. Based on these earlier studies, and those reported here, it appears that GroEL binds folding intermediates of both subunits that resemble in many respects the structures of the subunits in the heterodimer, except that they are less stable.…”

The Mycobacterium marinum mel2 locus displays similarity to bacterial bioluminescence systems and plays a role in defense against reactive oxygen and nitrogen species

“…We obtained a 100% alignment (E = 1 × 10 -28 ) for the 323 amino acid (a.a.) bacterial luciferase-like monooxygenase motif (pfam00296.11; Figure 1A). This motif is conserved in all bacterial luciferase genes, including the luxA and luxB genes from Vibrio harveyi , for which crystal structures have been previously determined [40,41]. Many of the residues responsible for catalytic activity and FMNH 2 binding for LuxA and LuxB are also present in MelF [42,43], suggesting that these proteins have related activities.…”

Molecular Modeling Study on the Three-dimensional Structure of the Luciferase Protein inVibrio-qinghaiensissp.-Q67

“…在 Mg 2＋ 、 ATP 和 O 2 存在时, 催化荧光素(Luciferin)氧化脱羧, 发 出可见光. 而细菌荧光素酶由 α 和 β 两个多肽亚基组成 异源二聚体, α 和 β 亚基分别由荧光素酶基因 luxA 和 luxB 编码而成 [6] . 细菌荧光素酶的底物是还原性黄素单 核苷酸(FMNH 2 )和长链脂肪醛, 后者与荧光素酶具有微 摩尔级的高亲和力.…”

“…从 Q67 荧光素酶序列出发搜索 PDB 蛋白质结构数据库, 找 到 与 模 板 序 列 一 致 性 高 于 30% 的 有 3fgc [8] , 1bsl [6] , 1nfp [29] 和 1fvp [30] 等蛋白质结构, 其中 Vibrio harveyi 荧光 素酶 3fgc 与 Q67 序列一致性最高(α 亚基 84%, β 亚基 60%). 选取 3fgc 作为最佳模版利用 HOMCOS (http:// strcomp.protein.osaka-u.ac.jp/homcos/) 进 行 异 源 二 聚 体 同源建模.…”