氏名　Narayanasamy　Nandhagopal
学位の種類　博士（工学）
学位記番号　博甲第168号
学位授与の日付　平成10年3月25日
学位論文の題目　Three-dimensional structure and function of a microbial hydrolase BphD in the biphenyl/PCB degradation pathway.　（微生物のビフェニル/PCB分解代謝経路に存在する加水分解酵素BphDの立体構造と機能）
論文審査委員
　主査　教授　曽田　邦嗣
　副査　教授　三井　幸雄
　副査　教授　山田　良平
　副査　教授　福田　雅夫
　副査　助教授　野中　孝昌

• 論文目次
• 論文要旨

［平成9（1997）年度博士論文題名一覧］ ［博士論文題名一覧］に戻る．

Contents

1.Introduction　p.1

Materials and Methods
2.Purification and Crystallization
　2-1. Purification　p.5
　2-1-1.　Large scale purification　p.6
　2-2.　Crystallization　p.8
　2-3.　Detemination of cell constants and space group　p.10
　2-4.　Data collection　p.10
　2-5.　Production of Heavy atom derivatives　p.11
　2-6.　Production of Selenomethionyl BphD （Se-Met BphD）　p.15
　2-6-1.　Purification and crystallization　p.15

3.Phase determination
　3-1.　Determination of heavy atom sites using difference Patterson map and difference Fourier map　p.17
　3-2.　Heavy atom position refinement and MIR phase determination　p.18

4.MIR phasing and initial model building　p.19

5.Structure refinement　p.21

6.Results and discussion
　6-1.　Characteristics of the refined model　p.24
　6-2.　Over all structure of octameric BphD enzyme　p.25
　6-3.　Subunit structure
　6-3-1.　Topology of the subunit structure　p.25
　6-3-2.　Domain structure of the subunit　p.26
　6-3-3.　Interaction between the subunits　p.28
　6-4.　Analysis of temperature factors　p.30
　6-5.　Active site　p.31
　6-6.　Catalytic action of the BphD enzyme　p.34
　6-7.　Comparison of the folding in the BphD enzyme with Haloalkane dehalogenase,Dienelactone hydrolase and lipase
　6-7-1.　Comparison with Haloalkane dehalogenase　p.37
　6-7-2.　Comparison with Dienelactone hydrolase　p.38
　6-7-3.　Comparison with Lipase　p.39
　6-8.　Comparisons of the active site cleft
　6-8-1.　Comparison of the active site cleft with Haloalkane dehalogenase　p.40
　6-8-2.　Comparison of the active site cleft with Dienelactone hydrolase　p.40
　6-8-3.　Comparison of the active site cleft with Lipase　p.41
　6-9.　Comparison of the catalytic triad residues in all the structures　p.41
　6-10.　Functional implications of the common features　p.42
　6-11.　Multiple sequence alignment　p.44
　6-11-1. Comparison of various motifs identified in the sequence alignment　p.46
　6-12.　Evolutionary relationship　p.48

7.Conclusion　p.50

References　p.53

Appendix-1

Appendix-2

Acknowledgement

The product of the bphD gene, BphD enzyme, from Rhodococcus sp.
strain RHA1 is involved in the PCB degradation pathway, which hydrolytically converts one of the highly reactive intermediates , the meta-cleavage product, into benzoic acid and 2-hydroxypenta-2,4-dienoate or its corresponding chlorinated compounds . This enzyme sometimes determines the substrate specificity of the degradation pathway as the whole. This project was undertaken with the object of solving the three-dimensional structure using X-ray crystallography, analyzing reaction mechanism in detail and to derive the evolutionary relationship with related hydrolases on the basis of the three-dimensional structure of the BphD enzyme. The BphD enzyme is composed of eight identical subunits consisting of 285 amino acid residues each and with a molecular weight of ca. 32 kDa.
The BphD enzyme expressed on E.coli strain, MV1190, was purified by three anion exchange chromatographic steps. Crystals suitable for crystallographic analyses were grown using the batch method. The crystal structure was solved by Mulitiple Isomorphous Replacement （ MIR ） method using the Hg, Au. Pt and Se-BphD derivatives. Refinement of the structure was carried out and the current R-factor is 18.1% with the free-R being 24.8%. The octameric enzyme has a dimension of 105Å along the four-fold axis and 82Å along the two fold axis running perpendicular to the four-fold axis. The octameric structure can be regarded as a stack of two planar rings each of which consists of four subunits related by four-fold rotational symmetry.
The subunit structure of the BphD enzyme was found to belong to the αβ-hydrolase fold family of enzymes, consisting of two domains （i） α-domain and （ii） αβ-domain. The active site was found to be situated between the two domains and the catalytic triad residues at the active site were identified as Ser11O, Asp235 and His263. The active site cavity was analyzed and it was found that one side of the active site constitute the hydrophobic residues while the other side consists of the polar residues. Docking experiment with the substrate showed that there is not enough space for the substrate to bind at the active site cleft. Therefore it can be concluded that conformational changes occur at the active site in order to accommodate the substrate.
Structural comparison were done with other hydrolases, whose three-dimensional structure were already known. lt revealed that （i） The catalytic triad residues are found to be in a similar arrangement and these residues are situated in the loop regions in all the four structures. （ii） All the structures have a similar αβ-hydrolase fold. It showed that this fold is important for the positioning of the catalytic triad residues in a particular three-dimensional geomety. This arrangement is necessary for the hydrolytic reaction mechanism to occur. （iii） A comparison of the active site cleft and the arrangement of the catalytic triad residues, reveals that the mechanism of hydrolytic activity are very similar in all these enzymes.
Sequence alignment of the hydrolases involved in degradation pathway of aromatic compound were done bases on the known three-dimensional structures. Based on this analysis, it can be concluded that most of these enzymes have evolved from a common ancestral gene, implying a divergent evolution.