Regulation Of Sugar Transport And Metabolism In Oral Bacteria

口腔细菌中糖运输和代谢的调节

• 批准号: 8148613
• 负责人: john thompson
• 金额: $ 36.1万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148613
• 关键词: Metabolism; Regulation; oral bacteria; sugar

Evolution and Biochemistry of Family 4 Glycosyl Hydrolases Glycosyl hydrolase Family 4 (GH4) is exceptional among the 114 families in this enzyme superfamily. Members of GH4 exhibit unusual cofactor requirements for activity, and an essential cysteine residue is present at the active site. Of greatest significance, is the fact that members of GH4 employ a unique catalytic mechanism for cleavage of the glycosidic bond. By phylogenetic analysis, and from available substrate specificities, we have assigned a majority of the enzymes of GH4 to five sub-groups. Our classification revealed an unexpected relationship between substrate specificity and the presence, in each sub-group, of a motif of four amino acids that includes the active-site Cys residue: alpha-glucosidase, CHE(I/V); alpha-galactosidase, CHSV; alpha-glucuronidase, CHGx; 6-phospho-alpha-glucosidase, CDMP; and 6-phospho-beta-glucosidase, CN(V/I)P. The question arises: does the presence of a particular motif sufficiently predict the catalytic function of an unassigned GH4 protein? To test this hypothesis, we have purified and characterized the alpha-glucoside specific GH4 enzyme (PalH) from the phytopathogen, Erwinia rhapontici.The CHEI motif in this protein has been changed by site-directed mutagenesis, and the effects upon substrate specificity have been determined. The change to CHSV caused the loss of all alpha-glucosidase activity, but the mutant protein exhibited none of the anticipated alpha-galactosidase activity. In conclusion, while the Cys-containing motif may be suggestive of enzyme specificity, and phylogenetic placement can greatly increase confidence in that specificity, the annotation and unambiguous assignment of function of a GH4 protein can be made only on the basis of experimental evidence. Enzymology and Pathogenicity of Streptococcus suis 1. Mannonate dehydratase (ManD) is found only in certain bacterial species, where it participates in the dissimilation of glucuronate. ManD catalyzes the dehydration of D-mannonate to yield 2-keto-3-deoxygluconate (2-KDG), the carbon and energy source for growth. Selective inactivation of ManD by drug targeting, is of therapeutic interest in the treatment of human Streptococcus suis infections. In this collaboration we have over-expressed, purified, functionally characterized and determined the crystallographic structure of ManD from S. suis. Importantly, by Fourier transform mass spectrometry (FTMS),we have shown that 2-KDG is formed when the chemically synthesized substrate (D-mannonate) is incubated with ManD. Inductively coupled plasma-mass spectrometry (ICP-MS) revealed the presence of Mn2+ in the purified protein, and in the solution state catalytically active ManD exists as a homodimer of 41-kDa subunits. The crystal structures of S.suis ManD in native form, and in complex with its substrate and Mn2+ ion, have been solved at a resolution of 2.9 Angstroms. The structure of S. suis ManD is that of a modified form of TIM barrel, similar that of other members of xylose isomerase-like superfamily. Structural analyses, and comparative amino acid sequence alignments, provide evidence for the importance of His311 and Tyr325 in ManD activity. The results of site-directed mutagenesis confirmed the functional role(s) of these residues in the dehydration reaction, and a plausible mechanism for the ManD-catalyzed reaction is proposed. 2. Gluconate 5-dehydrogenase (Ga5DH) is an NADP(H)-dependent enzyme that catalyzes a reversible oxido-reduction between D-gluconate and 5-keto- D-gluconate, thereby regulating the flux of this important carbon and energy source in bacteria. However, despite the considerable amount of physiological and biochemical knowledge of G5DH, there is little physical or structural information available for this enzyme. To this end, we have determined the crystal structures of Ga5DH from the pathogenic organism Streptococcus suis serotype 2 in both native and liganded (NADP+/D-gluconate/metal ion) quaternary complex forms at 1.9 and 1.8 Angstroms resolution, respectively. Structural analysis reveals that Ga5DH adopts a protein fold similar to that found in members of the short chain dehydrogenase/reductase (SDR) family, while the enzyme itself represents a previously uncharacterized member of this family. In solution, Ga5DH exists as a tetramer comprised of four identical 26 kDA subunits. The catalytic site of Ga5DH shows considerable architectural similarity to that found in other enzymes of the SDR family, but the S. suis protein contains an additional residue (Arg104) that plays an important role in substrate binding. The quaternary complex structure provides the first crystallographic evidence for the role of a catalytically important serine residue, and also reveals an amino acid tetrad RSYK that differs from the SYK triad found in the majority of SDR enzymes. Inspection of the crystal structures also reveals the important contributions of metal ions in active site formation, and of residues at the C-termini of subunits to tetramer assembly. Ga5DH is a potential target for therapy, and our findings provide insight not only of the catalytic mechanism, but also for structure-based design of inactivating drugs. Enzymology of Clostridium botulinum Sequencing of the genome of Clostridium botulinum strain Hall A revealed a gene (CBO0515), whose putative amino acid sequence was suggestive of the rare enzyme N5-(1-carboxyethyl) ornithine synthase. To test this hypothesis, CBO0515 has been cloned, and the encoded polypeptide was purified and characterized. This unusual gene appears to be confined to proteolytic strains assigned to Group 1 of C. botulinum.

家族 4 糖基水解酶的进化和生物化学 糖基水解酶家族 4 (GH4) 是该酶超家族 114 个家族中的佼佼者。 GH4 的成员表现出不寻常的活性辅助因子要求，并且活性位点存在必需的半胱氨酸残基。最重要的是，GH4 成员采用独特的催化机制来裂解糖苷键。通过系统发育分析，并根据可用的底物特异性，我们将 GH4 的大多数酶分配到五个亚组。 我们的分类揭示了底物特异性与每个亚组中包含活性位点半胱氨酸残基的四个氨基酸基序的存在之间存在意想不到的关系：α-葡萄糖苷酶，CHE(I/V)； α-半乳糖苷酶，CHSV； α-葡萄糖醛酸酶，CHGx； 6-磷酸-α-葡萄糖苷酶，CDMP；和 6-磷酸-β-葡萄糖苷酶，CN(V/I)P。问题出现了：特定基序的存在是否足以预测未分配的 GH4 蛋白的催化功能？为了检验这一假设，我们纯化并表征了植物病原体欧文氏菌 (Erwinia rhapontici) 中的 α-葡萄糖苷特异性 GH4 酶 (PalH)。该蛋白质中的 CHEI 基序已通过定点诱变发生改变，并且对底物特异性的影响已得到证实。决定。 CHSV 的改变导致所有 α-葡萄糖苷酶活性丧失，但突变蛋白没有表现出预期的 α-半乳糖苷酶活性。总之，虽然含有 Cys 的基序可能暗示酶的特异性，并且系统发育的放置可以大大增加对该特异性的置信度，但 GH4 蛋白的注释和功能的明确分配只能基于实验证据。 猪链球菌的酶学和致病性 1. 甘露酸脱水酶 (ManD) 仅存在于某些细菌物种中，它参与葡萄糖醛酸的异化。 ManD 催化 D-甘露糖酸脱水生成 2-酮-3-脱氧葡萄糖酸 (2-KDG)，这是生长的碳和能源。通过药物靶向选择性灭活ManD对于治疗人类猪链球菌感染具有治疗意义。在这次合作中，我们对猪链球菌中的 ManD 进行了过度表达、纯化、功能表征和晶体结构测定。重要的是，通过傅里叶变换质谱（FTMS），我们发现当化学合成的底物（D-甘露糖酸盐）与ManD一起孵育时，会形成2-KDG。电感耦合等离子体质谱 (ICP-MS) 显示纯化蛋白中存在 Mn2+，并且在溶液状态下，具有催化活性的 ManD 以 41 kDa 亚基的同二聚体形式存在。天然形式的猪链球菌 ManD 及其底物和 Mn2+ 离子复合物的晶体结构已以 2.9 埃的分辨率解析。猪链球菌ManD的结构是TIM桶的修饰形式，与木糖异构酶样超家族的其他成员相似。结构分析和比较氨基酸序列比对为 His311 和 Tyr325 在 ManD 活性中的重要性提供了证据。定点诱变的结果证实了这些残基在脱水反应中的功能作用，并提出了 ManD 催化反应的合理机制。 2. 葡萄糖酸 5-脱氢酶 (Ga5DH) 是一种 NADP(H) 依赖性酶，可催化 D-葡萄糖酸和 5-酮-D-葡萄糖酸之间的可逆氧化还原，从而调节这一重要碳和能量来源的通量。细菌。然而，尽管有大量关于 G5DH 的生理和生化知识，但关于这种酶的物理或结构信息却很少。为此，我们分别以 1.9 和 1.8 埃的分辨率测定了致病微生物猪链球菌血清型 2 的天然和配体（NADP+/D-葡萄糖酸盐/金属离子）四元复合物形式的 Ga5DH 晶体结构。结构分析表明，Ga5DH 采用与短链脱氢酶/还原酶 (SDR) 家族成员相似的蛋白质折叠，而该酶本身代表了该家族中以前未表征的成员。在溶液中，Ga5DH 以四聚体形式存在，由四个相同的 26 kDA 亚基组成。 Ga5DH 的催化位点与 SDR 家族其他酶的催化位点具有相当大的结构相似性，但猪链球菌蛋白含有一个额外的残基 (Arg104)，该残基在底物结合中发挥重要作用。四元复合物结构为催化重要丝氨酸残基的作用提供了第一个晶体学证据，并且还揭示了与大多数 SDR 酶中发现的 SYK 三联体不同的氨基酸四联体 RSYK。对晶体结构的检查还揭示了金属离子在活性位点形成中的重要贡献，以及亚基 C 末端残基对四聚体组装的重要贡献。 Ga5DH 是一个潜在的治疗靶点，我们的研究结果不仅提供了催化机制的见解，而且还为基于结构的失活药物设计提供了见解。 肉毒梭菌的酶学 肉毒杆菌 Hall A 菌株的基因组测序揭示了一个基因 (CBO0515)，其推定的氨基酸序列暗示了稀有酶 N5-(1-羧乙基) 鸟氨酸合酶。为了检验这一假设，克隆了 CBO0515，并对编码的多肽进行了纯化和表征。这种不寻常的基因似乎仅限于属于肉毒杆菌第 1 组的蛋白水解菌株。