Regulation Of Sugar Transport And Metabolism In Oral Bacteria

糖基水解酶家族4的进化与生物化学 糖基水解酶家族4(GH4)是该酶超家族114个家族中的一个例外。GH4的成员表现出不寻常的辅因子活性要求，活性部位存在必要的半胱氨酸残基。最重要的是，GH4的成员使用了一种独特的催化机制来裂解糖苷键。通过系统发育分析，根据可用的底物特异性，我们将GH4的大部分酶分配给了五个亚组。 我们的分类揭示了底物特异性和每个亚群中包括活性部位Cys残基的四个氨基酸基序的存在之间的意外关系：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。问题产生了：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？为了验证这一假设，我们从植物病原菌欧文氏杆菌中纯化并鉴定了α-葡萄糖苷特异性GH4酶(PalH)，通过定点突变改变了该蛋白中的Chei基序，并确定了其对底物专一性的影响。CHSV的改变导致了所有α-葡萄糖苷酶活性的丧失，但突变蛋白没有表现出预期的α-半乳糖苷酶活性。总之，虽然含有半胱氨酸的基序可能暗示了酶的特异性，并且系统发育位置可以大大增加这种特异性的置信度，但只有在实验证据的基础上，才能对GH4蛋白的功能进行注释和明确的分配。 猪链球菌的酶学特性及致病性研究 1.甘露酸脱水酶(Mand)只存在于某些细菌中，参与葡萄糖醛酸异化。Mand催化D-甘露糖脱水生成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，在治疗人类猪链球菌感染方面具有重要的治疗意义。在这次合作中，我们从猪链霉菌中过表达、纯化、功能鉴定并确定了Mand的晶体结构。重要的是，通过傅里叶变换质谱仪(FTMS)，我们证明了化学合成的底物(D-甘露糖酸盐)与Mand孵育时生成了2-KDG。电感耦合等离子体质谱(ICPMS)分析表明，纯化蛋白中存在Mn2+，在溶液中催化活性的Mand以41 kDa亚基的同源二聚体形式存在。在2.9埃的分辨率下，对猪链霉菌天然形态及其与其底物和Mn2+离子形成的络合物的晶体结构进行了解析。猪链霉菌的结构是一种改良形式的Tim Barrel，类似于木糖异构酶超家族的其他成员。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。定点突变的结果证实了这些残基在脱水反应中的功能作用(S)，并对Mand催化的反应提出了一个合理的机理。 2.葡萄糖酸5-脱氢酶是一种依赖于NADP(H)的酶，它催化D-葡萄糖酸和5-酮-D-葡萄糖酸之间的可逆氧化还原，从而调节细菌中这一重要碳源和能源的通量。然而，尽管G5DH有相当多的生理生化知识，但关于这种酶的物理或结构信息却很少。为此，我们测定了病原菌猪链球菌2型Ga5DH在天然和配体(NADP+/D-葡萄糖酸/金属离子)四元络合物中的晶体结构，分别在1.9埃和1.8埃分辨率下形成。结构分析表明，Ga5DH采用了与短链脱氢酶/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一个以前未描述的成员。在溶液中，Ga5DH以四聚体形式存在，由四个相同的26个KDA亚基组成。Ga5DH的催化位点与SDR家族的其他酶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。四元络合物结构为重要的丝氨酸残基的作用提供了第一个结晶学证据，并揭示了一个不同于大多数SDR酶中发现的SYK三联体的氨基酸四联体RSYK。对晶体结构的检查也揭示了金属离子在活性中心形成中的重要作用，以及亚基C-末端的残基对四聚体组装的重要贡献。Ga5DH是一个潜在的治疗靶点，我们的发现不仅为了解催化机制，也为失活药物的基于结构的设计提供了洞察力。 肉毒梭菌的酶学研究 对肉毒梭菌Hall A株的基因组测序发现了一个基因(CBO0515)，其推测的氨基酸序列与罕见的酶N5-(1-羧乙基)鸟氨酸合成酶有关。为了验证这一假设，我们克隆了CBO0515，并对编码的多肽进行了纯化和鉴定。这种不同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。

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.