Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Kiel, Germany.
J Bacteriol. 2010 Feb;192(4):964-74. doi: 10.1128/JB.01281-09. Epub 2009 Dec 18.
The pathway of glucose degradation in the thermoacidophilic euryarchaeon Picrophilus torridus has been studied by in vivo labeling experiments and enzyme analyses. After growth of P. torridus in the presence of [1-(13)C]- and [3-(13)C]glucose, the label was found only in the C-1 and C-3 positions, respectively, of the proteinogenic amino acid alanine, indicating the exclusive operation of an Entner-Doudoroff (ED)-type pathway in vivo. Cell extracts of P. torridus contained all enzyme activities of a nonphosphorylative ED pathway, which were not induced by glucose. Two key enzymes, gluconate dehydratase (GAD) and a novel 2-keto-3-deoxygluconate (KDG)-specific aldolase (KDGA), were characterized. GAD is a homooctamer of 44-kDa subunits, encoded by Pto0485. KDG aldolase, KDGA, is a homotetramer of 32-kDa subunits. This enzyme was highly specific for KDG with up to 2,000-fold-higher catalytic efficiency compared to 2-keto-3-deoxy-6-phosphogluconate (KDPG) and thus differs from the bifunctional KDG/KDPG aldolase, KD(P)GA of crenarchaea catalyzing the conversion of both KDG and KDPG with a preference for KDPG. The KDGA-encoding gene, kdgA, was identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) as Pto1279, and the correct translation start codon, an ATG 24 bp upstream of the annotated start codon of Pto1279, was determined by N-terminal amino acid analysis. The kdgA gene was functionally overexpressed in Escherichia coli. Phylogenetic analysis revealed that KDGA is only distantly related to KD(P)GA, both enzymes forming separate families within the dihydrodipicolinate synthase superfamily. From the data we conclude that P. torridus degrades glucose via a strictly nonphosphorylative ED pathway with a novel KDG-specific aldolase, thus excluding the operation of the branched ED pathway involving a bifunctional KD(P)GA as a key enzyme.
嗜热嗜酸古菌皮氏硫珠菌葡萄糖降解途径的研究采用了体内标记实验和酶分析方法。在[1-(13)C]-和[3-(13)C]葡萄糖存在的条件下培养皮氏硫珠菌后，发现标记仅出现在蛋白质氨基酸丙氨酸的 C-1 和 C-3 位，表明体内仅存在一条 Entner-Doudoroff（ED）途径。皮氏硫珠菌细胞提取物含有非磷酸化 ED 途径的所有酶活性，这些酶活性不受葡萄糖诱导。两种关键酶，葡萄糖酸脱水酶（GAD）和一种新型 2-酮-3-脱氧葡萄糖（KDG）特异性醛缩酶（KDGA），已被鉴定。GAD 是由 Pto0485 编码的 44kDa 亚基的同型八聚体。KDG 醛缩酶，KDGA，是一个由 32kDa 亚基组成的同型四聚体。该酶对 KDG 具有高达 2000 倍的催化效率，与 2-酮-3-脱氧-6-磷酸葡萄糖（KDPG）相比，因此与古菌中的双功能 KDG/KDPG 醛缩酶 KD(P)GA 不同，后者催化 KDG 和 KDPG 的转化，对 KDPG 具有偏好。通过基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱（MS）鉴定，KDGA 编码基因 kdgA 被鉴定为 Pto1279，正确的翻译起始密码子位于 Pto1279 注释起始密码子上游 24bp 的 ATG，并通过 N-末端氨基酸分析确定。kdgA 基因在大肠杆菌中功能过表达。系统发育分析表明，KDGA 与 KD(P)GA 只有远亲关系，这两种酶在二氢二吡啶羧酸合酶超家族中形成独立的家族。根据这些数据，我们得出结论，皮氏硫珠菌通过一种严格的非磷酸化 ED 途径降解葡萄糖，该途径涉及一种新型的 KDG 特异性醛缩酶，因此排除了涉及双功能 KD(P)GA 的分支 ED 途径作为关键酶的作用。
