Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA.
Microbiology (Reading). 2013 Aug;159(Pt 8):1748-1759. doi: 10.1099/mic.0.066357-0. Epub 2013 May 31.
Bacterial β-class carbonic anhydrases (CAs) are zinc metalloenzymes catalysing reversible hydration of CO2. They maintain the intracellular balance of CO2/bicarbonate required for biosynthetic reactions and represent a new group of antimicrobial drug targets. Genome sequence analysis of Pseudomonas aeruginosa PAO1, an opportunistic human pathogen causing life threatening infections, identified three genes, PAO102, PA2053 and PA4676, encoding putative β-CAs that share 28-45 % amino acid sequence identity and belong to clades A and B. The genes are conserved among all sequenced pseudomonads. The CAs were cloned, heterologously expressed and purified. Metal and enzymic analyses confirmed that the proteins contain Zn(2+) and catalyse hydration of CO2 to bicarbonate. PAO102 (psCA1) was 19-26-fold more active, and together with PA2053 (psCA2) showed CA activity at both pH 7.5 and 8.3, whereas PA4676 (psCA3) was active only at pH 8.3. Circular dichroism spectroscopy suggested that psCA2 and psCA3 undergo pH-dependent structural changes. Taken together, the data suggest that psCA1 may belong to type I and psCA3 to type II β-CAs. Immunoblot analysis showed that all three CAs are expressed in PAO1 cells when grown in ambient air and at 5 % CO2; psCA1 appeared more abundant under both conditions. Growth studies of transposon mutants showed that the disruption of psCA1 impaired PAO1 growth in ambient air and caused a minor defect at high CO2. Thus, psCA1 contributes to the adaptation of P. aeruginosa to low CO2 conditions and will be further studied for its role in virulence and as a potential antimicrobial drug target in this organism.
细菌β-类碳酸酐酶（CAs）是催化 CO2 可逆水合的锌金属酶。它们维持细胞内 CO2/碳酸氢盐的平衡，这是生物合成反应所必需的，并且代表了一类新的抗菌药物靶标。机会性病原体铜绿假单胞菌 PAO1 的基因组序列分析，该病原体可引起危及生命的感染，鉴定出三个基因，PAO102、PA2053 和 PA4676，分别编码假定的β-CA，它们具有 28-45%的氨基酸序列同一性，属于 A 和 B 两个分支。这些基因在所有测序的假单胞菌中都保守。CA 被克隆、异源表达和纯化。金属和酶分析证实，这些蛋白质含有 Zn(2+)并催化 CO2 水合生成碳酸氢盐。PAO102（psCA1）的活性高 19-26 倍，与 PA2053（psCA2）一起在 pH 7.5 和 8.3 下均具有 CA 活性，而 PA4676（psCA3）仅在 pH 8.3 下具有活性。圆二色性光谱表明，psCA2 和 psCA3 发生 pH 依赖性结构变化。综合来看，数据表明 psCA1 可能属于 I 型，psCA3 属于 II 型β-CA。免疫印迹分析表明，当在环境空气中和 5%CO2 下生长时，这三种 CA 都在 PAO1 细胞中表达；在这两种条件下，psCA1 似乎更为丰富。转座子突变体的生长研究表明，psCA1 的缺失会损害 PAO1 在环境空气中的生长，并在高 CO2 下导致轻微缺陷。因此，psCA1 有助于铜绿假单胞菌适应低 CO2 条件，并且将进一步研究其在该生物体中的毒力作用和作为潜在的抗菌药物靶标。
