Chen Siyun, White Catharine E, diCenzo George C, Zhang Ye, Stogios Peter J, Savchenko Alexei, Finan Turlough M
Department of Biology, McMaster University, Hamilton, Ontario, Canada.
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.
J Bacteriol. 2016 Feb 1;198(7):1171-81. doi: 10.1128/JB.00961-15.
Sinorhizobium meliloti forms N2-fixing root nodules on alfalfa, and as a free-living bacterium, it can grow on a very broad range of substrates, including l-proline and several related compounds, such as proline betaine, trans-4-hydroxy-l-proline (trans-4-l-Hyp), and cis-4-hydroxy-d-proline (cis-4-d-Hyp). Fourteen hyp genes are induced upon growth of S. meliloti on trans-4-l-Hyp, and of those, hypMNPQ encodes an ABC-type trans-4-l-Hyp transporter and hypRE encodes an epimerase that converts trans-4-l-Hyp to cis-4-d-Hyp in the bacterial cytoplasm. Here, we present evidence that the HypO, HypD, and HypH proteins catalyze the remaining steps in which cis-4-d-Hyp is converted to α-ketoglutarate. The HypO protein functions as a d-amino acid dehydrogenase, converting cis-4-d-Hyp to Δ(1)-pyrroline-4-hydroxy-2-carboxylate, which is deaminated by HypD to α-ketoglutarate semialdehyde and then converted to α-ketoglutarate by HypH. The crystal structure of HypD revealed it to be a member of the N-acetylneuraminate lyase subfamily of the (α/β)8 protein family and is consistent with the known enzymatic mechanism for other members of the group. It was also shown that S. meliloti can catabolize d-proline as both a carbon and a nitrogen source, that d-proline can complement l-proline auxotrophy, and that the catabolism of d-proline is dependent on the hyp cluster. Transport of d-proline involves the HypMNPQ transporter, following which d-proline is converted to Δ(1)-pyrroline-2-carboxylate (P2C) largely via HypO. The P2C is converted to l-proline through the NADPH-dependent reduction of P2C by the previously uncharacterized HypS protein. Thus, overall, we have now completed detailed genetic and/or biochemical characterization of 9 of the 14 hyp genes.
Hydroxyproline is abundant in proteins in animal and plant tissues and serves as a carbon and a nitrogen source for bacteria in diverse environments, including the rhizosphere, compost, and the mammalian gut. While the main biochemical features of bacterial hydroxyproline catabolism were elucidated in the 1960s, the genetic and molecular details have only recently been determined. Elucidating the genetics of hydroxyproline catabolism will aid in the annotation of these genes in other genomes and metagenomic libraries. This will facilitate an improved understanding of the importance of this pathway and may assist in determining the prevalence of hydroxyproline in a particular environment.
苜蓿中华根瘤菌在苜蓿上形成固氮根瘤，作为一种自由生活的细菌，它能在非常广泛的底物上生长，包括L-脯氨酸和几种相关化合物，如脯氨酸甜菜碱、反式-4-羟基-L-脯氨酸（反式-4-L-Hyp）和顺式-4-羟基-D-脯氨酸（顺式-4-D-Hyp）。苜蓿中华根瘤菌在反式-4-L-Hyp上生长时会诱导14个hyp基因，其中hypMNPQ编码一种ABC型反式-4-L-Hyp转运蛋白，hypRE编码一种表异构酶，该酶在细菌细胞质中将反式-4-L-Hyp转化为顺式-4-D-Hyp。在此，我们提供证据表明HypO、HypD和HypH蛋白催化顺式-4-D-Hyp转化为α-酮戊二酸的其余步骤。HypO蛋白作为一种D-氨基酸脱氢酶发挥作用，将顺式-4-D-Hyp转化为Δ(1)-吡咯啉-4-羟基-2-羧酸盐，该物质被HypD脱氨生成α-酮戊二酸半醛，然后由HypH转化为α-酮戊二酸。HypD的晶体结构表明它是(α/β)8蛋白家族中N-乙酰神经氨酸裂解酶亚家族的成员，这与该组其他成员已知的酶促机制一致。还表明苜蓿中华根瘤菌可以将D-脯氨酸作为碳源和氮源进行分解代谢，D-脯氨酸可以补充L-脯氨酸营养缺陷型，并且D-脯氨酸的分解代谢依赖于hyp基因簇。D-脯氨酸的转运涉及HypMNPQ转运蛋白，之后D-脯氨酸主要通过HypO转化为Δ(1)-吡咯啉-2-羧酸盐（P2C）。P2C通过先前未鉴定的HypS蛋白依赖NADPH的还原作用转化为L-脯氨酸。因此，总体而言，我们现在已经完成了14个hyp基因中9个基因的详细遗传和/或生化特征分析。
羟脯氨酸在动植物组织的蛋白质中含量丰富，在包括根际、堆肥和哺乳动物肠道在内的各种环境中作为细菌的碳源和氮源。虽然细菌羟脯氨酸分解代谢的主要生化特征在20世纪60年代就已阐明，但遗传和分子细节直到最近才确定。阐明羟脯氨酸分解代谢的遗传学将有助于在其他基因组和宏基因组文库中对这些基因进行注释。这将有助于更好地理解该途径的重要性，并可能有助于确定特定环境中羟脯氨酸的普遍性。
