Sinorhizobium melilotiforms N2-fixing root nodules on alfalfa, and as a free-living bacterium, it can grow on a very broad range of substrates, includingl-proline and several related compounds, such as proline betaine,trans-4-hydroxy-l-proline (trans-4-l-Hyp), andcis-4-hydroxy-d-proline (cis-4-d-Hyp). Fourteenhypgenes are induced upon growth ofS. melilotiontrans-4-l-Hyp, and of those,hypMNPQencodes an ABC-typetrans-4-l-Hyp transporter andhypREencodes an epimerase that convertstrans-4-l-Hyp tocis-4-d-Hyp in the bacterial cytoplasm. Here, we present evidence that the HypO, HypD, and HypH proteins catalyze the remaining steps in whichcis-4-d-Hyp is converted to α-ketoglutarate. The HypO protein functions as ad-amino acid dehydrogenase, convertingcis-4-d-Hyp to Δ1-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 theN-acetylneuraminate lyase subfamily of the (α/β)8protein family and is consistent with the known enzymatic mechanism for other members of the group. It was also shown thatS. melilotican catabolized-proline as both a carbon and a nitrogen source, thatd-proline can complementl-proline auxotrophy, and that the catabolism ofd-proline is dependent on thehypcluster. Transport ofd-proline involves the HypMNPQ transporter, following whichd-proline is converted to Δ1-pyrroline-2-carboxylate (P2C) largely via HypO. The P2C is converted tol-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 14hypgenes.
IMPORTANCEHydroxyproline 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.