The coenzyme M biosynthetic pathway in proteobacterium Xanthobacter autotrophicus Py2
Partovi, Sarah Eve
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The metabolically versatile bacterium Xanthobacter autotrophicus Py2 has been the focus of many studies within the field of bioenergy sciences, as it contains two unique CO 2 fixing enzymes, and can utilize unconventional substrates such as propylene and acetone as the sole supplemented carbon source while fixing CO 2 in the process. Unexpectedly, coenzyme M (CoM) was found to play a crucial role as a C 3 carrier in the pathway for propylene metabolism in the late 1990s. Previously, CoM was thought to be present solely as a C 1 carrier in methanogenic archaea for nearly 30 years. Though CoM biosynthesis has been characterized in methanogenic archaea, bacterial CoM biosynthesis remained uncharacterized. In X. autotrophicus Py2, four putative CoM biosynthetic enzymes encoded by xcbB1, C1, D1, and E1 have been identified through informatics and proteomic approaches. XcbB1 is homologous to the archaeal ComA which catalyzes the addition of sulfite to phosphoenolpyruvate, and forms the initial intermediate, phosphosulfolactate, in one of the methanogen CoM biosynthetic pathways. The remaining genes do not encode homologues of any of the previously characterized enzymes in methanogen CoM biosynthesis, suggesting bacteria have a unique pathway. The production of phosphosulfolactate by ComA homolog XcbB1 was verified, indicating that bacterial CoM biosynthesis is initiated in an analogous fashion to the PEP-dependent methanogenic archaeal CoM biosynthesis pathway. XcbC1 and D1 are members of the aspartase/fumarase superfamily (AFS), and XcbE1 is a pyridoxal 5'-phosphate-containing enzyme with homology to D-cysteine desulfhydrases. Direct demonstration of activities for XcbB1 and C1 strengthens their hypothetical assignment to a CoM biosynthetic pathway, and puts firm contraints on our proposed functions for XcbD1 and E1. Known AFS members catalyze beta-elimination reactions of succinyl-containing substrates, yielding fumarate as the common unsaturated elimination product. We demonstrate herein that XcbC1 catalyzes a beta-elimination reaction on the substrate phosphosulfolactate to yield sulfoacrylic acid and inorganic phosphate. To our knowledge, beta-elimination reactions releasing phosphate is unprecedented among the AFS, indicating XcbC1 is a unique phosphatase. This work will serve as the framework for future studies aimed at uncovering the final stages of the biosynthetic pathway. By elucidating the XcbB1 and XcbC1 reactions, we have made significant strides towards understanding bacterial CoM biosynthesis which evaded characterization in previous years.