Solution, Crystal and in Silico Structures of the Organometallic Vitamin B 12 ‐Derivative Acetylcobalamin and of its Novel Rhodium‐Analogue Acetylrhodibalamin

The natural vitamin B12‐derivatives are intriguing complexes of cobalt that entrap the metal within the strikingly skewed and ring‐contracted corrin ligand. Here, we describe the synthesis of the Rh(III)‐corrin acetylrhodibalamin (AcRhbl) from biotechnologically produced metal‐free hydrogenobyric acid and analyze the effect of the replacement of the cobalt‐center of the organometallic vitamin B12‐derivative acetylcobalamin (AcCbl) with its group‐IX homologue rhodium, to give AcRhbl. The structures of AcCbl and AcRhbl were thoroughly analyzed in aqueous solution, in crystals and by in silico methods, in order to gain detailed insights into the structural adaptations to the two homologous metals. Indeed, the common, nucleotide‐appended corrin‐ligand in these two metal corrins features extensive structural similarity. Thus, the rhodium‐corrin AcRhbl joins the small group of B12‐mimics classified as ‘antivitamins B12’, isostructural metal analogues of the natural cobalt‐corrins that hold significant potential in biological and biomedical applications as selective inhibitors of key cellular processes

SNZ3 Encodes a PLP Synthase Involved in Thiamine Synthesis in Saccharomyces cerevisiae

Pyridoxal 5′-phosphate (the active form of vitamin B6) is a cofactor that is important for a broad number of biochemical reactions and is essential for all forms of life. Organisms that can synthesize pyridoxal 5′-phosphate use either the deoxyxylulose phosphate-dependent or -independent pathway, the latter is encoded by a two-component pyridoxal 5′-phosphate synthase. Saccharomyces cerevisiae contains three paralogs of the two-component SNZ/SNO pyridoxal 5′-phosphate synthase. Past work identified the biochemical activity of Snz1p, Sno1p and provided in vivo data that SNZ1 was involved in pyridoxal 5′-phosphate biosynthesis. Snz2p and Snz3p were considered redundant isozymes and no growth condition requiring their activity was reported. Genetic data herein showed that either SNZ2 or SNZ3 are required for efficient thiamine biosynthesis in Saccharomyces cerevisiae. Further, SNZ2 or SNZ3 alone could satisfy the cellular requirement for pyridoxal 5′-phosphate (and thiamine), while SNZ1 was sufficient for pyridoxal 5′-phosphate synthesis only if thiamine was provided. qRT-PCR analysis determined that SNZ2,3 are repressed ten-fold by the presence thiamine. In total, the data were consistent with a requirement for PLP in thiamine synthesis, perhaps in the Thi5p enzyme, that could only be satisfied by SNZ2 or SNZ3. Additional data showed that Snz3p is a pyridoxal 5′-phosphate synthase in vitro and is sufficient to satisfy the pyridoxal 5′-phosphate requirement in Salmonella enterica when the medium has excess ammonia

Getting the payload in place - Unravelling the complexities of making a bacterial microcompartment

Bacterial microcompartments (BMCs) are complex macromolecular assemblies composed of any outer protein shell that encases a specific metabolic pathway cargo. Recent research is now starting to unravel some of the processes that are involved in directing the enzyme cargo to the inside of the BMC. In particular, an article in this issue of J Bacteriol by N. W. Kennedy, C. E. Mills, C. H. Abrahamson, A. Archer, et al. (J Bacteriol 204:e00576-21, 2022, https://doi.org/10.1128/jb.00576-21) highlights the role played by the shell protein PduB in coordinating this internalization process

Functional characterization of the HMP‐P synthase of Legionella pneumophila

The production of the pyrimidine moiety in thiamine synthesis, 2‐methyl‐4‐amino‐5‐hydroxymethylpyrimidine phosphate (HMP‐P), has been described to proceed through the Thi5‐dependent pathway in Saccharomyces cerevisiae and other yeast. Previous work found that ScThi5 functioned poorly in a heterologous context. Here we report a bacterial ortholog to the yeast HMP‐P synthase (Thi5) was necessary for HMP synthesis in Legionella pneumophila. Unlike ScThi5, LpThi5 functioned in vivo in Salmonella enterica under multiple growth conditions. The protein LpThi5 is a dimer that binds pyridoxal‐5′‐phosphate (PLP), apparently without a solvent‐exposed Schiff base. A small percentage of LpThi5 protein co‐purifies with a bound molecule that can be converted to HMP. Analysis of variant proteins both in vivo and in vitro confirmed that residues in sequence motifs conserved across bacterial and eukaryotic orthologs modulate the function of LpThi5.ImportanceThiamine is an essential vitamin for the vast majority of organisms. There are multiple strategies to synthesize and salvage this vitamin. The predominant pathway for synthesis of the pyrimidine moiety of thiamine involves the Fe‐S cluster protein ThiC. An alternative pathway utilizes Thi5, a novel enzyme that uses PLP as a substrate. The Thi5‐dependent pathway is poorly characterized in yeast and has not been characterized in Bacteria. Here we demonstrate that a Thi5‐dependent pathway is necessary for thiamine biosynthesis in Legionella pneumophila and provide biochemical data to extend knowledge of the Thi5 enzyme, the corresponding biosynthetic pathway, and the role of metabolic network architecture in optimizing its function.HMP‐P synthase (Thi5) is a critical enzyme in the biosynthesis of thiamine pyrophosphate in yeast. A Thi5 homolog from Legionella pneumophila, which contributes to HMP synthesis in its native host, is an enzyme that binds pyridoxal‐5′‐phosphate and releases HMP when the purified protein is incubated with iron. In a heterologous system, Thi5 from Saccharomyces cerevisiae and L. pneumophila have functional differences that could reflect structural differences between the enzymes or metabolic differences between organismal hosts.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/167425/1/mmi14622-sup-0001-Supinfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167425/2/mmi14622.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167425/3/mmi14622_am.pd

Functional characterization of the HMP‐P synthase of Legionella pneumophila (Lpg1565)

The production of the pyrimidine moiety in thiamine synthesis, 2‐methyl‐4‐amino‐5‐hydroxymethylpyrimidine phosphate (HMP‐P), has been described to proceed through the Thi5‐dependent pathway in Saccharomyces cerevisiae and other yeast. Previous work found that ScThi5 functioned poorly in a heterologous context. Here we report a bacterial ortholog to the yeast HMP‐P synthase (Thi5) was necessary for HMP synthesis in Legionella pneumophila. Unlike ScThi5, LpThi5 functioned in vivo in Salmonella enterica under multiple growth conditions. The protein LpThi5 is a dimer that binds pyridoxal‐5′‐phosphate (PLP), apparently without a solvent‐exposed Schiff base. A small percentage of LpThi5 protein co‐purifies with a bound molecule that can be converted to HMP. Analysis of variant proteins both in vivo and in vitro confirmed that residues in sequence motifs conserved across bacterial and eukaryotic orthologs modulate the function of LpThi5.ImportanceThiamine is an essential vitamin for the vast majority of organisms. There are multiple strategies to synthesize and salvage this vitamin. The predominant pathway for synthesis of the pyrimidine moiety of thiamine involves the Fe‐S cluster protein ThiC. An alternative pathway utilizes Thi5, a novel enzyme that uses PLP as a substrate. The Thi5‐dependent pathway is poorly characterized in yeast and has not been characterized in Bacteria. Here we demonstrate that a Thi5‐dependent pathway is necessary for thiamine biosynthesis in Legionella pneumophila and provide biochemical data to extend knowledge of the Thi5 enzyme, the corresponding biosynthetic pathway, and the role of metabolic network architecture in optimizing its function.HMP‐P synthase (Thi5) is a critical enzyme in the biosynthesis of thiamine pyrophosphate in yeast. A Thi5 homolog from Legionella pneumophila, which contributes to HMP synthesis in its native host, is an enzyme that binds pyridoxal‐5′‐phosphate and releases HMP when the purified protein is incubated with iron. In a heterologous system, Thi5 from Saccharomyces cerevisiae and L. pneumophila have functional differences that could reflect structural differences between the enzymes or metabolic differences between organismal hosts.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/167425/1/mmi14622-sup-0001-Supinfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167425/2/mmi14622.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167425/3/mmi14622_am.pd