Chenguang Fan

Short N-terminal sequences package proteins into bacterial microcompartments

Hundreds of bacterial species produce proteinaceous microcompartments (MCPs) that act as simple organelles by confining the enzymes of metabolic pathways that have toxic or volatile intermediates. A fundamental unanswered question about bacterial MCPs is how enzymes are packaged within the protein shell that forms their outer surface. Here, we report that a short N-terminal peptide is necessary and sufficient for packaging enzymes into the lumen of an MCP involved in B12-dependent 1,2-propanediol utilization (Pdu MCP). Deletion of 10 or 14 amino acids from the N terminus of the propionaldehyde dehydrogenase (PduP) enzyme, which is normally found within the Pdu MCP, substantially impaired packaging, with minimal effects on its enzymatic activity. Fusion of the 18 N-terminal amino acids from PduP to GFP, GST, or maltose-binding protein resulted in their encapsulation within MCPs. Bioinformatic analyses revealed N-terminal extensions in two additional Pdu proteins and three proteins from two unrelated MCPs, suggesting that N-terminal peptides may be used to package proteins into diverse MCPs. The potential uses of MCP assembly principles in nature and in biotechnology are discussed.

Interactions between the termini of lumen enzymes and shell proteins mediate enzyme encapsulation into bacterial microcompartments.

Bacterial microcompartments (MCPs) are a widespread family of proteinaceous organelles that consist of metabolic enzymes encapsulated within a protein shell. For MCPs to function specific enzymes must be encapsulated. We recently reported that a short N-terminal targeting sequence of propionaldehyde dehydrogenase (PduP) is necessary and sufficient for the packaging of enzymes into a MCP that functions in 1,2-propanediol (1,2-PD) utilization (Pdu) by Salmonella enterica. Here we show that encapsulation is mediated by binding of the PduP targeting sequence to a short C-terminal helix of the PduA shell protein. In vitro studies indicated binding between PduP and PduA (and PduJ) but not other MCP shell proteins. Alanine scanning mutagenesis determined that the key residues involved in binding are E7, I10, and L14 of PduP and H81, V84, and L88 of PduA. In vivo targeting studies indicated that the binding between the N terminus of PduP and the C terminus of PduA is critical for encapsulation of PduP within the Pdu MCP. Structural models suggest that the N terminus of PduP and C terminus of PduA both form helical structures that bind one another via the key residues identified by mutagenesis. Cumulatively, these results show that the N-terminal targeting sequence of PduP promotes its encapsulation by binding to MCP shell proteins. This is a unique report determining the mechanism by which a MCP targeting sequence functions. We propose that specific interactions between the termini of shell proteins and lumen enzymes have general importance for guiding the assembly and the higher level organization of bacterial MCPs.

Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids

Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technology has been largely restricted to proteins containing a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation. We evolved chromosomal aminoacyl-tRNA synthetases (aaRSs) with up to 25-fold increased protein production for p-acetyl-L-phenylalanine and p-azido-L-phenylalanine (pAzF). We also evolved aaRSs with tunable specificities for 14 nsAAs, including an enzyme that efficiently charges pAzF while excluding 237 other nsAAs. These variants enabled production of elastin-like-polypeptides with 30 nsAA residues at high yields (∼50 mg/L) and high accuracy of incorporation (>95%). This approach to aaRS evolution should accelerate and expand our ability to produce functionalized proteins and sequence-defined polymers with diverse chemistries.

The N-Terminal Region of the Medium Subunit (PduD) Packages Adenosylcobalamin-Dependent Diol Dehydratase (PduCDE) into the Pdu Microcompartment

Salmonella enterica produces a proteinaceous microcompartment for B(12)-dependent 1,2-propanediol utilization (Pdu MCP). The Pdu MCP consists of catabolic enzymes encased within a protein shell, and its function is to sequester propionaldehyde, a toxic intermediate of 1,2-propanediol degradation. We report here that a short N-terminal region of the medium subunit (PduD) is required for packaging the coenzyme B(12)-dependent diol dehydratase (PduCDE) into the lumen of the Pdu MCP. Analysis of soluble cell extracts and purified MCPs by Western blotting showed that the PduD subunit mediated packaging of itself and other subunits of diol dehydratase (PduC and PduE) into the Pdu MCP. Deletion of 35 amino acids from the N terminus of PduD significantly impaired the packaging of PduCDE with minimal effects on its enzyme activity. Western blotting showed that fusing the 18 N-terminal amino acids of PduD to green fluorescent protein or glutathione S-transferase resulted in the association of these fusion proteins with the MCP. Immunoprecipitation tests indicated that the fusion proteins were encapsulated inside the MCP shell.

The PduQ enzyme is an alcohol dehydrogenase used to recycle NAD+ internally within the Pdu microcompartment of Salmonella enterica.

Salmonella enterica uses a bacterial microcompartment (MCP) for coenzyme B12-dependent 1,2-propanediol (1,2-PD) utilization (Pdu). The Pdu MCP consists of a protein shell that encapsulates enzymes and cofactors required for metabolizing 1,2-PD as a carbon and energy source. Here we show that the PduQ protein of S. enterica is an iron-dependent alcohol dehydrogenase used for 1,2-PD catabolism. PduQ is also demonstrated to be a new component of the Pdu MCP. In addition, a series of in vivo and in vitro studies show that a primary function of PduQ is to recycle NADH to NAD+ internally within the Pdu MCP in order to supply propionaldehyde dehydrogenase (PduP) with its required cofactor (NAD+). Genetic tests determined that a pduQ deletion mutant grew slower than wild-type Salmonella on 1,2-PD and that this phenotype was not complemented by a non-MCP associated Adh2 from Zymomonas that catalyzes the same reaction. This suggests that PduQ has a MCP-specific function. We also found that a pduQ deletion mutant had no growth defect in a genetic background having a second mutation that prevents MCP formation which further supports a MCP-specific role for PduQ. Moreover, studies with purified Pdu MCPs demonstrated that the PduQ enzyme can convert NADH to NAD+ to supply the PduP reaction in vitro. Cumulatively, these studies show that the PduQ enzyme is used to recycle NADH to NAD+ internally within the Pdu MCP. To our knowledge, this is the first report of internal recycling as a mechanism for cofactor homeostasis within a bacterial MCP.

The PduX Enzyme of Salmonella enterica Is an l-Threonine Kinase Used for Coenzyme B12 Synthesis

Here, the PduX enzyme of Salmonella enterica is shown to be an l-threonine kinase used for the de novo synthesis of coenzyme B12 and the assimilation of cobyric acid (Cby). PduX with a C-terminal His tag (PduX-His6) was produced at high levels in Escherichia coli, purified by nickel affinity chromatography, and partially characterized. 31P NMR spectroscopy established that purified PduX-His6 catalyzed the conversion of l-threonine and ATP to l-threonine-O-3-phosphate and ADP. Enzyme assays showed that ATP was the preferred substrate compared with GTP, CTP, or UTP. PduX displayed Michaelis-Menten kinetics with respect to both ATP and l-threonine and nonlinear regression was used to determine the following kinetic constants: Vmax = 62.1 ± 3.6 nmol min–1 mg of protein–1; Km, ATP = 54.7 ± 5.7 μm and Km,Thr = 146.1 ± 8.4 μm. Growth studies showed that pduX mutants were impaired for the synthesis of coenzyme B12 de novo and from Cby, but not from cobinamide, which was the expected phenotype for an l-threonine kinase mutant. The defect in Cby assimilation was corrected by ectopic expression of pduX or by supplementation of growth medium with l-threonine-O-3-phosphate, providing further support that PduX is an l-threonine kinase. In addition, a bioassay showed that a pduX mutant was impaired for the de novo synthesis of coenzyme B12 as expected. Collectively, the genetic and biochemical studies presented here show that PduX is an l-threonine kinase used for AdoCbl synthesis. To our knowledge, PduX is the first enzyme shown to phosphorylate free l-threonine and the first l-threonine kinase shown to function in coenzyme B12 synthesis.

The PduM Protein Is a Structural Component of the Microcompartments Involved in Coenzyme B12-Dependent 1,2-Propanediol Degradation by Salmonella enterica

Diverse bacteria use proteinaceous microcompartments (MCPs) to optimize metabolic pathways that have toxic or volatile intermediates. MCPs consist of metabolic enzymes encased within a protein shell that provides a defined environment. In Salmonella enterica, a MCP is involved in B(12)-dependent 1,2-propanediol utilization (Pdu MCP). In this report, we show that the protein PduM is required for the assembly and function of the Pdu MCP. The results of tandem mass spectrometry and Western blot analyses show that PduM is a component of the Pdu MCP. Electron microscopy shows that a pduM deletion mutant forms MCPs with abnormal morphology. Growth tests and metabolite measurements establish that a pduM deletion mutant is unable to form functional MCPs. PduM is unrelated in sequence to proteins of known function and hence may represent a new class of MCP structural proteins. We also report a modified protocol for the purification of Pdu MCP from Salmonella which allows isolation of milligram amounts of MCPs in about 4 h. We believe that this protocol can be extended or modified for the purification of MCPs from diverse bacteria.

Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids

Genetic encoding of noncanonical amino acids (ncAAs) into proteins is a powerful approach to study protein functions. Pyrrolysyl-tRNA synthetase (PylRS), a polyspecific aminoacyl-tRNA synthetase in wide use, has facilitated incorporation of a large number of different ncAAs into proteins to date. To make this process more efficient, we rationally evolved tRNAPyl to create tRNAPyl-opt with six nucleotide changes. This improved tRNA was tested as substrate for wild-type PylRS as well as three characterized PylRS variants (Nϵ-acetyllysyl-tRNA synthetase [AcKRS], 3-iodo-phenylalanyl-tRNA synthetase [IFRS], a broad specific PylRS variant [PylRS-AA]) to incorporate ncAAs at UAG codons in super-folder green fluorescence protein (sfGFP). tRNAPyl-opt facilitated a 5-fold increase in AcK incorporation into two positions of sfGFP simultaneously. In addition, AcK incorporation into two target proteins (Escherichia coli malate dehydrogenase and human histone H3) caused homogenous acetylation at multiple lysine residues in high yield. Using tRNAPyl-opt with PylRS and various PylRS variants facilitated efficient incorporation of six other ncAAs into sfGFP. Kinetic analyses revealed that the mutations in tRNAPyl-opt had no significant effect on the catalytic efficiency and substrate binding of PylRS enzymes. Thus tRNAPyl-opt should be an excellent replacement of wild-type tRNAPyl for future ncAA incorporation by PylRS enzymes.

Exploring the Substrate Range of Wild-Type Aminoacyl- tRNA Synthetases

We tested the substrate range of four wild‐type E. coli aminoacyl‐tRNA synthetases (AARSs) with a library of nonstandard amino acids (nsAAs). Although these AARSs could discriminate efficiently against the other canonical amino acids, they were able to use many nsAAs as substrates. Our results also show that E. coli tryptophanyl‐tRNA synthetase (TrpRS) and tyrosyl‐tRNA synthetase have overlapping substrate ranges. In addition, we found that the nature of the anticodon sequence of tRNATrp altered the nsAA substrate range of TrpRS; this implies that the sequence of the anticodon affects the TrpRS amino acid binding pocket. These results highlight again that inherent AARS polyspecificity will be a major challenge in the aim of incorporating multiple different amino acids site‐specifically into proteins.

Kinetic and Functional Analysis of l-Threonine Kinase, the PduX Enzyme of Salmonella enterica

The PduX enzyme of Salmonella enterica is an l-threonine kinase used for the de novo synthesis of coenzyme B12 and the assimilation of cobyric acid. PduX with an N-terminal histidine tag (His8-PduX) was produced in Esch e richia coli and purified. The recombinant enzyme was soluble and active. Kinetic analysis indicated a steady-state Ordered Bi Bi complex mechanism in which ATP is the first substrate to bind. Based on a multiple sequence alignment of PduX homologues and other GHMP (galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase) family members, 14 PduX variants having changes at 10 conserved serine/threonine and aspartate/glutamate sites were constructed by site-directed mutagenesis. Each variant was produced in E. coli and purified. Comparison of the circular dichroism spectra and kinetic properties of the PduX variants with those of the wild-type enzyme indicated that Glu-24 and Asp-135 are needed for proper folding, Ser-99 and Glu-132 are used for ATP binding, and Ser-253 and Ser-255 are critical to l-threonine binding whereas Ser-100 is essential to catalysis, but its precise role is uncertain. The studies reported here are the first to investigate the kinetic and catalytic mechanisms of l-threonine kinase from any organism.