Atsushi Yahashiri

The Bacterial Septal Ring Protein RlpA is a Lytic Transglycosylase that Contributes to Rod Shape and Daughter Cell Separation in Pseudomonas aeruginosa

Rare lipoprotein A (RlpA) is a widely conserved outer membrane protein of unknown function that has previously only been studied in Escherichia coli, where it localizes to the septal ring and scattered foci along the lateral wall, but mutants have no phenotypic change. Here we show rlpA mutants of Pseudomonas aeruginosa form chains of short, fat cells when grown in low osmotic strength media. These morphological defects indicate RlpA is needed for efficient separation of daughter cells and maintenance of rod shape. Analysis of peptidoglycan sacculi from an rlpA deletion mutant revealed increased tetra and hexasaccharides that lack stem peptides (hereafter called ‘naked glycans’). Incubation of these sacculi with purified RlpA resulted in release of naked glycans containing 1,6‐anhydro N‐acetylmuramic acid ends. RlpA did not degrade sacculi from wild‐type cells unless the sacculi were subjected to a limited digestion with an amidase to remove some of the stem peptides. Thus, RlpA is a lytic transglycosylase with a strong preference for naked glycan strands. We propose that RlpA activity is regulated in vivo by substrate availability, and that amidases and RlpA work in tandem to degrade peptidoglycan in the division septum and lateral wall.

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

Significance Cell division in bacteria is mediated by a large collection of proteins that assemble into a contractile ring at the division site. Understanding how these proteins are targeted to that site is important for understanding how division is spatially and temporally regulated. Here we demonstrate that a septal-targeting domain found in many bacterial cell-division proteins works by binding to a cell-wall structure that is present only transiently during cell division. These findings support a model for how different cell-wall hydrolases work together during separation of daughter cells and suggest a mechanism for coordinating synthesis and degradation of the cell wall during division. Bacterial SPOR domains bind peptidoglycan (PG) and are thought to target proteins to the cell division site by binding to “denuded” glycan strands that lack stem peptides, but uncertainties remain, in part because septal-specific binding has yet to be studied in a purified system. Here we show that fusions of GFP to SPOR domains from the Escherichia coli cell-division proteins DamX, DedD, FtsN, and RlpA all localize to septal regions of purified PG sacculi obtained from E. coli and Bacillus subtilis. Treatment of sacculi with an amidase that removes stem peptides enhanced SPOR domain binding, whereas treatment with a lytic transglycosylase that removes denuded glycans reduced SPOR domain binding. These findings demonstrate unequivocally that SPOR domains localize by binding to septal PG, that the physiologically relevant binding site is indeed a denuded glycan, and that denuded glycans are enriched in septal PG rather than distributed uniformly around the sacculus. Accumulation of denuded glycans in the septal PG of both E. coli and B. subtilis, organisms separated by 1 billion years of evolution, suggests that sequential removal of stem peptides followed by degradation of the glycan backbone is an ancient feature of PG turnover during bacterial cell division. Linking SPOR domain localization to the abundance of a structure (denuded glycans) present only transiently during biogenesis of septal PG provides a mechanism for coordinating the function of SPOR domain proteins with the progress of cell division.

Triple Isotopic Labeling and Kinetic Isotope Effects: Exposing H-Transfer Steps in Enzymatic Systems

Kinetic isotope effect (KIE) studies can provide insight into the mechanism and kinetics of specific chemical steps in complex catalytic cascades. Recent results from hydrogen KIE measurements have examined correlations between enzyme dynamics and catalytic function, leading to a surge of studies in this area. Unfortunately, most enzymatic H-transfer reactions are not rate limiting, and the observed KIEs do not reliably reflect the intrinsic KIEs on the chemical step of interest. Given their importance to understanding the chemical step under study, accurate determination of the intrinsic KIE from the observed data is essential. In 1975, Northrop developed an elegant method to assess intrinsic KIEs from their observed values [Northrop, D. B. (1975) Steady-state analysis of kinetic isotope effects in enzymic reactions. Biochemistry 14, 2644-2651]. The Northrop method involves KIE measurements using all three hydrogen isotopes, where one of them serves as the reference isotope. This method has been successfully used with different combinations of observed KIEs over the years, but criteria for a rational choice of reference isotope have never before been experimentally determined. Here we compare different reference isotopes (and hence distinct experimental designs) using the reduction of dihydrofolate and dihydrobiopterin by two dissimilar enzymes as model reactions. A number of isotopic labeling patterns have been applied to facilitate the comparative study of reference isotopes. The results demonstrate the versatility of the Northrop method and that such experiments are limited only by synthetic techniques, availability of starting materials, and the experimental error associated with the use of distinct combinations of isotopologues.

Tuning of the H-transfer coordinate in primitive versus well-evolved enzymes.

The nature of an H-transfer reaction catalyzed by a primitive enzyme is examined and compared to the same reaction catalyzed by a mature (highly evolved) enzyme. The findings are evaluated using two different theoretical models. The tunneling correction model[1–3] suggests that the reaction catalyzed by the mature enzyme involves extensive tunneling, while that of the primitive enzyme involves no tunneling contribution. Marcus-like models,[2–5] on the other hand, suggest that the reaction catalyzed by the primitive enzyme has a poorly reorganized reaction coordinate, while the mature enzyme has tuned the reaction coordinate to near perfect reorganization. The latter interpretation does not indicate the degree of tunneling, but it does address the level of system preparation that brings the reaction coordinate to the tunneling conformation. Importantly, the findings indicate that, in contrast to the primitive enzyme, the mature one has evolved to catalyze a reaction with asignificant tunneling contribution or with a perfectly reorganized reaction coordinate for H-tunneling (using tunneling-correction or the Marcus-like models, respectively).

Identification of SPOR Domain Amino Acids Important for Septal Localization, Peptidoglycan Binding, and a Disulfide Bond in the Cell Division Protein FtsN

SPOR domains are about 75 amino acids long and probably bind septal peptidoglycan during cell division. We mutagenized 33 amino acids with surface-exposed side chains in the SPOR domain from an Escherichia coli cell division protein named FtsN. The mutant SPOR domains were fused to Tat-targeted green fluorescent protein ((TT)GFP) and tested for septal localization in live E. coli cells. Lesions at the following 5 residues reduced septal localization by a factor of 3 or more: Q251, S254, W283, R285, and I313. All of these residues map to a β-sheet in the published solution structure of FtsN(SPOR). Three of the mutant proteins (Q251E, S254E, and R285A mutants) were purified and found to be defective in binding to peptidoglycan sacculi in a cosedimentation assay. These results match closely with results from a previous study of the SPOR domain from DamX, even though these two SPOR domains share <20% amino acid identity. Taken together, these findings support the proposal that SPOR domains localize by binding to septal peptidoglycan and imply that the binding site is associated with the β-sheet. We also show that FtsN(SPOR) contains a disulfide bond between β-sheet residues C252 and C312. The disulfide bond contributes to protein stability, cell division, and peptidoglycan binding.

Effects of Cavities at the Nicotinamide Binding Site of Liver Alcohol Dehydrogenase on Structure, Dynamics and Catalysis

A role for protein dynamics in enzymatic catalysis of hydrogen transfer has received substantial scientific support, but the connections between protein structure and catalysis remain to be established. Valine residues 203 and 207 are at the binding site for the nicotinamide ring of the coenzyme in liver alcohol dehydrogenase and have been suggested to facilitate catalysis with “protein-promoting vibrations” (PPV). We find that the V207A substitution has small effects on steady-state kinetic constants and the rate of hydrogen transfer; the introduced cavity is empty and is tolerated with minimal effects on structure (determined at 1.2 Å for the complex with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol). Thus, no evidence is found to support a role for Val-207 in the dynamics of catalysis. The protein structures and ligand geometries (including donor–acceptor distances) in the V203A enzyme complexed with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol or 2,2,2-trifluoroethanol (determined at 1.1 Å) are very similar to those for the wild-type enzyme, except that the introduced cavity accommodates a new water molecule that contacts the nicotinamide ring. The structures of the V203A enzyme complexes suggest, in contrast to previous studies, that the diminished tunneling and decreased rate of hydride transfer (16-fold, relative to that of the wild-type enzyme) are not due to differences in ground-state ligand geometries. The V203A substitution may alter the PPV and the reorganization energy for hydrogen transfer, but the protein scaffold and equilibrium thermal motions within the Michaelis complex may be more significant for enzyme catalysis.

Nuclear magnetic resonance solution structure of the peptidoglycan-binding SPOR domain from Escherichia coli DamX: insights into septal localization.

SPOR domains are present in thousands of bacterial proteins and probably bind septal peptidoglycan (PG), but the details of the SPOR-PG interaction have yet to be elucidated. Here we characterize the structure and function of the SPOR domain for an Escherichia coli division protein named DamX. Nuclear magnetic resonance revealed the domain comprises a four-stranded antiparallel β-sheet buttressed on one side by two α-helices. A third helix, designated α3, associates with the other face of the β-sheet, but this helix is relatively mobile. Site-directed mutagenesis revealed the face of the β-sheet that interacts with α3 is important for septal localization and binding to PG sacculi. The position and mobility of α3 suggest it might regulate PG binding, but although α3 deletion mutants still localized to the septal ring, they were too unstable to use in a PG binding assay. Finally, to assess the importance of the SPOR domain in DamX function, we constructed and characterized E. coli mutants that produced DamX proteins with SPOR domain point mutations or SPOR domain deletions. These studies revealed the SPOR domain is important for multiple activities associated with DamX: targeting the protein to the division site, conferring full resistance to the bile salt deoxycholate, improving the efficiency of cell division when DamX is produced at normal levels, and inhibiting cell division when DamX is overproduced.

The Effect of Electrostatic Shielding on H Tunneling in R67 Dihydrofolate Reductase

Dihydrofolate reductase (DHFR) catalyzes the hydride (H) transfer reaction between NADPH and dihydrofolate, and produces tetrahydrofolate and NADP+. R67 DHFR is a plasmid encoded enzyme, and is considered a “primitive enzyme” due to its genomic, structural, and kinetic properties.[1, 2] Interestingly, kinetic studies of R67 DHFR show an enhancement in H-transfer rate with increasing ionic strength.[3] To evaluate the source of this rate enhancement, the temperature dependency of intrinsic kinetic isotope effects (KIEs) was measured and the nature of the H-transfer step was evaluated at low and high ionic strengths. At high ionic strength, the KIEs were less temperature dependent than at lower ionic strength. These findings were evaluated using a Marcus-like model, which suggests that at higher ionic strength, the donor and acceptor of the hydride were better oriented for H-tunneling than the same system at lower ionic strength. This comparison addresses the level of system preparation that brings the reaction coordinate into a tunneling-ready conformation. While the effect is small, it is statistically significant, as apparent from the comparative data and standard deviations presented in the Supplementary Information (SI – Table S2). These data demonstrate the high sensitivity of the methodology that was developed to study this system (see detailed methods in the SI). The differences in electrostatic potential surface between low and high ionic strengths were calculated, and the theoretical findings add a molecular perspective to the experimental data.