Production and properties of enzymes that activate and produce carbon monoxide.

The chapter focuses on the methods involved in producing and characterizing two key nickel-iron-sulfur enzymes in the Wood-Ljungdahl pathway (WLP) of anaerobic conversion of carbon dioxide fixation into acetyl-CoA: carbon monoxide dehydrogenase (CODH) and acetyl-CoA synthase (ACS). The WLP is used for biosynthesis of cell material and energy conservation by anaerobic bacteria and archaea, and it is central to several industrial biotechnology processes aimed at using syngas and waste gases for the production of fuels and chemicals. The pathway can run in reverse to allow organisms, e.

Heterohexamers Formed by CcmK3 and CcmK4 Increase the Complexity of Beta Carboxysome Shells.

Bacterial microcompartments (BMCs) encapsulate enzymes within a selectively permeable, proteinaceous shell. Carboxysomes are BMCs containing ribulose-1,5-bisphosphate carboxylase oxygenase and carbonic anhydrase that enhance carbon dioxide fixation. The carboxysome shell consists of three structurally characterized protein types, each named after the oligomer they form: BMC-H (hexamer), BMC-P (pentamer), and BMC-T (trimer).

In Vitro Assembly of Diverse Bacterial Microcompartment Shell Architectures.

Bacterial microcompartments (BMCs) are organelles composed of a selectively permeable protein shell that encapsulates enzymes involved in CO fixation (carboxysomes) or carbon catabolism (metabolosomes). Confinement of sequential reactions by the BMC shell presumably increases the efficiency of the pathway by reducing the crosstalk of metabolites, release of toxic intermediates, and accumulation of inhibitory products. Because BMCs are composed entirely of protein and self-assemble, they are an emerging platform for engineering nanoreactors and molecular scaffolds.

Engineering the Bacterial Microcompartment Domain for Molecular Scaffolding Applications.

As synthetic biology advances the intricacy of engineered biological systems, the importance of spatial organization within the cellular environment must not be marginalized. Increasingly, biological engineers are investigating means to control spatial organization within the cell, mimicking strategies used by natural pathways to increase flux and reduce cross-talk. A modular platform for constructing a diverse set of defined, programmable architectures would greatly assist in improving yields from introduced metabolic pathways and increasing insulation of other heterologous systems.

The Q-Cycle Mechanism of the bc Complex: A Biologist's Perspective on Atomistic Studies.

The Q-cycle mechanism of the bc complex is now well enough understood to allow application of advanced computational approaches to the study of atomistic processes. In addition to the main features of the mechanism, these include control and gating of the bifurcated reaction at the Q-site, through which generation of damaging reactive oxygen species is minimized. We report a new molecular dynamics model of the Rhodobacter sphaeroides bc complex implemented in a native membrane, and constructed so as to eliminate blemishes apparent in earlier Rhodobacter models.

The mechanism of ubihydroquinone oxidation at the Qo-site of the cytochrome bc1 complex.

1. Recent results suggest that the major flux is carried by a monomeric function, not by an intermonomer electron flow. 2.

Role of the -PEWY-glutamate in catalysis at the Q(o)-site of the Cyt bc(1) complex.

We re-examine the pH dependence of partial processes of ubihydroquinone (QH(2)) turnover in Glu-295 mutants in Rhodobacter sphaeroides to clarify the mechanistic role. In more crippled mutants, the bell-shaped pH profile of wildtype was replaced by dependence on a single pK at ~8.5 favoring electron transfer.

Transient kinetic analysis of the interaction of L-serine with Escherichia coli D-3-phosphoglycerate dehydrogenase reveals the mechanism of V-type regulation and the order of effector binding.

Pre-steady state stopped-flow analysis of Escherichia coli d-3-phosphoglycerate dehydrogenase (PGDH) reveals that the physiological inhibitor, l-serine, exerts its effect on at least two steps in the kinetic mechanism, but to very different degrees. First, there is a small but significant effect on the dissociation constant of NADH, the first substrate to bind in the ordered mechanism. The effect of serine is mainly on the binding off rate, increasing the K(d) to 5 and 23 muM from 0.

Role of the anion-binding site in catalysis and regulation of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase.

D-3-Phosphoglycerate dehydrogenase from Mycobacterium tuberculosis displays substantial substrate inhibition in the direction of NADH oxidation by its physiological substrate, hydroxypyruvic acid phosphate (HPAP). Previous investigations showed that plots of substrate concentration versus activity derived from steady state assays could be fit with the equation for complete uncompetitive inhibition and that the mechanism may be allosteric. This investigation uses a simulation of transient kinetic data to demonstrate that the mechanism is consistent with the interaction of substrate at a second site called the anion-binding site.

A stopped flow transient kinetic analysis of substrate binding and catalysis in Escherichia coli D-3-phosphoglycerate dehydrogenase.

Pre-steady state, stopped flow analysis of Escherichia coli D-3-phosphoglycerate dehydrogenase was performed by following the fluorescence of protein tryptophan and the fluorescence resonance energy transfer from protein tryptophan to bound NADH. The results indicate that binding of substrates is ordered, with coenzyme, NADH, binding first. Furthermore, the analysis indicated that there are two sets of sites on the tetrameric enzyme that can be differentiated by their kinetic behavior.

Structural analysis of substrate and effector binding in Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase.

The crystal structure of Mycobacterium tuberculosis d-3-phosphoglycerate dehydrogenase has been solved with bound effector, l-serine, and substrate, hydroxypyruvic acid phosphate, at resolutions of 2.7 and 2.4 A, respectively.

A novel mechanism for substrate inhibition in Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase.

Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase undergoes significant inhibition of activity with increasing concentrations of its substrate, hydroxypyruvic acid phosphate. The enzyme also displays an unusual dual pH optimum. A significant decrease in the K(i) for substrate inhibition at pH values corresponding to the valley between these optima is responsible for this phenomena.