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Chlorophyll, the most abundant pigment in nature, is degraded during normal plant growth, when leaves change color, and at specific developmental stages. Chlorophyllase catalyzes the first chemical reaction in this process, that is, the hydrolysis of chlorophyll into chlorophyllide. Here, we describe a series of laboratory sessions designed to illustrate a sequence of experiments used as part of the scientific research process and to convey key biochemical concepts. The format guides students through the process of biochemical protein analysis, starting from a recombinant protein expression vector and working through a kinetic analysis of the purified protein. Over the course of these experiments, students learn protocols in basic protein chemistry that allow them to design and conduct a related experiment of their own interest. The described set of laboratories can be tailored to fit either a 4- or an 8-week series of experiments for use in either introductory or advanced biochemistry laboratory courses, respectively. (Contains 4 figures and 1 footnote.)

Bacteria and yeast rely on either homoserine transsuccinylase (HIS, metA) or homoserine transacetylase (HTA; met2) for the biosynthesis of methionine. Although HTS and HTA catalyze similar chemical reactions, these proteins are typically unrelated in both sequence and three-dimensional structure. Here we present the 2.0 Å resolution x-ray crystal structure of the Bacillus cereus metA protein in complex with homoserine, which provides the first view of a ligand bound to either HTA or HTS. Surprisingly, functional analysis of the B. cereus metA protein shows that it does not use succinyl-CoA as a substrate. Instead, the protein catalyzes the transacetylation of homoserine using acetyl-CoA. Therefore, the B. cereus metA protein functions as an HTA despite greater than 50% sequence identity with bonafide HIS proteins. This result emphasizes the need for functional confirmation of annotations of enzyme function based on either sequence or structural comparisons. Kinetic analysis of site-directed mutants reveals that the B. cereus metA protein and the E. coli HTS share a common catalytic mechanism. Structural and functional examination of the B. cereus metA protein reveals that a single amino acid in the active site determines acetyl-CoA (Glu-111) versus succinyl-CoA (Gly-111) specificity in the metA-like of acyltransferases. Switching of this residue provides a mechanism for evolving substrate specificity in bacterial methionine biosynthesis. Within this enzyme family, HTS and HTA activity likely arises from divergent evolution in a common structural scaffold with conserved catalytic machinery and homoserine binding sites. [ABSTRACT FROM AUTHOR]

Glutathione is found in mammals, plants, and bacteria and is synthesized by glutathione synthetase (GS). Some plants synthesize glutathione analogs in which ?-alanine replaces glycine. The chemical diversity of these molecules suggests that the substrate specificity of the GS-like peptide ligases differs from GS, but the molecular basis for this variety remains unclear. To better understand the reaction mechanism of Arabidopsis GS (AtGS) site-directed mutagenesis was performed. Residues interacting with bound magnesium ions (E148, N150, E371, and E429) were essential for activity. K313 and K367 were important for ATP binding. Kinetic analysis suggests that R132 provides a key catalytic role. To define the determinants of substrate specificity, we compared AtGS (glycine specific) with Glycine max (soybean) homoglutathione synthetase (GmhGS; ?-alanine specific). Sequence alignments suggest that two active site residues (AtGS: A466 and A467; GmhGS: L466 and P467) modulate substrate preference between glycine and ?-alanine. The interconversion of AtGS to GmhGS and vice versa is described. GmhGS displays a 700-fold preference for ?-alanine, whereas, the double mutant accepts either substrate with equal efficiency. This suggests that differences outside the active site further define substrate specificity in this family of enzymes, and emphasize the need for crystallographic studies. [ABSTRACT FROM AUTHOR]

Aspartate kinase (AK) and homoserine dehydrogenase (HSD) function as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback-inhibited by threonine. In plants the biochemical features of AK and bifunctional AK-HSD enzymes have been characterized, but the molecular properties of the monofunctional HSD remain unexamined. To investigate the role of HSD, we have cloned the cDNA and gene encoding the monofunctional HSD (GmHSD) from soybean. Using heterologously expressed and purified GmHSD, initial velocity and product inhibition studies support an ordered bi bi kinetic mechanism in which nicotinamide cofactor binds first and leaves last in the reaction sequence. Threonine inhibition of GmHSD occurs at concentrations (Kí = 160 -240 mM) more than 1000-fold above physiological levels. This is in contrast to the two AK-HSD isoforms in soybean that are sensitive to threonine inhibition (Kí150 ILM). In addition, GmHSD is not inhibited by other aspartate-derived amino acids. The ratio of threonine-resistant to threonine-sensitive HSD activity in soybean tissues varies and likely reflects different demands for amino acid biosynthesis. This is the first cloning and detailed biochemical characterization of a monofunctional feedback-insensitive HSD from any plant. Threonine-resistant HSD offers a useful biotechnology tool for manipulating the aspartate amino acid pathway to increase threonine and methionine production in plants for improved nutritional content. [ABSTRACT FROM AUTHOR]

In the degradation of chlorophyll, chlorophyllase catalyzes the initial hydrolysis of the phytol moiety from the pigment. Since chlorophyll degradation is a defining feature of plant senescence, compounds inhibiting chlorophyllase activity may delay senescence, thereby improving shelf life and appearance of plant products. Here we describe the development of a 96-well plate-based purification and assay system for measuring chlorophyllase activity. Integrated lysis and immobilized metal affinity chromatography plates were used for purifying recombinant hexahistidine-tagged Triticum aestivum (wheat) chlorophyllase from Escherichia coli. Chlorophyllase assays using chlorophyll as a substrate showed that the immobilized fusion protein displayed kinetic parameters similar to those of recombinant enzyme purified by affinity chromatography; however, the need to extract reaction products from a multiwell plate limits the value of this assay for high-throughput screening applications. Replacing chlorophyll with p-nitrophenyl-ester substrates eliminates the extraction step and allows for continuous measurement of chlorophyllase activity in a multiwell plate format. Determination of steady state kinetic constants, pH rate profile, the inhibitory effects of metal ions and esterase inhibitors, and the effect of functional group-modifying reagents validated the utility of the plate-based system. The combined purification and assay system provides a convenient and rapid method for the assessment of chlorophyllase activity.

Chlorophyllase catalyzes the initial step in the degradation of chlorophyll and plays a key role in leaf senescence and fruit ripening. Here, we report the cloning of chlorophyllase from Triticum aestivum (wheat) and provide a detailed mechanistic analysis of the enzyme. Purification of recombinant chlorophyllase from an Escherichia coli expression system indicates that the enzyme functions as a dimeric protein. Wheat chlorophyllase hydrolyzed the phytol moiety from chlorophyll (k(cat) = 566 min(-1); K(m) = 63 microM) and was active over a broad temperature range (10-75 degrees C). In addition, the enzyme displays carboxylesterase activity toward p-nitrophenyl (PNP)-butyrate, PNP-decanoate, and PNP-palmitate. The pH-dependence of the reaction showed the involvement of an active site residue with a pK(a) of approximately 6.5 for both k(cat) and k(cat)/K(m) with chlorophyll, PNP-butyrate, and PNP-decanoate. Using these substrates, solvent kinetic isotope effects ranging from 1.5 to 1.9 and from 1.4 to 1.9 on k(cat) and k(cat)/K(m), respectively, were observed. Proton inventory experiments suggest the transfer of a single proton in the rate-limiting step. Our analysis of wheat chlorophyllase indicates that the enzyme uses a charge-relay mechanism similar to other carboxylesterases for catalysis. Understanding the activity and mechanism of chlorophyllase provides insight on the biological and chemical control of senescence in plants and lays the groundwork for biotechnological improvement of this enzyme.