Starch branching enzymes from immature rice seeds.

Four forms of branching enzyme, termed RBE1, RBE2 (a mixture of RBE2A and RBE2B), RBE3, and RBE4, were apparently separated by DEAE-cellulose column chromatography of soluble extract from immature rice seeds, and each of these four forms was further purified by gel-filtration. RBE1, RBE2A, and RBE2B were the predominant forms of the enzyme. The molecular size, amino-terminal amino acid sequence, and immunoreactivity with anti-maize branching enzyme-I (BE-I) antibody were identical among these three forms, except that the molecular mass of RBE2A was almost 3 kDa higher than those of RBE1 and RBE2B. These results indicate that RBE1, RBE2A, and RBE2B are the same (termed rice BE-I). The cDNA clones coding for rice BE-I have been identified from a rice seed library in lambda gt11, using the maize BE-I cDNA as a probe. The nucleotide sequence indicates that rice BE-I is initially synthesized as an 820-residue precursor protein, including a putative 64- or 66-residue transit peptide at the amino terminus. The rice mature BE-I contains 756 (or 754) amino acids with a calculated molecular mass of 86,734 (or 86,502) Da, and shares a high degree of sequence identity (86%) with the maize protein. The consensus sequences of the four regions that form the catalytic sites of amylolytic enzymes are conserved in the central region of the rice BE-I sequence. Thus, rice BE-I as well as the maize protein belongs to a family of amylolytic enzymes.     

Immunological comparison of the starch branching enzymes from potato tubers and maize kernels

Starch branching enzyme was purified from potato (Solanum tuberosum L.) tubers as a single species of 79 kilodaltons and specific antibodies were prepared against both the native enzyme and against the gel-purified, denatured enzyme. The activity of potato branching enzyme could only be neutralized by antinative potato branching enzyme, whereas both types of antibodies reacted with denatured potato branching enzyme. Starch branching enzymes were also isolated from maize (Zea mays L.) kernels. All of the denatured forms of the maize enzyme reacted with antidenatured potato branching enzyme, whereas recognition by antinative potato branching enzyme was limited to maize branching enzymes I and IIb. Antibodies directed against the denatured potato enzyme were unable to neutralize the activity of any of the maize branching enzymes. Antinative potato branching enzyme fully inhibited the activity of maize branching enzyme I; the neutralized maize enzyme was identified as a 82 kilodalton protein. It is concluded that potato branching enzyme (M_r = 79,000) shares a high degree of similarity with maize branching enzyme I (M_r = 82,000), in the native as well as the denatured form. Cross-reactivity between potato branching enzyme and the other forms of maize branching enzyme was observed only after denaturation, which suggests mutual sequence similarities between these species.     

Expression of branching enzyme I of maize endosperm in Escherichia coli.

The gene encoding for mature branching enzyme (BE) I (BEI) of maize (Zea mays L.) endosperm has been expressed in Escherichia coli using the T7 promoter. The expressed BEI was purified to near homogeneity so that amylolytic activity and bacterial BE could be completely eliminated from the BE preparation. The recombinant enzyme showed properties very similar to those of BEI purified from developing maize endosperm with respect to branching amylose and amylopectin. This result confirmed our earlier report that maize endosperm BEI had a higher rate of branching amylose and a much lower rate (less than 10% of that of branching amylose) of branching amylopectin. This study also showed a great advantage in purifying BEI from the bacterial expression system rather than from developing maize endosperm. Most important, this study has established the system with which to study the structure-function relationships of the maize BEI using site-directed mutagenesis.     

Cloning and sequencing of a cDNA encoding 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from maize. Possible relationship to the alkaline phosphatase family.

The primary sequence of maize 2,3-bisphosphoglycerate-independent phosphoglycerate mutase was deduced from cDNAs isolated from maize cDNA libraries by screening with specific antibodies to the cofactor-independent enzyme and from a maize genomic clone. The genomic clone provided the 5'-nucleotide sequence encoding the N-terminal amino acids which could not be obtained from the cDNA. Confirmation that the nucleotide sequence was for the cofactor-independent phosphoglycerate mutase was obtained by sequencing the peptides generated from cyanogen bromide cleavage of the purified protein. This is the first report of the amino acid sequence of a 2,3-bisphosphoglycerate cofactor-independent phosphoglycerate mutase, which consists of 559 amino acids and is twice the molecular size of the mammalian cofactor-dependent enzyme subunit. Analysis of the cofactor-independent phosphoglycerate mutase amino acid sequence revealed no identity with the cofactor-dependent mutase types. Northern blot analysis confirmed this difference since the maize cofactor-independent phosphoglycerate mutase cDNA did not hybridize with mRNA of the cofactor-dependent mutase. The lack of amino acid identity between cofactor-dependent and -independent enzymes is consistent with their different catalytic mechanisms and suggests that both enzymes are unrelated evolutionarily and arose from two independent ancestral genes. However, a constellation of residues which are involved in metal ion binding in various alkaline phosphatases is conserved in the maize cofactor-independent phosphoglycerate mutase, which suggests that the enzyme is a member of the alkaline phosphatase family of enzymes.     

Limited proteolysis of branching enzyme from Escherichia coli

Branching enzyme is involved in determining the structure of starch and glycogen. It catalyzes the formation of branch points by cleavage and transfer of alpha-1,4-glucan chains to alpha-1,6 branch points. Branching enzyme belongs to the amylolytic family of enzymes containing four conserved regions in a central (alpha/beta)8-barrel. Limited proteolysis of the branching enzyme from Escherichia coli (84 kDa) by proteinase K produced a truncated protein of 70-kDa, which still retained 40-60% of branching activity, depending on the type of assay used. Amino acid sequencing showed that the 70-kDa protein lacked 111 or 113 residues at the amino terminal, whereas the carboxy terminal was still intact. We purified this truncated enzyme to homogeneity and analyzed its properties. The enzyme had a three- to fourfold lower catalytic efficiency than the native enzyme, whereas the substrate specificity was unaltered. Furthermore, a branching enzyme with 112 residues deleted at the amino terminal was constructed by recombinant technology and found to have properties identical to those of the proteolyzed enzyme.     

Analysis of the active center of branching enzyme II from maize endosperm

Analysis of the primary structure of mBEII, with those of other branching and amylolytic enzymes as reference, identifies four highly conserved regions which may be involved in substrate binding and in catalysis. When one of the amino acid residues corresponding to the putative catalytic sites of mBEII, i.e., Asp-386, Glu-441, and Asp-509, was replaced, activity disappeared. These putative catalytic residues are located in three different regions (regions 2–4) of the four highly conserved regions (regions 1–4) which exist in the primary structure of most starch hydrolases and related enzymes, including branching enzymes. Region 3, which contains Glu-441 as one of the putative catalytic residues, was located downstream of the carboxyl-terminal position previously reported. The importance of the carboxyl amino acid residues was also demonstrated by chemical modification of the branching enzyme protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.     

Starch- and glycogen-debranching and branching enzymes: Prediction of structural features of the catalytic (β/α)8-barrel domain and evolutionary relationship to other amylolytic enzymes

Sequence alignment and structure prediction are used to locate catalytic α-amylase-type (β/α)₈-barrel domains and the positions of their β-strands and α-helices in isoamylase, pullulanase, neopullulanase, α-amylase-pullulanase, dextran glucosidase, branching enzyme, and glycogen branching enzymes—all enzymes involved in hydrolysis or synthesis of α-1,6-glucosidic linkages in starch and related polysaccharides. This has allowed identification of the transferase active site of the glycogen debranching enzyme and the locations of β ? α loops making up the active sites of all enzymes studied. Activity and specificity of the enzymes are discussed in terms of conserved amino acid residues and loop variations. An evolutionary distance tree of 47 amylolytic and related enzymes is built on 37 residues representing the four best conserved β-strands of the barrel. It exhibits clusters of enzymes close in specificity, with the branching and glycogen debranching enzymes being the most distantly related.     

Localization of C-terminal domains required for the maximal activity or for determination of substrate preference of maize branching enzymes

Previous analysis of a chimeric enzyme mBEII-IBspHI, in which the C-terminal 229 amino acids of maize endosperm branching enzyme isoform II (mBEII) are replaced by the corresponding 284 amino acids of isoform I (mBEI), suggested that the carboxyl terminus of maize branching enzymes may be involved in catalytic efficiency and substrate preference. In the present study, additional hybrids of mBEI and mBEII were generated and expressed in Escherichia coli BL21 (DE3) to dissect the structure/function relationships of the C-terminal regions of maize branching enzymes. A truncated form of purified mBEII-IBspHI, which lacks the C-terminal 58 amino acids, retained similar levels of V(max) in branching activity, K(m) for reduced amylose AS 320, and substrate preference for amylose than amylopectin when compared to mBEII-IBspHI. This indicates that the C-terminal extension derived from mBEI is not required for either catalysis or substrate preference. However, deletion of an additional 87 amino acids from the carboxyl terminus resulted in complete loss of activity. Replacement of the deleted C-terminal additional 87 amino acids with the corresponding 79 amino acids from mBEII restored 25% of the mBEII-IBspHI branching activity without altering substrate preference. It thus appears that a C-terminal region encompassing Leu649-Asp735 of mBEII-IBspHI is required for maximum catalytic efficiency. Another C-terminal region, residues Gln510-Asp648, of mBEII-IBspHI (Gln476-Asp614 of mBEI) may be involved in substrate-preference determination.     

Starch branching enzymes belonging to distinct enzyme families are differentially expressed during pea embryo development

cDNA clones for two isoforms of starch branching enzyme (SBEI and SBEII) have been isolated from pea embryos and sequenced. The deduced amino acid sequences of pea SBEI and SBEII are closely related to starch branching enzymes of maize, rice, potato and cassava and a number of glycogen branching enzymes from yeast, mammals and several prokaryotic species. In comparison with SBEI, the deduced amino acid sequence of SBEII lacks a flexible domain at the N-terminus of the mature protein. This domain is also present in maize SBEII and rice SBEIII and resembles one previously reported for pea granule-bound starch synthase II (GBSSII). However, in each case it is missing from the other isoform of SBE from the same species. On the basis of this structural feature (which exists in some isoforms from both monocots and dicots) and other differences in sequence, SBEs from plants may be divided into two distinct enzyme families. There is strong evidence from our own and other work that the amylopectin products of the enzymes from these two families are qualitatively different. Pea SBEI and SBEII are differentially expressed during embryo development. SBEI is relatively highly expressed in young embryos whilst maximum expression of SBEII occurs in older embryos. The differential expression of isoforms which have distinct catalytic properties means that the contribution of each SBE isoform to starch biosynthesis changes during embryo development. Qualitative measurement of amylopectin from developing and maturing embryos confirms that the nature of amylopectin changes during pea embryo development and that this correlates with the differential expression of SBE isoforms.     

Primary structure of the maize NADP-dependent malic enzyme

Chloroplast-localized NADP-dependent malic enzyme (EC 1.1.1.40) (NADP-ME) provides a key activity for the carbon 4 fixation pathway. In maize, nuclear encoded NADP-ME is synthesized in the cytoplasm as a precursor with a transit peptide that is removed upon transport into the chloroplast stroma. We present here the complete nucleotide sequence for a 2184-base pair full-length maize NADP-ME cDNA. The predicted precursor protein is 636 amino acids long with a Mr of 69,800. There is a strong codon bias found in the amino-terminal portion of NADP-ME that is present in genes for the other enzymes of the C-4 photosynthetic pathway. The NADP-ME transit peptide has general features common to other known chloroplast stroma transit peptides. Comparison of mature maize NADP-ME to the amino acid sequences of known malic enzymes shows two conserved dinucleotide-binding sites. There is a third highly conserved region of unknown function. On the basis of amino acid sequence similarity, the maize chloroplastic enzyme is more closely related to eukaryotic cytosolic isoforms of malic enzyme than to prokaryotic isoforms. We discuss the functional and evolutionary relationship between the chloroplastic and cytosolic forms of NADP-ME.     

Immunological characterization of maize starch branching enzymes

Highly purified fractions of three starch branching enzymes from developing maize (Zea mays L.) endosperm were used to prepare antisera in rabbits. In double diffusion experiments, no immunoprecipitate was observed when branching enzyme IIa or IIb was tested against branching enzyme I antiserum. No immunoprecipitate was formed when branching enzyme I was tested against branching enzyme IIa or IIb antiserum. Increasing amounts of antisera in the above combinations also failed to inhibit enzyme activity. Branching enzyme IIa antiserum cross-reacted and formed spurs with branching enzyme IIb when compared with branching enzyme IIa antigen. Comparison of branching enzyme IIb antiserum with branching enzyme IIa also resulted in an immunoprecipitate. Increasing levels of branching enzyme IIa antiserum inhibited branching enzyme IIb as did the reciprocal combination. The data indicated that branching enzymes IIa and IIb are immunologically similar while branching enzyme I is distinct. The data supports the classification of starch branching enzymes based on genetic, kinetic, and chromatographic properties.     

Structural features of the glycogen branching enzyme encoding genes from aspergilli

A maltose binding protein, p78, was purified to homogeneity from Aspergillus nidulans by a single column chromatography step on cross-linked amylose. The partial amino acid sequence was highly homologous to the glycogen branching enzymes (GBEs) of human and yeast, and p78 did show branching enzyme activity. The genomic gene and its cDNA encoding GBE (p78) were isolated from the A. nidulans genomic and cDNA libraries. Furthermore, a cDNA encoding A. oryzae GBE was entirely sequenced. A. nidulans GBE shared overall and significant amino acid sequence identity with GBEs from A. oryzae (83.9%), Saccharomyces cerevisiae (61.1%) and human (63.0%), and with starch branching enzymes from green plants (55–56%).     

Analysis of the amino terminus of maize branching enzyme II by polymerase chain reaction random mutagenesis

Maize endosperm branching enzyme II (mBEII) plays a pivotal role in determining the quality of starch by catalyzing the synthesis of the alpha-1,6-branch points. While the central (alpha/beta)8-barrel and the C-terminal domains of mBEII have been analyzed previously, the possible role of its amino terminus in catalysis is still poorly understood. Because the amino terminus of mBEII shares very little sequence homology with other amylolytic enzymes, the Met1-Gly276 region of mBEII was randomly mutagenized under error-prone PCR conditions. Subsequent screening by a heterologous complementation system, utilizing an Escherichia coli strain devoid of the endogenous glycogen branching enzyme (glgB-), led to the recovery of mBEII mutants with altered iodine-staining patterns and reduced branching enzyme activities. The NR-625 mutant enzyme, which lacks the N-terminal 39 residues of mBEII due to a frameshift mutation introduced during the random mutagenesis, retained more than 70% of the wild-type activity. The chain transfer pattern and substrate preference of the truncated enzyme were almost identical to those of the wild-type mBEII. It appears that the N-terminal 39 residues of mBEII are neither required for catalysis nor involved in chain transfer. On the other hand, the Gln-to-Arg substitution at position 270 of mBEII resulted in the loss of more than 90% of branching activity. The Gln270 of mBEII, located at the beginning of the (alpha/beta)8-barrel domain, may be required for maximum enzyme activity.     

Purification and characterization of fructose bisphosphate aldolase from the ground squirrel,Spermophilus lateralis: enzyme role in mammalian hibernation

Previous work has reported the production of an Escherichia coli branching enzyme with a 112-residue deletion at the amino terminal by limited proteolysis. Here, we study the chain transfer pattern of this enzyme. Gel-permeation chromatography of in vitro branched amylose shows that the truncated branching enzyme transfers fewer short chains (degree of polymerization [d.p.] <20) and a greater proportion of intermediate size chains (d.p. 30-90) than the native enzyme. High-performance anion-exchange chromatography (HPAEC) of the branching limited alpha-glucan product indicates that the truncated branching enzyme transfers a smaller proportion of chains with d.p. 4-11 and more chains longer than d.p. 12. Also, the genes encoding native or truncated branching enzyme were individually expressed in a branching enzyme-deficient mutant, AC71 (glgB(-)). By HPAEC analysis of the purified alpha-glucans we find that truncated branching enzyme transfers fewer chains of d.p. 5-11 and more chains longer than d.p. 12 relative to the full-length enzyme. These observations allow us to conclude that truncation of the amino-terminal domain has altered the branching pattern of the enzyme. Our results are consistent with the construction of hybrid branching enzymes from the maize isoforms.     

The degree of branching in (alpha 1,4)-(alpha 1,6)-linked glucopolysaccharides is dependent on intrinsic properties of the branching enzymes

1. Branching enzymes from rat and rabbit liver, as well as from potato and maize were prepared. They were almost free from contaminating glucan-degrading enzymes. 2. In 'sweet corn' maize, two separate fractions with (alpha 1,4)glucan: (alpha 1,4)glucan alpha 6-glycosyltransferase activities were obtained. One of them synthesized amylopectin, the branched component of starch, in the presence of phosphorylase and Glc1P, while the other fraction synthesized phytoglycogen. Furthermore, in a maize variety which does not accumulate phytoglycogen, only one fraction of branching activity was found, that formed amylopectin under the above-mentioned conditions. 3. Comparative analyses performed with native (alpha 1,4)-(alpha 1,6)glucopolysaccharides, and those synthesized in vitro with the branching enzyme from the same tissue, demonstrated a close similarity between both glucans. 4. It may be concluded that the branching enzyme is responsible for the specific degree of (alpha 1,6) branch linkages found in the native polysaccharide.     

Multiple forms of starch branching enzyme of maize: Evidence for independent genetic control

Purification of starch branching enzymes from kernels of two nonlinked mutants of maize, $\text{sugary}$ and $\text{amylose-extender}$, showed the basis of the two mutations to be associated with branching enzymes I and IIb, respectively. Branching enzyme I from $\text{sugary}$ kernels purified as nonmutant branching enzyme I, but had an altered pattern of activity when amylose was used as a substrate. In addition to the typical fall in absorbance at high wavelengths (550–700 nm) of the amylose-iodine complex, branching of amylose by $\text{sugary}$ branching enzyme I caused an increase in absorbance at low wavelengths (400–550 nm). Branching enzyme IIb was undetected in extracts of $\text{amylose-extender}$ kernels, while branching enzymes I and IIa appeared unaltered. Low umprimed starch synthase activity was also observed in DEAE-cellulose fractions of $\text{amylose-extender}$ maize, but this activity was regenerated by the addition of any branching enzyme.