Maltose/maltodextrin system of Escherichia coli: transport, metabolism, and regulation

Abstract

The maltose system of Escherichia coli offers an unusually rich set of enzymes, transporters, and regulators as objects of study. This system is responsible for the uptake and metabolism of glucose polymers (maltodextrins), which must be a preferred class of nutrients for E. coli in both mammalian hosts and in the environment. Because the metabolism of glucose polymers must be coordinated with both the anabolic and catabolic uses of glucose and glycogen, an intricate set of regulatory mechanisms controls the expression of mal genes, the activity of the maltose transporter, and the activities of the maltose/maltodextrin catabolic enzymes. The ease of isolating many of the mal gene products has contributed greatly to the understanding of the structures and functions of several classes of proteins. Not only was the outer membrane maltoporin, LamB, or the phage lambda receptor, the first virus receptor to be isolated, but also its three-dimensional structure, together with extensive knowledge of functional sites for ligand binding as well as for phage lambda binding, has led to a relatively complete description of this sugar-specific aqueous channel. The periplasmic maltose binding protein (MBP) has been studied with respect to its role in both maltose transport and maltose taxis. Again, the combination of structural and functional information has led to a significant understanding of how this soluble receptor participates in signaling the presence of sugar to the chemosensory apparatus as well as how it participates in sugar transport. The maltose transporter belongs to the ATP binding cassette family, and although its structure is not yet known at atomic resolution, there is some insight into the structures of several functional sites, including those that are involved in interactions with MBP and recognition of substrates and ATP. A particularly astonishing discovery is the direct participation of the transporter in transcriptional control of the mal regulon. The MalT protein activates transcription at all mal promoters. A subset also requires the cyclic AMP receptor protein for transcription. The MalT protein requires maltotriose and ATP as ligands for binding to a dodecanucleotide MalT box that appears in multiple copies upstream of all mal promoters. Recent data indicate that the ATP binding cassette transporter subunit MalK can directly inhibit MalT when the transporter is inactive due to the absence of substrate. Despite this wealth of knowledge, there are still basic issues that require clarification concerning the mechanism of MalT-mediated activation, repression by the transporter, biosynthesis and assembly of the outer membrane and inner membrane transporter proteins, and interrelationships between the mal enzymes and those of glucose and glycogen metabolism.

FIG. 1

Regulatory region upstream of malT. The unboxed shaded area represents the cAMP/CAP binding site for the malT promoter. The boxed shaded area represents the binding site of Mlc, the regulator of malT expression, as determined by footprint analysis. The arrows at malT +1 and +1 malP indicate the transcriptional startpoints of the malT and malP transcripts, respectively. The arrows upstream of the malP promoter show the single and tandem MalT boxes needed for malP expression. −10 and −35 regions are indicated for the malT promoter only. The changes of AT to GC at position 39 (malTp7) and of GC to TA at position 100 (malTp1) are mutations that increase malT expression at the translational and transcriptional levels, respectively. malTp1 renders malT expression independent of the cAMP/CAP complex (34). The extent of the deletions upstream of the malT promoter, Δ511, Δ512, Δ513, and Δ514, are indicated. Their malT expression relative to the wild type is increased by a factor of 2.8 (Δ511), 2.2 (Δ512), 1.1 (Δ513), and 1.0 (Δ514) (200). The Tn10(Cam) insertion at nucleotide 418 leads to a 2.5-fold increase in malT expression. Dotted lines indicate homology to sites that have been identified as OmpR binding sites. In particular, I and III indicate homology to site FII and II indicates site FI as defined in reference . Modified from reference with permission of the publisher.

FIG. 2

Scheme for the promoter structures of the different mal operons. Arrows indicate transcriptional start points. Solid large arrow-shaped bullets represent MalT binding sites (MalT boxes), and open rectangles represent cAMP/CAP binding sites. The open arrow-shaped bullet indicates a MalT box that has been identified by footprinting but seems dispensable for Mal-dependent transcriptional activation of malP (46). This site overlaps a CAP binding site (dashed cAMP/CAP binding site) as determined by footprint analysis. Again, mutation of this site does not affect the transcription of malP (46). Similarly, the cAMP/CAP binding site most proximal to the malK promoter (dashed rectangle) has been identified by footprint analysis but is not essential for transcription of malK (279). Having two MalT binding sites in a direct repeat is a recurrent feature of mal promoters and plays a crucial role in their activation (281). The dashed arrow in the malZ promoter indicates a second transcriptional start site that is independent of MalT and more frequently used at high osmolarity. The cAMP/CAP binding site in the malS promoter has not been tested for its functional importance. The three cAMP/CAP binding sites between malK and malE are essential for the transcription of both genes (279).

FIG. 3

Crystal structures of the open and closed forms of MBP. (A) Structural representation of MBP in the open (top) and closed (bottom) forms. The yellow structure represents maltose bound within the binding site. Courtesy of Sherry Mowbray (247); reprinted with permission of the publisher. (B) Perspective view of the superimposed backbone structure of unliganded (mauve) and maltose-bound (blue) MBP (looking into the binding cleft from the side) with bound maltose (ball-and-stick model). The direction and magnitude of the conformational change when going from the unliganded to the ligand-bound form are shown. Reprinted from reference with permission of the publisher.

FIG. 3

Crystal structures of the open and closed forms of MBP. (A) Structural representation of MBP in the open (top) and closed (bottom) forms. The yellow structure represents maltose bound within the binding site. Courtesy of Sherry Mowbray (247); reprinted with permission of the publisher. (B) Perspective view of the superimposed backbone structure of unliganded (mauve) and maltose-bound (blue) MBP (looking into the binding cleft from the side) with bound maltose (ball-and-stick model). The direction and magnitude of the conformational change when going from the unliganded to the ligand-bound form are shown. Reprinted from reference with permission of the publisher.

FIG. 4

Two-dimensional structure of MalF and MalG. A topological model of MalF and MalG based on the analysis of malF::phoA and malG::phoA fusions is shown. MBP-independent mutants always require two mutations, one in the p (proximal) region and one in the d (distal) region. The G338R mutation in MalF alone causes MBP-dependent lactose transport. The EAAXXLG consensus motif is located in the cytoplasmic loop between MSS 6 and 7 of MalF and MSS 4 and 5 of MalG. Reprinted from reference with permission of the publisher.

FIG. 5

Crystal structure of LamB. A schematic drawing of the λ-receptor (maltoporin) monomer is shown. The cell exterior is at the top, and the periplasmic space at the bottom. The area of the subunit in trimer contacts is facing the viewer. The 18 antiparallel β strands of the barrel are represented by arrows. Strands are connected to their nearest neighbors by loops or regular turns. Loops L1 (blue), L3 (red), and L6 (green) fold inward toward the barrel. L3 is the major determinant of the constriction site. The yellow bond symbolizes the disulfide bridge Cys22-Cys38 within loop 1. Loop 2, facing the viewer, latches onto an adjacent subunit in the trimer. Loops L4 to L6 and L9 form a large protrusion. The horizontal lines delineate the boundaries of the hydrophobic core of the membrane as inferred from the hydrophobic area found on the molecular surface. Reprinted from reference with permission of the publisher.

FIG. 6

Maltose degradation by the maltose enzymes. The enzymes amylomaltase (malQ), maltodextrin phosphorylase (malP), and maltodextrin glucosidase (malZ) are indicated by their genes. After transport of maltose by the binding protein-dependent ABC transporter, a maltosyl, maltotriosyl, or maltotetraosyl (and so on) residue is transferred from maltotriose, maltotetraose, or maltopentaose (and so on) onto the incoming maltose releasing glucose in the process. Maltopentaose and longer maltodextrins are recognized by the maltodextrin phosphorylase, forming glucose-1-phosphate and a maltodextrin that is smaller by one glucosyl residue. Maltodextrin glucosidase recognizes maltotriose and longer maltodextrins (up to maltoheptaose), releasing glucose consecutively from the reducing end of the maltodextrin. This scheme demonstrates that maltose degradation by the maltose enzymes requires the endogenous presence of a maltodextrin primer with the minimal size of maltotriose. The activity of the last maltose enzyme, the periplasmic α-amylase, is not considered in this scheme. Adapted from reference with permission of the American Society for Microbiology.

FIG. 7

MalK cycle of transport and repression. A proposed model for the function of MalK in transport and regulation is shown. When maltose (solid circle) is present in the medium, it is recognized by MBP (MalE) and then transported through the membrane via MalFGK₂. In the process, the signal of substrate recognition is related to MalK on the cytoplasmic side of the membrane and ATP is hydrolyzed. Under these conditions, MalK is tightly associated with the MalFG complex and has no affinity for MalT. In the absence of maltose, the affinity of MalK for MalFG is lowered and it becomes available to interact with MalT, keeping MalT in its inactive state as a mal gene transcriptional activator. The key point in controlling the function of MalK is its association with ATP. When ATP is bound to MalK (but not hydrolyzed due to the absence of triggering substrate-loaded MBP), the protein is a repressor and interacts with MalT. This interaction is counteracted by the inducer (maltotriose). Thus, MalK cycles between an exclusively transporting and an exclusively MalT-inhibiting state, the predominance of transport or inhibition being determined by exogenous substrate and endogenous inducer. It should be stressed that there is no compelling evidence that MalK actually dissociates from MalFG during MalT inhibition. However, it is known that overproduced, i.e., unbound, MalK can exert repression.

FIG. 8

Structures of maltose and trehalose. (A) Maltose (α-d-glucopyranosyl-1→4-glucopyranose) in its predominant α enantiomeric form. (B) Trehalose, shown as the naturally occurring α-d-glucopyranosyl-1→1-α-d-glucopyranoside. The structures were calculated by minimizing the free energy of the axial and equatorial hydroxyl positions. The structures are shown in their chair conformation, with all substituents (except the anomeric oxygens) in the equatorial configuration.