Structural and kinetic characterization of (S)-1-amino-2-propanol kinase from the aminoacetone utilization microcompartment of Mycobacterium smegmatis

Abstract

Bacterial microcompartments encapsulate enzymatic pathways that generate small, volatile, aldehyde intermediates. The Rhodococcus and Mycobacterium microcompartment (RMM) operon from Mycobacterium smegmatis encodes four enzymes, including (S)-1-amino-2-propanol dehydrogenase and a likely propionaldehyde dehydrogenase. We show here that a third enzyme (and its nonmicrocompartment-associated paralog) is a moderately specific (S)-1-amino-2-propanol kinase. We determined the structure of apo-aminopropanol kinase at 1.35 Å, revealing that it has structural similarity to hexosamine kinases, choline kinases, and aminoglycoside phosphotransferases. We modeled substrate binding, and tested our model by characterizing key enzyme variants. Bioinformatics analysis established that this enzyme is widespread in Actinobacteria, Proteobacteria, and Firmicutes, and is very commonly associated with a candidate phospholyase. In Rhizobia, aminopropanol kinase is generally associated with aromatic degradation pathways. In the RMM (and the parallel pathway that includes the second paralog), aminopropanol kinase likely degrades aminoacetone through a propanolamine-phosphate phospho-lyase-dependent pathway. These enzymatic activities were originally described in Pseudomonas, but the proteins responsible have not been previously identified. Bacterial microcompartments typically co-encapsulate enzymes which can regenerate required co-factors, but the RMM enzymes require four biochemically distinct co-factors with no overlap. This suggests that either the RMM shell can uniquely transport multiple co-factors in stoichiometric quantities, or that all enzymes except the phospho-lyase reside outside of the shell. In summary, aminopropanol kinase is a novel enzyme found in diverse bacteria and multiple metabolic pathways; its presence in the RMM implies that this microcompartment degrades aminoacetone, using a pathway that appears to violate some established precepts as to how microcompartments function.

Keywords: aminoacetone utilization microcompartment; aminopropanol kinase; biodegradation; bioinformatics; enzyme kinetics; microbiology; protein structure.

Figure 1.

Enzyme kinetics. APK_MSM0270 initial reaction rates were measured by changes in the NADH concentration due to ADP production coupled with pyruvate kinase/lactate dehydrogenase, and fit to the Michaelis-Menten equation by nonlinear regression. A, kinetics data for S-(+)-1-amino-2-propanol and B, kinetics data for ATP.

Figure 2.

Structure of APK_MSM270. A, structure of the APK_MSM0270 monomer. Secondary structure elements are labeled. B, superposition of APK_MSM0270 with representative structural homologs. APK_MSM0270 is shown in dark blue. N-Acetylhexosamine-1-phosphate kinase (PDB 4ocq) is shown in pale yellow. Choline kinase (PDB 4r78) in pale orange. Spectinomycin phosphotransferase (PDB 3i0o) is shown in pale cyan. C, details of the catalytic site of APK_MSM0270. D, model of the ternary complex of APK_MSM0270 with ATP and propanolamine. E, multiple sequence alignment of APK with structural homologs. The secondary structure is colored as described in A. APK_MSM0270 is aligned with APK_MSM0780 (greyed residues are present in UniProt but are likely a misannotation), S. pneumoniae bacterial choline kinase (BCHK, UniProt Q8DPI4), Homo sapiens choline kinase B (HCHKB, UniProt Q9Y259), B. longum N-acetylhexosamine 1-kinase (NAHK, UniProt E8MF12), and Enterococcus casseliflavus aminoglycoside phosphotransferase (2′)-Iva. The secondary structure and residue numbering of APK_MSM0270 are aligned with the multiple sequence alignment. Colored circles below the multiple sequence alignment indicate residues of the hydrophobic methyl binding pocket (gray), substrate α-amino binding (pink), conserved Brenner motif (green), and the phosphate-product stabilizing residue (light blue).

Figure 3.

Bioinformatics analysis of APK homologs. A, sequence similarity network of APK homologs. Sequences are connected by edges if E < e-50. Sequences are colored by phyla, as noted in the key. Phyla marked (c) are candidate phyla. APK homologs highlighted in B are indicated. B, gene organization of representative examples of APK homologs. Gene labels: BMC, bacterial microcompartment shell proteins; AT3, aminotransferase III; trans., transporter; 4HPH, 4-hydroxyphenylacetate 3-hydroxylase; FD, flavin reductase; HHMI, 5-carboxymethyl-2-hydroxymuconatedelta-isomerase; CHMD, 5-carboxy-2-hydroxymuconate semialdehyde dehydrogenase; DHPD, 3,4-dihydroxyphenylacetate 2,3-dioxygenase; FAAH, fumarylacetoacetate hydrolase; 2KPH, 2-keto-4-pentenoate hydratase; OHDH, 2-oxo-hept-3-ene-1,7-dioate hydratase; CTTD, catechol 2,3-dioxygenase.

Figure 4.

Proposed organization of, and pathway mediated by, the aminoacetone utilization microcompartment. Aminoacetone is imported into the cell via a aminopropanol permease (AAP). It is then reduced to (S)-aminopropanol by APDH, which is in turn phosphorylated APK. Both of these reactions occur in the cytosol. Aminopropanol-phosphate then enters the microcompartment through the shell, and is transformed into propionaldehyde by (S)-1-amino-2-propanol-phosphate phospho-lyase (APPL). Propionaldehyde escapes through the shell, and is captured by propionaldehyde dehydrogenase (AldDH), which adheres to the exterior face of the microcompartment via its encapsulation peptide.