Catalytic Module Research Articles

Catalytic cleave of an RNA substrate that bypasses the reorganization of its secondary structure during substrate recognition by a trans-acting VS ribozyme

Varkud satellite ribozyme (VS ribozyme) is a class of catalytic RNA with self-cleavage activity. The wild-type VS ribozyme has structural modularity with a relatively large catalytic module (H2–H6 elements) and a small substrate module (H1 element). The two modules can be dissected physically, and the substrate H1 RNA is recognized and then cleaved by the rest of the parent ribozyme serving as catalytic RNA. We characterized the catalytic properties of a bimolecular VS ribozyme developed and employed for an in-droplet evolution experiment of the VS ribozyme. We examined the effects of polyamines and several divalent metal ions. The results obtained in this study would be useful for the optimization of laboratory evolution of the VS ribozyme.

A comparative study of the cryo-EM structures of Saccharomyces cerevisiae and human anaphase-promoting complex/cyclosome (APC/C)

The anaphase-promoting complex/cyclosome (APC/C) is a large multi-subunit E3 ubiquitin ligase that controls progression through the cell cycle by orchestrating the timely proteolysis of mitotic cyclins and other cell cycle regulatory proteins. Although structures of multiple human APC/C complexes have been extensively studied over the past decade, the Saccharomyces cerevisiae APC/C has been less extensively investigated. Here, we describe medium resolution structures of three S. cerevisiae APC/C complexes: unphosphorylated apo-APC/C and the ternary APC/CCDH1-substrate complex, and phosphorylated apo-APC/C. Whereas the overall architectures of human and S. cerevisiae APC/C are conserved, as well as the mechanism of CDH1 inhibition by CDK-phosphorylation, specific variations exist, including striking differences in the mechanism of coactivator-mediated stimulation of E2 binding, and the activation of APC/CCDC20 by phosphorylation. In contrast to human APC/C in which coactivator induces a conformational change of the catalytic module APC2:APC11 to allow E2 binding, in S. cerevisiae apo-APC/C the catalytic module is already positioned to bind E2. Furthermore, we find no evidence of a phospho-regulatable auto-inhibitory segment of APC1, that in the unphosphorylated human APC/C, sterically blocks the CDC20C-box binding site of APC8. Thus, although the functions of APC/C are conserved from S. cerevisiae to humans, molecular details relating to their regulatory mechanisms differ.

A comparative study of the cryo-EM structures of Saccharomyces cerevisiae and human anaphase-promoting complex/cyclosome (APC/C).

The anaphase-promoting complex/cyclosome (APC/C) is a large multi-subunit E3 ubiquitin ligase that controls progression through the cell cycle by orchestrating the timely proteolysis of mitotic cyclins and other cell cycle regulatory proteins. Although structures of multiple human APC/C complexes have been extensively studied over the past decade, the Saccharomyces cerevisiae APC/C has been less extensively investigated. Here, we describe medium resolution structures of three S. cerevisiae APC/C complexes: unphosphorylated apo-APC/C and the ternary APC/CCDH1-substrate complex, and phosphorylated apo-APC/C. Whereas the overall architectures of human and S. cerevisiae APC/C are conserved, as well as the mechanism of CDH1 inhibition by CDK-phosphorylation, specific variations exist, including striking differences in the mechanism of coactivator-mediated stimulation of E2 binding, and the activation of APC/CCDC20 by phosphorylation. In contrast to human APC/C in which coactivator induces a conformational change of the catalytic module APC2:APC11 to allow E2 binding, in S. cerevisiae apo-APC/C the catalytic module is already positioned to bind E2. Furthermore, we find no evidence of a phospho-regulatable auto-inhibitory segment of APC1, that in the unphosphorylated human APC/C, sterically blocks the CDC20C-box binding site of APC8. Thus, although the functions of APC/C are conserved from S. cerevisiae to humans, molecular details relating to their regulatory mechanisms differ.

Visible-light-driven photobiocatalytic synthesis of chiral sulfoxides over immobilized chloroperoxidase on magnetic yolk–shell Fe3O4@Cu-H-TiO2 photocatalyst

Chloroperoxidase (CPO) is promising for the asymmetric synthesis of sulfoxides from sulfides, owing to its high enantioselectivity under mild reaction conditions. However, suffering from the stability and recyclability issue, the application of free CPO is heavily restricted in current stage. In this work, an integrated photobiocatalyst was designed and fabricated by immobilizing CPO on a magnetically recyclable photocatalyst, Fe3O4@Cu-H-TiO2, which can supply H2O2 for CPO in situ continuously and suppress the destruction of CPO caused by excess H2O2. The obtained CPO@Fe3O4@Cu-H-TiO2 can catalyze the synthesis of enantiopure (R)-sulfoxides from sulfides with high yield up to 88% and high ee value up to >99% over this TiO2-based photobiocatalyst. The enhanced performance of the integrated photobiocatalyst is attributed to the controllable supply of H2O2, the improved stability of immobilized CPO and the enhanced proximity effect between photo- and bio- catalytic modules.

Plant BCL-Domain Homologues play a conserved role in SWI/SNF complex stability.

The SWItch/Sucrose Non-Fermenting (SWI/SNF) complexes are evolutionarily conserved, ATP-dependent chromatin remodelers crucial for multiple nuclear functions in eukaryotes. Recently, plant BCL-Domain Homolog (BDH) proteins were identified as shared subunits of all plant SWI/SNF complexes, significantly impacting chromatin accessibility and various developmental processes in Arabidopsis. In this study, we performed a comprehensive characterization of bdh mutants, revealing a previously overlooked impact on hypocotyl cell elongation. Through detailed analysis of BDH domains, we identified a plant-specific N-terminal domain that facilitates the interaction between BDH and the rest of the complex. Additionally, we uncovered the critical role of the BDH β-hairpin domain, which is phylogenetically related to metazoan BCL7 SWI/SNF subunits. While phylogenetic analyses did not identify BDH/BCL7 orthologs in fungi, structure prediction modeling demonstrated strong similarities between the SWI/SNF catalytic modules of plants, animals, and fungi, and revealed the yeast Rtt102 protein as a structural homolog of BDH and BCL7. This finding is supported by the ability of Rtt102 to interact with the Arabidopsis catalytic module subunit ARP7 and partially rescue the bdh mutant phenotypes. Further experiments revealed that BDH promotes the stability of the ARP4-ARP7 heterodimer, leading to the partial destabilization of ARP4 in the SWI/SNF complexes. In summary, our study unveils the molecular function of BDH proteins in plant SWI/SNF complexes and suggests that β-hairpin-containing proteins are evolutionarily conserved subunits crucial for ARP heterodimer stability and SWI/SNF activity across eukaryotes.

Screening and modification of (+)-germacrene A synthase for the production of the anti-tumor drug (−)-β-elemene in engineered Saccharomyces cerevisiae

(−)-β-Elemene is a primary bioactive compound derived from Curcuma wenyujin and has been widely utilized as an anti-tumor agent for various types of cancer. Due to the inefficiency of plant extraction methods for β-elemene, significant efforts have been directed toward the heterogeneous biosynthesis of β-elemene using microbial cell factories. However, there has been less emphasis on the stereochemical configuration of germacrene A and its rearranged product, β-elemene. In this study, we constructed a yeast cell factory to produce (−)-β-elemene by optimizing the mevalonate pathway and screening for germacrene A synthases (GASs) from both plant and microbial sources. Notably, we discovered that the rearranged products of GASs exhibited different conformations, and only (+)-germacrene A produced by plant-derived GASs could rearrange to form (−)-β-elemene. Building on this discovery, we further investigated the catalytic mechanisms of GASs and developed an efficient catalytic gene module for generating (+)-germacrene A. Ultimately, the engineered yeast produced 1152 mg/L of (−)-β-elemene, marking the highest titer reported in yeast to date. Overall, this work highlights the differences in the stereoconformations of catalytic products between plant- and microbial-derived germacrene A synthases and establishes a foundation for the green and efficient production of β-elemene with a specific stereochemical configuration.

Signaling by a bacterial phytochrome histidine kinase involves a conformational cascade reorganizing the dimeric photoreceptor

Phytochromes (Phys) are a divergent cohort of bili-proteins that detect light through reversible interconversion between dark-adapted Pr and photoactivated Pfr states. While our understandings of downstream events are emerging, it remains unclear how Phys translate light into an interpretable conformational signal. Here, we present models of both states for a dimeric Phy with histidine kinase (HK) activity from the proteobacterium Pseudomonas syringae, which were built from high-resolution cryo-EM maps (2.8–3.4-Å) of the photosensory module (PSM) and its following signaling (S) helix together with lower resolution maps for the downstream output region augmented by RoseTTAFold and AlphaFold structural predictions. The head-to-head models reveal the PSM and its photointerconversion mechanism with strong clarity, while the HK region is interpretable but relatively mobile. Pr/Pfr comparisons show that bilin phototransformation alters PSM architecture culminating in a scissoring motion of the paired S-helices linking the PSMs to the HK bidomains that ends in reorientation of the paired catalytic ATPase modules relative to the phosphoacceptor histidines. This action apparently primes autophosphorylation enroute to phosphotransfer to the cognate DNA-binding response regulator AlgB which drives quorum-sensing behavior through transient association with the photoreceptor. Collectively, these models illustrate how light absorption conformationally translates into accelerated signaling by Phy-type kinases.

Structural insights into the assembly of type IIA topoisomerase DNA cleavage-religation center.

The ability to catalyze reversible DNA cleavage and religation is central to topoisomerases' role in regulating DNA topology. In type IIA topoisomerases (Top2), the formation of its DNA cleavage-religation center is driven by DNA-binding-induced structural rearrangements. These changes optimally position key catalytic modules, such as the active site tyrosine of the WHD domain and metal ion(s) chelated by the TOPRIM domain, around the scissile phosphodiester bond to perform reversible transesterification. To understand this assembly process in detail, we report the catalytic core structures of human Top2α and Top2β in an on-pathway conformational state. This state features an in trans formation of an interface between the Tower and opposing TOPRIM domain, revealing a groove for accommodating incoming G-segment DNA. Structural superimposition further unveils how subsequent DNA-binding-induced disengagement of the TOPRIM and Tower domains allows a firm grasp of the bound DNA for cleavage/religation. Notably, we identified a previously undocumented protein-DNA interaction, formed between an arginine-capped C-terminus of an α-helix in the TOPRIM domain and the DNA backbone, significantly contributing to Top2 function. This work uncovers a previously unrecognized role of the Tower domain, highlighting its involvement in anchoring and releasing the TOPRIM domain, thus priming Top2 for DNA binding and cleavage.

Ultrathin silica-tiling on living cells for chemobiotic catalysis

Harnessing the power of cell biocatalysis for sustainable chemical synthesis requires rational integration of living cells with the modern synthetic catalysts. Here, we develop silica-tiling strategy that constructs a hierarchical, inorganic, protocellular confined nanospace around the individual living cell to accommodate molecularly accessible abiotic catalytic sites. This empowers the living microorganisms for new-to-nature chemical synthesis without compromising the cellular regenerative process. Yeast cell, a widely used biocatalyst, is upgraded via highly controlled self-assembly of 2D-bilayer silica-based catalytic modules on cell surfaces, opening the avenues for diverse chemobiotic reactions. For example, combining [AuPt]-catalyzed NADH regeneration, light-induced [Pd]-catalyzed C-C cross-coupling or lipase-catalyzed esterification reactions—with the natural ketoreductase activity inside yeast cell. The conformal silica bilayer provides protection while allowing proximity to catalytic sites and preserving natural cell viability and proliferation. These living nanobiohybrids offer to bridge cell’s natural biocatalytic capabilities with customizable heterogeneous metal catalysis, enabling programmable reaction sequences for sustainable chemical synthesis.

Bacterial microcompartments as a next-generation metabolic engineering tool: utilizing nature's solution for confining challenging catabolic pathways.

Advancements in synthetic biology have facilitated the incorporation of heterologous metabolic pathways into various bacterial chassis, leading to the synthesis of targeted bioproducts. However, total output from heterologous production pathways can suffer from low flux, enzyme promiscuity, formation of toxic intermediates, or intermediate loss to competing reactions, which ultimately hinder their full potential. The self-assembling, easy-to-modify, protein-based bacterial microcompartments (BMCs) offer a sophisticated way to overcome these obstacles by acting as an autonomous catalytic module decoupled from the cell's regulatory and metabolic networks. More than a decade of fundamental research on various types of BMCs, particularly structural studies of shells and their self-assembly, the recruitment of enzymes to BMC shell scaffolds, and the involvement of ancillary proteins such as transporters, regulators, and activating enzymes in the integration of BMCs into the cell's metabolism, has significantly moved the field forward. These advances have enabled bioengineers to design synthetic multi-enzyme BMCs to promote ethanol or hydrogen production, increase cellular polyphosphate levels, and convert glycerol to propanediol or formate to pyruvate. These pioneering efforts demonstrate the enormous potential of synthetic BMCs to encapsulate non-native multi-enzyme biochemical pathways for the synthesis of high-value products.

Klebsiella pneumoniae O-polysaccharide biosynthesis highlights the diverse organization of catalytic modules in ABC transporter-dependent glycan assembly

Klebsiella pneumoniae provides influential prototypes for lipopolysaccharide O antigen (OPS) biosynthesis in Gram-negative bacteria. Sequences of OPS-biosynthesis gene clusters in serotypes O4 and O7 suggest fundamental differences in the organization of required enzyme modules compared to other serotypes. Furthermore, some required activities were not assigned by homology shared with characterized enzymes. The goal of this study was therefore to resolve the serotype O4 and O7 pathways, to expand our broader understanding of glycan polymerization and chain termination processes. The O4 and O7 antigens were produced from cloned genetic loci in recombinant Escherichia coli. Systematic in vivo and in vitro approaches were then applied to assign each enzyme in each of the pathways, defining the necessary components for polymerization and chain termination. OPS assembly is accomplished by multiprotein complexes formed by interactions between polymerase components variably distributed in single and multi-module proteins. In each complex, a terminator function is present in a protein containing a characteristic coiled-coil molecular ruler, which determines glycan chain-length. In serotype O4, we discovered a CMP-α-3-deoxy-ᴅ-manno-octulosonic acid (Kdo)-dependent chain-terminating glycosyltransferase that is the founding member of a new glycosyltransferase family (GT137), and potentially identifies a new glycosyltransferase fold. The O7 OPS is terminated by a methylphosphate moiety, like the K. pneumoniae O3 antigen, but the methyltransferase-kinase enzyme pairs responsible for termination in these serotypes differ in sequence and predicted structures. Together, the characterization of O4 and O7 has established unique enzyme activities and provided new insight into glycan-assembly strategies that are widely distributed in bacteria.

Molecular basis of bacterial DSR2 anti-phage defense and viral immune evasion

Defense-associated sirtuin 2 (DSR2) systems are widely distributed across prokaryotic genomes, providing robust protection against phage infection. DSR2 recognizes phage tail tube proteins and induces abortive infection by depleting intracellular NAD+, a process that is counteracted by another phage-encoded protein, DSR Anti Defense 1 (DSAD1). Here, we present cryo-EM structures of Bacillus subtilis DSR2 in its apo, Tube-bound, and DSAD1-bound states. DSR2 assembles into an elongated tetramer, with four NADase catalytic modules clustered in the center and the regulatory-sensing modules distributed at four distal corners. Interestingly, monomeric Tube protein, rather than its oligomeric states, docks at each corner of the DSR2 tetramer to form a 4:4 DSR2-Tube assembly, which is essential for DSR2 NADase activity. DSAD1 competes with Tube for binding to DSR2 by occupying an overlapping region, thereby inhibiting DSR2 immunity. Thus, our results provide important insights into the assembly, activation and inhibition of the DSR2 anti-phage defense system.

Engineering the substrate channeling interface between the heterologous modules for a highly efficient FAD synthetase

Flavin adenine dinucleotide synthetase (FADS) orchestrates the biosynthesis of flavin adenine dinucleotide (FAD) with two consecutive catalytic modules. The process entails the release and transfer of the reactant from the initial module to the subsequent one, while the transport process of the intermediate flavin mononucleotide (FMN) between these modules remains unclear. This study focuses on the oligomeric assembly of CaFADS from Corynebacterium ammoniagenes, revealing insights into substrate channeling interface between the two heterologous modules. Employing a semi-rational mutagenesis for residues in the interfaces of the heterologous modules, L204V is screened out and exhibited a 2.3-fold improvement in FAD productivity compared to the wild type. Molecular dynamics simulations indicate the L204V mutant facilitates a larger exit for the intermediate FMN, expediting its release from the first catalytic module. While the mutant showed minor changes of binding affinity with the riboflavin calculated by surface plasmon resonance assay, steered MD simulations of FMN from the first catalytic module demonstrate that the L204V mutant reduces the binding free energy for FMN departure, substantiating its role in improving substrate transport efficiency. The findings shed light on the intricate interplay between different modules of bifunctional enzymes and emphasize the importance of substrate channeling in enhancing catalytic efficiency.

Value‐Added Cascade Synthesis Mediated by Paired‐Electrolysis Using an Ultrathin Microenvironment‐Inbuilt Metalized Covalent Organic Framework Heterojunction

AbstractEnergy‐saving and value‐added management in advanced catalysis is highly desirable but is challenged by the limitations of multifunctional catalysts and catalytic modules. Herein, an azo‐linked phthalocyanine‐porphyrin covalent organic framework (COF) with the ultrathin layered nanostructure grown on carbon nanotubes (NiPc‐azo‐H2Pp@CNTs) has been designed and synthesized, which can serve as a highly active and stable bifunctional heterojunction electrocatalyst for selective paired‐electrosynthesis through coupling anodic iodide oxidation reaction and cathodic CO2 conversion. Particularly, the inbuilt local microenvironment conferred by the dihydroporphyrin moieties in COF can act as a proton reservoir to promote the proton relay at the heterojunction interface during the electrocatalytic process. Moreover, through the cascade construction of dual electrocatalytic/organocatalytic modules, the cathode‐generated CO can be further converted to dimethyl carbonate with a yield of 6.21 mmol L−1 h−1, while the anode‐produced iodine can be derived into iodoform on the hundred‐milligram scale. It is worth noting that the value‐added cascade synthesis mediated by paired‐electrolysis using the distinctive and high‐powered electrocatalysts will help advance the sustainable development of industrial intelligent manufacturing.

Structural characterization of lytic transglycosylase MltD of Pseudomonas aeruginosa, a target for the natural product bulgecin A

Natural product bulgecin A potentiates the activity of β-lactam antibiotics by inhibition of three lytic transglycosylases in Pseudomonas aeruginosa, of which MltD is one. MltD exhibits both endolytic and exolytic reactions in the turnover of the cell-wall peptidoglycan and tolerates the presence or absence of stem peptides in its substrates. The present study reveals structural features of the multimodular MltD, presenting a catalytic module and four cell-wall-binding LysM modules that account for these attributes. Three X-ray structures are reported herein for MltD that disclose one unpredicted LysM module tightly attached to the catalytic domain, whereas the other LysM modules are mobile, and connected to the catalytic domain through long flexible linkers. The formation of crystals depended on the presence of bulgecin A. The expansive active-site cleft is highlighted by the insertion of a helical region, a hallmark of the family 1D of lytic transglycosylases, which was mapped out in a ternary complex of MltD:bulgecinA:chitotetraose, revealing at the minimum the presence of eight subsites (from −4 to +4, with the seat of reaction at subsites −1 and + 1) for binding of sugars of the substrate for the endolytic reaction. The mechanism of the exolytic reaction is revealed in one of the structures, showing how the substrate's terminal anhydro-NAM moiety could be sequestered at subsite +2. Our results provide the structural insight for both the endolytic and exolytic activities of MltD during cell-wall-turnover events.

The CBM91 module enhances the activity of β-xylosidase/α-L-arabinofuranosidase PphXyl43B from Paenibacillus physcomitrellae XB by adopting a unique loop conformation at the top of the active pocket

Carbohydrate-binding module (CBM) family 91 is a novel module primarily associated with glycoside hydrolase (GH) family 43 enzymes. However, our current understanding of its function remains limited. PphXyl43B is a β-xylosidase/α-L-arabinofuranosidase bifunctional enzyme from physcomitrellae patens XB belonging to the GH43_11 subfamily and containing CBM91 at its C terminus. To fully elucidate the contributions of the CBM91 module, the truncated proteins consisting only the GH43_11 catalytic module (rPphXyl43B-dCBM91) and only the CBM91 module (rCBM91) of PphXyl43B were constructed, respectively. The result showed that rPphXyl43B-dCBM91 completely lost hydrolysis activity against both p-nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside; it also exhibited significantly reduced activity towards xylobiose, xylotriose, oat spelt xylan and corncob xylan compared to the control. Thus, the CBM91 module is crucial for the β-xylosidase/α-L-arabinofuranosidase activities in PphXyl43B. However, rCBM91 did not exhibit any binding capability towards corncob xylan. Structural analysis indicated that CBM91 of PphXyl43B might adopt a loop conformation (residues 496–511: ILSDDYVVQSYGGFFT) to actively contribute to the catalytic pocket formation rather than substrate binding capability. This study provides important insights into understanding the function of CBM91 and can be used as a reference for analyzing the action mechanism of GH43_11 enzymes and their application in biomass energy conversion.

The first crystal structure of a family 45 glycoside hydrolase from a brown-rot fungus, Gloeophyllum trabeum GtCel45A.

Here we describe the first crystal structure of a beta-1,4-endoglucanase from a brown-rot fungus, Gloeophyllum trabeum GtCel45A, which belongs to subfamily C of glycoside hydrolase family 45 (GH45). GtCel45A is ~ 18 kDa in size and the crystal structure contains 179 amino acids. The structure is refined at 1.30 Å resolution and Rfree 0.18. The enzyme consists of a single catalytic module folded into a six-stranded double-psi beta-barrel domain surrounded by long loops. GtCel45A is very similar in sequence (82% identity) and structure to PcCel45A from the white-rot fungus Phanerochaete chrysosporium. Surprisingly though, initial hydrolysis of barley beta-glucan was almost twice as fast in GtCel45A as compared to PcCel45A.

Computation-Aided Phylogeny-Oriented Engineering of β-Xylosidase: Modification of "Blades" to Enhance Stability and Activity for the Bioconversion of Hemicellulose to Produce Xylose.

Hemicellulose is a highly abundant, ubiquitous, and renewable natural polysaccharide, widely present in agricultural and forestry residues. The enzymatic hydrolysis of hemicellulose has generally been accomplished using β-xylosidases, but concomitantly increasing the stability and activity of these enzymes remains challenging. Here, we rationally engineered a β-xylosidase from Bacillus clausii to enhance its stability by computation-aided design combining ancestral sequence reconstruction and structural analysis. The resulting combinatorial mutant rXYLOM25I/S51L/S79E exhibited highly improved robustness, with a 6.9-fold increase of the half-life at 60 °C, while also exhibiting improved pH stability, catalytic efficiency, and hydrolytic activity. Structural analysis demonstrated that additional interactions among the propeller blades in the catalytic module resulted in a much more compact protein structure and induced the rearrangement of the opposing catalytic pocket to mediate the observed improvement of activity. Our work provides a robust biocatalyst for the hydrolysis of agricultural waste to produce various high-value-added chemicals and biofuels.

UBE2A and UBE2B are recruited by an atypical E3 ligase module in UBR4.

UBR4 is a 574 kDa E3 ligase (E3) of the N-degron pathway with roles in neurodevelopment, age-associated muscular atrophy and cancer. The catalytic module that carries out ubiquitin (Ub) transfer remains unknown. Here we identify and characterize a distinct E3 module within human UBR4 consisting of a 'hemiRING' zinc finger, a helical-rich UBR zinc-finger interacting (UZI) subdomain, and an N-terminal region that can serve as an affinity factor for the E2 conjugating enzyme (E2). The structure of an E2-E3 complex provides atomic-level insight into the specificity determinants of the hemiRING toward the cognate E2s UBE2A/UBE2B. Via an allosteric mechanism, the UZI subdomain modestly activates the Ub-loaded E2 (E2∼Ub). We propose attenuated activation is complemented by the intrinsically high lysine reactivity of UBE2A, and their cooperation imparts a reactivity profile important for substrate specificity and optimal degradation kinetics. These findings reveal the mechanistic underpinnings of a neuronal N-degron E3, its specific recruitment of UBE2A, and highlight the underappreciated architectural diversity of cross-brace domains with Ub E3 activity.

Development of large-flux laminated methanol reforming microreactor heated by methanol catalytic combustion

To achieve high flow hydrogen production, a laminated self-thermal methanol reforming microreactor was designed. Through simulation and experiments, the hydrogen production performance of the microreactor and the compatibility between the methanol catalytic combustion module and the methanol steam reforming module were investigated. The results show that the microreactor exhibits impressive hydrogen production performance, with a methanol conversion rate of 95.27%. When the inlet flow rate of the mixture of methanol and deionized water was increased to a high flow rate, the microreactor exhibits significant cold spots over a wide range in the reaction chamber plates, which could be effectively solved by enhancing the methanol catalytic combustion intensity. The methanol catalytic combustion module inside the microreactor can meet the heat demand of the methanol reforming module. The microreactor operated for 20 h with a methanol-deionized water mixture inlet flow rate of 12 mL/min and a methanol inlet flow rate of 7.5 mL/min; the methanol conversion rate finally stabilized at 90.1%, which was decreased by about 2.2% compared with the initial methanol conversion rate.